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WO2018101375A1 - Procédé de détection d'adn génomique humain - Google Patents

Procédé de détection d'adn génomique humain Download PDF

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WO2018101375A1
WO2018101375A1 PCT/JP2017/042943 JP2017042943W WO2018101375A1 WO 2018101375 A1 WO2018101375 A1 WO 2018101375A1 JP 2017042943 W JP2017042943 W JP 2017042943W WO 2018101375 A1 WO2018101375 A1 WO 2018101375A1
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primer
nucleotide sequence
probe
criteria
human
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英雄 明石
広大 船越
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Akita University NUC
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Akita University NUC
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

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  • the present invention relates to a method for designing a PCR primer pair for detecting human Alu, a PCR primer pair for detecting human Alu designed using the PCR primer pair, and human genomic DNA in a test sample using the PCR primer pair for detecting human Alu.
  • the present invention relates to a method for detection and / or quantification.
  • the present invention also provides a method of designing a PCR probe for detecting human Alu, a PCR probe for detecting human Alu designed using the same, a PCR probe for detecting human Alu, and the PCR primer for detecting human Alu.
  • the present invention relates to a method for detecting and / or quantifying human genomic DNA in a test sample.
  • the Alu sequence is a kind of SINE (short interspersed element) that is a retrotransposon, and is a repetitive sequence that exists specifically in the genome of primates including humans.
  • the Alu sequence is composed of two 7SL RNA-derived sequences called left monomer and right monomer, and an A-rich sequence sandwiched between them.
  • the Alu sequence is specific to the primate genome, it has been found that the 7SL RNA-derived sequence that forms part of it is also present in the rodent genome. Further, recent research has revealed that the Alu sequences in the human genome are not all composed of the same sequence, but are composed of 46 subfamilies (Non-patent Document 1).
  • the Alu sequence is known to be present in over 1 million copies in the human genome and occupy 10% or more of the human genome. Since the total length of the human genome is about 3 billion nucleotide pairs, a simple calculation means that there is an average of 1 copy of Alu sequence per 3000 nucleotide pairs of human genomic DNA. In terms of weight, since the weight of the whole haploid human genome is 3 pg, 0.003 fg of human genomic DNA is considered to contain an average of 1 copy of Alu sequence. Thus, the Alu sequence is always present in a very small amount of human genomic DNA, and since it is a nucleotide sequence specific to primates, it is considered an ideal target for detection and quantification of human cells. It has been.
  • Alu-qPCR quantitative real-time PCR method
  • Patent Document 1 A number of methods for detecting and quantifying a human genome using a quantitative real-time PCR method (hereinafter sometimes referred to as “Alu-qPCR”) targeting an Alu sequence have been reported (for example, Patent Document 1).
  • Non-patent document 2 As kits for Alu-qPCR, Innoquant (registered trademark) (manufactured by InnoGenomics Technologies) and Femto (registered trademark) Human DNA Quantification kit (manufactured by ZYMO RESEARCH) are already on the market.
  • Innoquant registered trademark
  • Femto registered trademark Human DNA Quantification kit
  • Non-patent Document 3 it is possible to confirm the degree of engraftment and proliferation of human stem cells xenografted into mice or the like (Non-patent Document 3), and the ability to diagnose cancer by measuring blood DNA concentration (non-patent document 3).
  • Patent Document 4 it has been reported that human genomic DNA in old samples used in the forensic field can be detected (Non-Patent Document 5).
  • Alu-qPCR reported so far has sufficient sensitivity. That is, theoretically, it should be possible to detect Alu from human genomic DNA of about 0.1 fg by real-time PCR, but known Alu-qPCR requires human genomic DNA of several pg or more (non-patented). Reference 5), extremely low sensitivity is obtained compared to the theoretical detection limit.
  • known Alu-qPCR primers contain the same nucleotide sequence as that of a part of the genome of a non-human species, and samples using a mixture of genomic DNA derived from other species and human genomic DNA are used. It was impossible to specifically detect trace amounts of Alu. From the above, development of Alu-qPCR having higher sensitivity and specificity has been demanded.
  • An object of the present invention is to specifically amplify human Alu sequences and to further minimize the formation of primer dimers (homodimers and heterodimers), and a PCR primer pair for detecting human Alu, and to detect the human Alu
  • a probe that specifically hybridizes to an amplification product by a PCR primer pair, and further capable of minimizing heterodimer formation and homodimer formation between the primer pair and the probe. It is in.
  • an object of the present invention is to provide a method for detecting and / or quantifying human genomic DNA in a test sample with high sensitivity using the PCR primer pair and probe for detecting human Alu.
  • the present inventors have intensively studied to solve the above-mentioned problems, and identified one “human Alu model sequence” that represents a consensus sequence of 46 human Alu subfamilies and used this Alu model sequence in the human genome.
  • the present inventors designed a primer pair and a probe from the above human Alu model sequence using a general primer / probe design program (Primer 3). Alu-qPCR was performed using such a primer pair and probe.
  • a sensitivity as low as that of the prior art detection limit value is more than the number of human genomic DNA pg
  • the second nucleotide from the 3 ′ end is G or C;
  • At least one of the nucleotides from the 3 'end to the 3rd end of the primer is A or T;
  • [Criteria 6] A nucleotide sequence complementary to a continuous nucleotide sequence of 5 or more nucleotides contained in any one molecule between the two primers, between the forward primers, between the reverse primers, and between the forward primer and the reverse primer.
  • the sequence does not contain the other molecule;
  • Tm value of the probe is designed to be about 10 ° C. higher than the Tm value of the primer.
  • the 3 ′ end of the primer should not be T (eg, Apte, A. and Daniel, S. (2009) PCR primer design. Cold Spring Harb Protoc, 2009).
  • Alu is a primate-specific sequence, the possibility that the primer and the probe hybridize to the genomic DNA of an organism other than humans has not been considered at all.
  • the present inventors provided the above criteria 1 and 8 in order to obtain a primer pair and a probe that can specifically detect human Alu from a sample in which genomic DNAs of organisms other than humans are mixed. Furthermore, the above criteria 3, 7, and 12 are completely new criteria that have not been reported so far.
  • the present inventors used the above criteria 1 to 7 to set a pair of primers (forward primer: GGTGAAACCCCGTCTCTACT (SEQ ID NO: 18), reverse primer: GGTTCAAGCGATTCTCCTG (SEQ ID NO: 19)), and four probes (ATACAAAAATAGTAGCGGGCG (sequence) No. 37), TACAAAAATTTAGCCGGGCGT (SEQ ID NO: 39), CGCCCGGCTAATTTTGTTAT (SEQ ID NO: 42), and ACGCCCGGCTAATTTTTGTA (SEQ ID NO: 47)).
  • the Tm value of the actually selected probe is the Tm value of the forward primer and the reverse primer. Compared to the value, it was 1-2 ° C. lower and the 3 ′ end of the forward primer was T.
  • the present inventors performed Alu-qPCR by combining these primer pairs and probes.
  • the test sample is a domestic animal / pet. Even when animal-derived genomic DNA is included, it is possible to quantitatively measure human genomic DNA in the range of 10 fg to 10 ng.
  • Clarified that human genomic DNA in the range of 10 fg to 10 ng can be quantitatively measured even if the test sample is an old sample (a sample derived from an ancient human bone several hundred years ago). .
  • the inventors improved the above criteria 1 to 11 in order to design a primer / probe set capable of quantifying highly fragmented human genomic DNA (100 bp), and the following primer criteria 1′ ⁇ 7 'and essential criteria for probes 8' to 11 'were provided.
  • the nucleotide sequence consisting of 17 or more consecutive nucleotides in the forward primer and the reverse primer is at two positions in the nucleotide sequence of one or more genomic DNAs selected from the group consisting of non-human organisms.
  • the second nucleotide from the 3 ′ end is G or C;
  • [Criteria 7 ′] A nucleotide sequence complementary to a continuous nucleotide sequence of 4 or more nucleotides consisting of G or C contained in either molecule between the two molecules of the forward primer and the reverse primer, Contains no molecules of [Criteria 8 ′] The nucleotide sequence consisting of 17 or more consecutive nucleotides in the probe does not exist in two or more places in the nucleotide sequence of one or more genomic DNAs selected from the group consisting of non-human organisms; [Criteria 9 ′] The probe has a nucleotide length of 18 or more; [Criteria 10 ′] The nucleotide sequence from the 3 ′ end to the 3rd position of either molecule between the forward primer and the probe and the reverse primer and the probe (the 1st to 3rd nucleotide sequence from the 3 ′ end) And the other molecule does not contain a complementary nucleotide sequence; [Criteria 7 ′] A nu
  • the present inventors selected a set of primer pairs (forward primer: GGCGGAGGTTGCAGTGAG (SEQ ID NO: 123), reverse primer: GTCTCGCTCTGTCGCCCA (SEQ ID NO: 148)) using the above criteria 1 ′ to 7 ′, and Using the criteria 8 ′ to 11 ′ and the above criteria 13 and 15, one probe (TGCAGTGGCCGCGATCTCG (SEQ ID NO: 149)) was selected. Then, the present inventors performed Alu-qPCR by combining these primer pairs and probes, and as a result, even human genomic DNA fragmented at 100 bp can be quantitatively measured in the range of 10 fg to 10 ng. It was revealed that.
  • the inventors have used a PCR primer / probe for detecting human Alu designed based on original criteria, and have a sensitivity close to the theoretical detection limit (that is, several hundred times more than the conventional technology).
  • the present inventors have found that Alu-qPCR having (sensitivity) is possible, and have completed the present invention.
  • the present invention (1) From the forward primer group and the reverse primer group capable of amplifying a part or all of the region of the human Alu model sequence consisting of the nucleotide sequence shown in SEQ ID NO: 4, among [Criteria 1 ′] forward primer and reverse primer 2 or more nucleotide sequences of 17 or more consecutive nucleotides are not present in the nucleotide sequence of one or more genomic DNAs selected from the group consisting of non-human organisms; [Criteria 2 ′] forward primer And the reverse primer each has a nucleotide length of 18 or more; [Criteria 3 ′] When at least the 5′-end or 3′-end nucleotide of the primer is G or C and the 5 ′ end of the primer is not G or C, The second nucleotide from the 5 'end is G or Is C and the 3 ′ end of the primer is not G or C, the second nucleotide from the 3 ′ end is G or C; [Criteria
  • a method for designing a PCR primer pair for detecting human Alu (2) [Criteria 1]
  • the nucleotide sequence consisting of 19 consecutive nucleotides in the forward primer and the reverse primer is in two places in the nucleotide sequence of one or more genomic DNAs selected from the group consisting of non-human organisms.
  • [Criteria 7] Forward ply -Complementary nucleotide sequence complementary to 4 or more consecutive nucleotide sequences consisting of G or C contained in either molecule, between two molecules, reverse primer, or between forward primer and reverse primer, A method of designing a PCR primer pair for detecting human Alu, including a step A for selecting a forward primer and a reverse primer that satisfy the criteria 1 to 7 of (3)
  • the present invention relates to a PCR primer pair for detecting human Alu designed by the method for designing a PCR primer pair for detecting human Alu according to any one of (1) to (4) above.
  • the present invention also provides: (6) A human Alu detection PCR primer pair comprising the following (a) or (a ′) forward primer and (b) or (b ′) reverse primer (a) the nucleotide shown in SEQ ID NO: 18 A nucleotide sequence consisting of at least 16 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO: 18, and one or several nucleotides are deleted, substituted or added in the nucleotide sequence shown in SEQ ID NO: 18
  • a forward primer comprising: (A ′) a nucleotide sequence represented by SEQ ID NO: 123; or a nucleotide sequence comprising at least 16 consecutive nucleotides in the nucleotide sequence represented by SEQ ID NO: 123, and one or a number in the nucleotide sequence represented by SEQ ID NO: 123
  • a forward primer consisting of a nucleotide sequence in which one nucleotide has been deleted, substituted, or added;
  • the present invention provides (11) Of the human Alu model sequence consisting of the nucleotide sequence shown in SEQ ID NO: 4, hybridizes to the region amplified by the human Alu detection PCR primer pair described in any of (5) to (8) above
  • a nucleotide sequence of 17 or more consecutive nucleotides in the [Criteria 8 ′] probe from the group of probes capable of being selected from the group consisting of non-human organisms, or one or more nucleotide sequences of genomic DNA [Criteria 9 ′]
  • the nucleotide length of the probe is 18 or more; [Criteria 10 ′] 3 of any one of the molecules between the forward primer and the probe and the reverse primer and the probe.
  • the other molecule does not contain a nucleotide sequence complementary to a nucleotide sequence of 4 nucleotides or more;
  • [Criteria 14] probes, forward primer and probe, and The change in free energy when the most stable dimer is formed between the two molecules of the reverse primer and the probe is -7 kcal / mol or more;
  • One or more than two The method for designing a PCR probe for detecting human Alu according to the above (11) or (12), further comprising a step C of selecting a soot probe, (14)
  • the present invention also provides: (17) a nucleotide sequence represented by SEQ ID NO: 37, 39, 42, 47 or 149; or a nucleotide sequence comprising at least 16 consecutive nucleotides in the nucleotide sequence represented by SEQ ID NO: 37, 39, 42, 47 or 149 A nucleotide probe in which one or several nucleotides are deleted, substituted, or added in the nucleotide sequence shown in SEQ ID NO: 37, 39, 42, 47, or 149; (18) The PCR probe for detecting human Alu according to claim 17, comprising the nucleotide sequence shown in SEQ ID NO: 42 or 149, (19) The PCR probe for detecting human Alu according to any one of the above (16) to (18), wherein the 5 ′ end of the probe is labeled with a fluorescent substance and the 3 ′ end is labeled with a quencher substance, (20) A human Alu detection PCR primer pair according to any one of (5) to (8) above, and
  • PCR primer / probe set for detecting human Alu (21) A method for detecting and / or quantifying human genomic DNA in a test sample using the PCR primer / probe set for detecting human Alu according to (20) above, (I) DNA extracted from the test sample A step of performing real-time PCR using the primer / probe set as a template; (II) using a standard sample prepared by serial dilution of a known amount of human genomic DNA as a template under the same conditions as in step (I) above Performing real-time PCR and preparing a calibration curve; (III) human genome DN in the test sample from the calibration curve Calculating the amount of A; a method comprising the steps (I) to (III) of: (22) The method according to (21) above, wherein the test sample is a biological sample derived from a xenograft model animal produced by transplanting human cells into a non-human animal, or an old sample derived from an ancient human bone, (23) The PCR primer pair for detecting human Alu according to
  • a PCR primer pair and probe for detecting human Alu that can detect human Alu sequences specifically and with high sensitivity.
  • Alu-qPCR using such a PCR primer pair and probe for detecting human Alu, even from samples with poor conditions (samples in which a large amount of genomic DNA of other organisms are mixed, samples in which DNA is highly fragmented, etc.) It becomes possible to specifically detect and / or measure human genomic DNA of about 0.1 to 10 fg.
  • FIG. 3 is a diagram showing the results of Alu-qPCR using a primer / probe set of Primer 3.
  • “Buf.” Indicates buffer only (template 1), and “Mouse” indicates mouse genomic DNA (template 2).
  • FIG. 7 is a diagram showing how many 19 nucleotide continuous nucleotide sequences starting from each position exist in the mouse, rat, and Guinea pig genomes for positions # 1 to # 264 of the Alu model sequence.
  • “Position #” indicates position # in the Alu model sequence
  • “Nucleotide” indicates the type of nucleotide at each position # in the Alu model sequence.
  • “MouselastBlast”, “Rat Blast”, and “Guinea piglastBlast” indicate where the nucleotide sequence of 19 nucleotides continuing from the position # exists in the genome of mouse, rat, and Guinea pig. . It is a figure which shows the secondary structure formed in the forward primer (sequence number 6) and reverse primer (sequence number 7) of Primer3. It is a figure which shows the secondary structure formed between two molecules of the primer probe probe set (forward primer; sequence number 1, reverse primer; sequence number 2, and probe; sequence number 3) of McBride. It is a figure which shows each position of the forward primer F10 of this invention, reverse primer R1, and probe P16 in an Alu model arrangement
  • FIG. 4 is a view showing the results of Alu-qPCR using the primer / probe set A of the present invention.
  • “Buf.” Indicates the buffer alone (template 1)
  • “Human genomic DNA (Log10pg)” indicates the amount of human genomic DNA contained in the standard sample (template 3).
  • FIG. 4 is a view showing the results of Alu-qPCR using the primer / probe set A of the present invention.
  • “Buf.” Indicates buffer only (template 1)
  • “Mouse” indicates mouse genomic DNA (template 2).
  • “Human genomic DNA (Log 10 pg)” indicates the amount of human genomic DNA contained in a standard sample (template 4) mixed with mouse genomic DNA.
  • FIG. 4 is a view showing the results of Alu-qPCR using the primer / probe set A of the present invention.
  • FIG. 4 is a view showing the results of Alu-qPCR using the primer / probe set C of the present invention.
  • “Buf.” Indicates buffer only (template 1), and “Rat” indicates rat genomic DNA (template 5).
  • “Human genomic DNA (Log 10 pg)” indicates the amount of human genomic DNA contained in the standard sample (template 6) mixed with rat genomic DNA. As a result of preparing a calibration curve based on the Ct value in each human genomic DNA amount, linearity was observed when the amount of human genomic DNA was between 1 fg and 10 ng.
  • FIG. 4 is a view showing the results of Alu-qPCR using the primer / probe set D of the present invention. In the figure, “Buf.” Indicates buffer only (template 1), and “Rat” indicates rat genomic DNA (template 5). “Human genomic DNA (Log 10 pg)” indicates the amount of human genomic DNA contained in the standard sample (template 6) mixed with rat genomic DNA.
  • FIG. 1 It is a figure which shows the result of having quantified human genomic DNA contained in the kidney of a xenograft model mouse using the primer and probe set A of the present invention.
  • A shows a calibration curve prepared using 0.5 fg / ⁇ L to 5 ng / ⁇ L of human genomic DNA, genomic DNA derived from the kidney of a control mouse, and a mixed standard sample (template 11).
  • B shows the result of calculating the number of human MSCs contained in the whole kidney of the xenograft model mouse based on the calibration curve. It is a figure which shows the result of having quantified the human genomic DNA contained in the liver of a xenograft model mouse using the primer probe set A of this invention.
  • FIG. 3 is a diagram showing the number of human cells in each mouse kidney calculated based on the quantitative value of human genomic DNA contained in the kidney of a subrenal capsule transplant model mouse using the primer / probe set of the present invention. .
  • the number of cells on the horizontal axis represents the number of human cells initially transplanted into the kidney of the model mouse.
  • the number of consecutive 16-20 nucleotide sequences contained in the primer / probe set A of the present invention in the genome of various biological species (mouse, rat, guinea pig, bear, cow, rabbit, chicken, pig, microorganism). It is a figure which shows whether it exists.
  • “101F” indicates the forward primer F10
  • “206R” indicates the reverse primer R1
  • “144RH” indicates the probe P16.
  • “20” indicates how many nucleotide sequences (full length) consisting of 20 consecutive nucleotides in the primer and probe exist in the genome of each species
  • “19” indicates in the primer and probe.
  • “18” indicates the number of nucleotide sequences consisting of 19 consecutive nucleotides in the genome of each species, and “18” indicates how many nucleotide sequences consisting of 18 consecutive nucleotides in the primer and probe are present in the genome of each species.
  • “17” indicates how many nucleotide sequences consisting of 17 consecutive nucleotides in the primer and probe exist in the genome of each species, and "16” indicates 16 consecutive nucleotides in the primer and probe. How many nucleotide sequences (full length) are present in the genome of each species. Show each one. It is a figure which shows distribution of the length of the fragmented human genomic DNA (20kbp) produced in Example 17.
  • FIG. 4 is a view showing the results of Alu-qPCR using the primer / probe set A of the present invention.
  • “Negative control” in the figure indicates only the buffer (template 1)
  • “human genomic DNA amount (Log 10 pg)” indicates a standard sample (template 18) containing human genomic DNA fragmented at 20 kbp.
  • FIG. 4 is a view showing the results of Alu-qPCR using the primer / probe set A of the present invention.
  • “Negative control” in the figure indicates only the buffer (template 1), and “human genomic DNA amount (Log 10 pg)” indicates a standard sample (template 19) containing human genomic DNA fragmented at 1000 bp.
  • “human genomic DNA amount (Log 10 pg)” indicates a standard sample (template 19) containing human genomic DNA fragmented at 1000 bp.
  • FIG. 4 is a view showing the results of Alu-qPCR using the primer / probe set A of the present invention.
  • FIG. 4 is a view showing the results of Alu-qPCR using the primer / probe set A of the present invention.
  • “Negative control” in the figure is only chicken genomic DNA (template 21), and “human genomic DNA amount (Log 10 pg)” is the amount of human genomic DNA contained in a standard sample (template 22) in which chicken genomic DNA and human genomic DNA are mixed. Indicates. As a result of preparing a calibration curve based on the Ct value in each human genomic DNA amount, linearity was observed when the amount of human genomic DNA was between 1 fg and 10 ng.
  • FIG. 4 is a view showing the results of Alu-qPCR using the primer / probe set A of the present invention.
  • FIG. 4 is a view showing the results of Alu-qPCR using the primer / probe set A of the present invention.
  • “Negative control” in the figure indicates only bovine genomic DNA (template 25), and “human genomic DNA amount (Log 10 pg)” indicates the amount of human genomic DNA contained in the standard sample (template 26) in which bovine genomic DNA and human genomic DNA are mixed. .
  • “Negative control” in the figure indicates only bovine genomic DNA (template 25), and “human genomic DNA amount (Log 10 pg)” indicates the amount of human genomic DNA contained in the standard sample (template 26) in which bovine genomic DNA and human genomic DNA are mixed. .
  • FIG. 4 is a view showing the results of Alu-qPCR using the primer / probe set A of the present invention.
  • FIG. 3 is a view showing an amplification curve obtained by Alu-qPCR using the primer / probe set A of the present invention.
  • FIG. 4 is a view showing the results of Alu-qPCR using the primer / probe set A of the present invention.
  • “Negative control” in the figure indicates only the buffer (template 1), and “human genomic DNA amount (Log 10 pg)” indicates the standard sample (template 29) containing only human genomic DNA.
  • FIG. 3 is a graph showing the results of quantifying the amount of human genomic DNA in ancient bones by Alu-qPCR using the primer / probe set A of the present invention.
  • the quantification of the ancient bone-derived samples (templates 30 to 32) was performed in duplicate, and the human genomic DNA concentration in the sample was calculated from the average value of the Ct values. It is a figure which shows the difference with the PCR primer * probe set for Alu-qPCR by a known probe method, and this invention.
  • “F” indicates a forward primer
  • “R” indicates a reverse primer
  • “H” indicates a hydrolysis probe.
  • the “Criteria #” column indicates whether or not each known primer / probe sequence satisfies the criteria 1 to 7 (for the primer) and the criteria 8 to 13 (for the probe) of the present invention. "X” if blank, blank if not filled). It is a figure which shows the difference with the PCR primer * probe set for Alu-qPCR by a known probe method, and this invention.
  • “F” indicates a forward primer
  • “R” indicates a reverse primer
  • “H” indicates a hydrolysis probe.
  • “0” in the “Mouse”, “Rat”, and “Guinea pig” columns represents a sequence consisting of 20 consecutive nucleotides contained in each known primer, or a full-length sequence (19 nucleotides or less) in mouse, rat , And how many guinea pigs are present in the genome, “ ⁇ 1” is a sequence consisting of 19 consecutive nucleotides contained in each known primer, or a full-length sequence (19 nucleotides or less) in mouse, rat, and It shows how many guinea pigs exist in the genome, and “ ⁇ 2” is a sequence consisting of 18 consecutive nucleotides contained in each known primer, or a full-length sequence (18 nucleotides or less) of mouse, rat, and guinea pigs.
  • “0” in the “Human” column indicates how many sequences consisting of 20 consecutive nucleotides contained in each known primer or a full-length sequence (19 nucleotides or less) exist in the human genome.
  • “*” in the figure indicates a Tm value calculated without taking into account the TaqMan-MGB effect
  • “**” indicates the number of hits including the sequence of positions # 807 to 1566 in the rat genome database NW_007906663. Show. It is a figure which shows the difference with the PCR primer probe set for Alu-qPCR by the known intercalator method, and this invention.
  • “Type” column in the figure “F” indicates a forward primer and “R” indicates a reverse primer.
  • the “Criteria” column indicates whether each known primer sequence satisfies the criteria 1 to 7 of the present invention (“X” when satisfied, or blank when not satisfied).
  • “0” in the “Mouse”, “Rat”, and “Guinea pig” columns represents a sequence consisting of 20 consecutive nucleotides contained in each known primer, or a full-length sequence (19 nucleotides or less) in mouse, rat , And how many guinea pigs are present in the genome
  • “ ⁇ 1” is a sequence consisting of 19 consecutive nucleotides contained in each known primer, or a full-length sequence (19 nucleotides or less) in mouse, rat, and It shows how many guinea pigs are present in the genome
  • “ ⁇ 2” indicates a sequence consisting of 18 consecutive nucleotides contained in each known primer, or a full-length sequence (18 nucleotides or less) of mouse, rat, and guinea pigs.
  • FIG. 6 is a diagram showing how many 18 nucleotide continuous nucleotide sequences starting from each position exist in mouse, rat, and Guinea pig genomes for positions # 1 to # 266 of the Alu model sequence.
  • “Position” indicates the position # in the Alu model array.
  • FIG. 6 is a diagram showing how many 17 nucleotide continuous nucleotide sequences starting from each position exist in mouse, rat, and Guinea pig genomes for positions # 1 to # 266 of the Alu model sequence.
  • “Position” indicates the position # in the Alu model array.
  • “Mouse Blast”, “Rat Blast”, and “Guinea pig Blast” indicate where the nucleotide sequence of 17 nucleotides consecutive from the position # is present in the genome of mouse, rat, and Guinea pig. . It is a figure which shows the secondary structure of the primer dimer formed in the forward primer F34 '(sequence number 123) and reverse primer R20' (sequence number 148) of this invention. It is a figure which shows distribution of the length of the fragmented human genomic DNA (100 bp) produced in Example 22.
  • FIG. 4 is a diagram showing the results of Alu-qPCR using the primer / probe set E of the present invention.
  • “Negative control” in the figure indicates only the buffer (template 1), and “human genomic DNA amount (Log 10 pg)” indicates a standard sample (template 33) containing human genomic DNA fragmented at 100 bp.
  • “Negative control” in the figure indicates only the buffer (template 1), and “human genomic DNA amount (Log 10 pg)” indicates a standard sample (template 33) containing human genomic DNA fragmented at 100 bp.
  • the “method for designing a PCR primer pair for detection of human Alu” of the present invention is an example of a human Alu model sequence consisting of the nucleotide sequence shown in SEQ ID NO: 4. There is no particular limitation as long as it includes a step A ′ for selecting a forward primer and a reverse primer satisfying the following criteria 1 ′ to 7 ′ from a forward primer group and a reverse primer group capable of amplifying a part or all of the region. .
  • the second nucleotide from the 3 ′ end is G or C;
  • [Criteria 7 ′] A nucleotide sequence complementary to a continuous nucleotide sequence of 4 or more nucleotides consisting of G or C contained in either molecule between the two molecules of the forward primer and the reverse primer, Contains no molecules of
  • a method including a step A for selecting a forward primer and a reverse primer satisfying the following criteria 1 to 7 can be mentioned.
  • [Criteria 2] Forward primer and reverse primer each have a nucleotide length of 20 or more;
  • [Criteria 3] When the 5 'end or 3' end nucleotide of the primer is G or C and the 5 'end of the primer is not G or C, the second nucleotide from the 5' end is G or C.
  • the second nucleotide from the 3 ′ end is G or C;
  • At least one of the nucleotides from the 3 'end to the 3rd end of the primer is A or T;
  • [Criteria 6] A nucleotide sequence complementary to a continuous nucleotide sequence of 5 or more nucleotides contained in any one molecule between the two primers, between the forward primers, between the reverse primers, and between the forward primer and the reverse primer.
  • the “human Alu model sequence” is not particularly limited as long as it consists of the nucleotide sequence shown in SEQ ID NO: 4, but is particularly preferably one consisting of the nucleotide sequence shown in SEQ ID NO: 5.
  • a “forward primer group capable of amplifying a part or all of the human Alu model sequence” (hereinafter also simply referred to as “forward primer group”) according to a conventional method.
  • a reverse primer group capable of amplifying a part or all of the human Alu model sequence” hereinafter also simply referred to as “reverse primer group”.
  • the above "forward primer group” And the above-mentioned “reverse primer group” can be selected.
  • the “step A ′” is not particularly limited as long as it is a step of selecting a forward primer and a reverse primer satisfying all of the criteria 1 ′ to 7 ′ from the “forward primer group” and the “reverse primer group”.
  • the criteria 1 ′ to 7 ′ may be applied to the “forward primer group” and the “reverse primer group” in any order, but the criteria 1 ′ to 7 ′ are sequentially applied and selected. It is preferable.
  • step A is not particularly limited as long as it is a step of selecting a forward primer and a reverse primer that satisfy all of the criteria 1 to 7 from the “forward primer group” and the “reverse primer group”.
  • the criteria 1 to 7 may be applied to the “forward primer group” and the “reverse primer group” in any order, but it is preferable that the criteria 1 to 7 are sequentially applied and selected. Specifically, as an example of the “step A”, it is determined whether or not each primer constituting the “forward primer group” and the “reverse primer group” satisfies the criteria 1 to 4, and the criteria 1 When there is one forward primer and one reverse primer satisfying ⁇ 4, it is determined whether or not criteria 5 ⁇ 7 are satisfied for the paired combination, and forward primer satisfying criteria 1 ⁇ 4 When there are a plurality of reverse primers, a step of determining whether or not all the combinations of the forward primer and the reverse primer satisfy the criteria 5 to 7 can be mentioned.
  • one or two or more genomic DNAs in which the nucleotide sequence consisting of 17 or more consecutive nucleotides contained in each primer is selected from the group consisting of non-human organisms The nucleotide sequence is not particularly limited as long as it does not exist in two or more places, and the forward primer and the reverse primer satisfying the above criteria 1 include a nucleotide sequence consisting of 19 consecutive nucleotides contained in each primer, There are no particular limitations as long as it does not exist in two or more nucleotide sequences of one or more genomic DNAs selected from the group consisting of non-human organisms.
  • non-human organism is not particularly limited as long as it is a non-human organism that can be partially mixed in a test sample to be detected by human Alu. Any organism such as a protozoan or a virus may be used, but a mammal other than a human is preferable, and a rodent is more preferable.
  • non-human organism examples include mice, rats, guinea pigs, dogs, rabbits, chickens, horses, cows, cats, bears, Xenopus, zebrafish, medaka, tiger puffers, squirts, lampreys, Examples include nematodes, sea urchins, planarians, Arabidopsis thaliana, rice, wheat, tobacco, Drosophila, silkworms, honey bees, Escherichia coli, Bacillus subtilis, cyanobacteria, among others, rodent animals such as mice, rats, and guinea pigs. Can be preferably mentioned.
  • a forward primer and a reverse primer satisfying the above criteria 1 ′ specifically, a sequence database (eg, GenBank, DDBJ, EMBL, etc.) and a homology search software (eg, BLAST (registered trademark)) Etc.) can be exemplified.
  • BLAST registered trademark
  • a region in the human Alu model sequence that can design a forward primer and a reverse primer satisfying the criterion 1 ′ is specified, and based on this information Thus, a forward primer and a reverse primer satisfying the criterion 1 ′ can be selected.
  • a sequence database eg, GenBank, DDBJ, EMBL, etc.
  • a homology search software eg, BLAST (registered trademark)
  • each of the forward primer and the reverse primer satisfying the above criteria 2 ′ are not particularly limited as long as each nucleotide length is 18 or more.
  • each of the forward primer and the reverse primer is preferably 20 to 60 nucleotides in length. Is 20 to 40 nucleotides long, more preferably 20 to 30 nucleotides long, more preferably 20 to 25 nucleotides long, further preferably 20 to 23 nucleotides long, more preferably 20 nucleotides long, still more preferably 19 nucleotides long, more preferably May include a primer pair 18 nucleotides long.
  • the primers may be the same nucleotide length or different nucleotide lengths.
  • each of the forward primer and the reverse primer satisfying the above criteria 2 are not particularly limited as long as each nucleotide length is 20 or more.
  • each of the forward primer and the reverse primer has a length of 20 to 60 nucleotides.
  • a primer pair that is preferably 20 to 40 nucleotides long, more preferably 20 to 30 nucleotides long, more preferably 20 to 25 nucleotides long, still more preferably 20 to 23 nucleotides long, and more preferably 20 nucleotides long.
  • the forward primer and the reverse primer may have the same nucleotide length or different nucleotide lengths.
  • At least one of the nucleotides from the 3 ′ end to the 3rd end of the primer (1st to 3rd nucleotide from the 3 ′ end) is A or T
  • it is more preferable that two of the first to third nucleotide sequences from the 3 ′ end of the primer are A or T.
  • the nucleotide sequence complementary to the third nucleotide sequence) is not particularly limited as long as the other molecule does not contain it, and the forward primer and the reverse primer satisfying the above criteria 5 include the forward primers, Nucleotide complementary to the nucleotide sequence from the 3 'end to the 3rd end of either molecule (the 1st to 3rd nucleotide sequence from the 3' end) between the reverse primers and between the forward primer and reverse primer molecules Arrangement
  • the nucleotide sequence complementary to the above nucleotide sequence is not particularly limited as long as the other molecule does not contain it.
  • the nucleotide more than 4 continuous nucleotides which consist of G or C contained in any one molecule
  • the nucleotide sequence complementary to the sequence is not particularly limited as long as the other molecule does not contain, and the forward primer and the reverse primer satisfying the above criteria 7 include the forward primer, the reverse primer, and the forward primer.
  • the reverse primer does not contain a nucleotide sequence complementary to the nucleotide sequence of 4 or more consecutive nucleotides consisting of G or C contained in one of the two molecules of the reverse primer. Not particularly limited, if any.
  • the method for selecting the forward primer and the reverse primer satisfying the above criteria 5 ′ to 7 ′ or 5 to 7 is not particularly limited. For example, download from “http://rna.urmc.rochester.edu/RNAstructure.html” RNA primers, forward primers, reverse primers, and forward primers using known programs such as mfold that can be downloaded from http://www.bioinfo.rpi.edu/applications/mfold/old/dna/form1.cgi Predicting the secondary structure formed between two molecules of the reverse primer and the reverse primer, and determining whether the forward primer and the reverse primer satisfy the criteria 5 ′ to 7 ′ or 5 to 7 based on the result Preferable examples can be given.
  • PCR primer pair for human Alu detection The “PCR primer pair for human Alu detection” of the present invention (hereinafter also simply referred to as “primer pair of the present invention”) is not particularly limited as long as it is designed by the primer pair design method of the present invention. Specifically, a primer pair composed of the following forward primer (a) or (a ′) and the reverse primer (b) or (b ′) can be preferably exemplified.
  • a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 18, or at least 16 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO: 18, preferably 17 nucleotides, more preferably 18 nucleotides, still more preferably 19 nucleotides, More preferably, it comprises a nucleotide sequence consisting of 20 nucleotides, and 1 or several (for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3, in the nucleotide sequence shown in SEQ ID NO: 18, A forward primer consisting of a nucleotide sequence in which 1 to 2, more preferably 1) nucleotides are deleted, substituted or added; (A ′) a nucleotide sequence shown in SEQ ID NO: 123; or a nucleotide sequence consisting of at least 16 consecutive nucleotides, preferably 17 nucleotides, more preferably 18 nucleotides in the nucleotide sequence shown in SEQ ID NO: 18,
  • a forward primer consisting of a nucleotide sequence with nucleotides deleted, substituted or added (B) a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 19, or at least 16 nucleotides, preferably 17 nucleotides, more preferably 18 nucleotides, more preferably 19 nucleotides in the nucleotide sequence shown in SEQ ID NO: 19, More preferably, it comprises a nucleotide sequence consisting of 20 nucleotides, and 1 or several nucleotides (for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3) in the nucleotide sequence shown in SEQ ID NO: 19.
  • Reverse primer consisting of a nucleotide sequence deleted, substituted, or added), more preferably 1 to 2, more preferably 1);
  • B ′ a nucleotide sequence represented by SEQ ID NO: 148; or a nucleotide sequence consisting of at least 16 consecutive nucleotides, preferably 17 nucleotides, more preferably 18 nucleotides in the nucleotide sequence represented by SEQ ID NO: 148, and 1 or several (for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3, further preferably 1 to 2, more preferably 1) in the nucleotide sequence represented by No. 148
  • a reverse primer comprising: a nucleotide sequence in which nucleotides are deleted, substituted, or added;
  • the forward primer and the reverse primer may be combined in any way, the combination of the forward primer of (a) and the reverse primer of (b), (a ′) A forward primer of (b ′), a forward primer of (a) and a reverse primer of (b ′), a forward primer of (a ′) and a reverse primer of (b) Any of the primer pairs in the combination of (a), the combination of the forward primer in (a ′) and the reverse primer in (b ′), or the forward primer in (a) and the above (b ′).
  • the combination of the reverse primer (1) can be particularly preferably exemplified.
  • the primer pair of the present invention includes a primer pair composed of a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 18 and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 19, or SEQ ID NO: 123.
  • a primer pair composed of a forward primer consisting of the nucleotide sequence shown and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 148 can be particularly preferably exemplified.
  • nucleotide sequences of 16 nucleotides or more contained in the nucleotide sequences shown in SEQ ID NOs: 18 and 19 are mouse, rat, guinea pig, bear, cow, rabbit, chicken, pig, It has been confirmed that it is hardly present in any genome of microorganisms.
  • the “containing a nucleotide sequence consisting of at least 16 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO: 18 described in (a) above, and one or several nucleotides in the nucleotide sequence shown in SEQ ID NO: 18 Including a nucleotide sequence consisting of at least 16 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO: 19 described in (b), and A primer pair consisting of a “reverse primer consisting of a nucleotide sequence in which one or several nucleotides have been deleted, substituted, or added in the nucleotide sequence shown in No. 19” can specifically amplify a human Alu sequence.
  • the “method for designing a PCR probe for detecting human Alu” of the present invention includes the above-mentioned human Alu model sequence consisting of the nucleotide sequence shown in SEQ ID NO: 4 above. There is no particular limitation as long as it includes step B ′ for selecting a probe satisfying the following criteria 8 ′ to 11 ′ from the group of probes capable of hybridizing to the region amplified by the primer pair of the present invention.
  • the probe designing method of the present invention there can be mentioned one including the step A of selecting a probe satisfying the following criteria 8 to 11.
  • the nucleotide sequence consisting of 19 consecutive nucleotides in the probe does not exist in two or more places in the nucleotide sequence of one or more genomic DNAs selected from the group consisting of non-human organisms;
  • the probe has a nucleotide length of 20 or more;
  • [Criteria 10] The nucleotide sequence from the 3 'end to the 3rd end of any one molecule between the probes, the forward primer and the probe, and the reverse primer and the probe (the first to third nucleotides from the 3' end) The other molecule does not contain a nucleotide sequence complementary to the sequence);
  • [Criteria 11] Between two probes, a forward primer and a probe, and a reverse primer and a probe, a nucleotide sequence complementary to a continuous nucle
  • the probe designing method of the present invention further includes a step C of selecting a probe that satisfies at least one or more of the following criteria 12 to 15 when a plurality of probes are selected in the above-described step B ′ or B. It may be included.
  • [Criteria 12] A nucleotide sequence complementary to a continuous nucleotide sequence of 4 or more nucleotides consisting of G or C contained in any one molecule between two molecules of the forward primer and the probe or the reverse primer and the probe Does not contain one molecule; [Criteria 13] The 5 ′ terminal nucleotide of the probe is not G; [Criteria 14] The change in free energy when the most stable dimer is formed between the two molecules of the probes, the forward primer and the probe, and the reverse primer and the probe is -7 kcal / mol or more; [Criteria 15] Shortest nucleotide length;
  • forward primer and “reverse primer” mean a forward primer and a reverse primer, respectively, constituting the primer pair of the present invention.
  • the “human Alu model sequence” is not particularly limited as long as it consists of the nucleotide sequence shown in SEQ ID NO: 4, but a nucleotide sequence shown in SEQ ID NO: 5 is particularly preferred.
  • a group of probes capable of hybridizing to the region amplified by the primer pair of the present invention (hereinafter also simply referred to as “a group of probes”). ) Can be selected.
  • a probe comprising a nucleotide sequence complementary to the probe can be selected as the “probe group”.
  • the “step B ′” is not particularly limited as long as it is a step of selecting probes satisfying the criteria 8 ′ to 11 ′ from the “probe group”, but the criteria 8 ′ to 11 ′ are included in the “probe group”. It is preferable that it is the process of selecting by applying sequentially.
  • the “step B” is not particularly limited as long as it is a step of selecting probes satisfying the criteria 8 to 11 from the “probe group”. However, the criteria 8 to 11 are sequentially applied to the “probe group”. It is preferable that it is a process selected.
  • the “step C” is not particularly limited as long as it is a step of selecting a probe satisfying at least one or more of the criteria 12 to 15 from a plurality of probes satisfying the criteria 8 ′ to 11 ′.
  • the labeling method, the number of selected probes, etc., one, preferably 2, more preferably 3, more preferably 4 criteria selected from the above criteria 12 to 15 are used. It is possible to particularly preferably exemplify a step of selecting a probe satisfying the criteria 13 and 15 from a plurality of probes satisfying the criteria 8 ′ to 11 ′.
  • the “step C” may be a step of selecting a probe satisfying at least one or more of the criteria 12 to 15 from a plurality of probes satisfying the criteria 8 to 11.
  • One, preferably two, more preferably three, and even more preferably four criteria selected from ⁇ 15 can be used. From a plurality of probes satisfying the criteria 8-11, the criteria 12-15 The step of selecting a probe satisfying all of the above can be particularly preferably exemplified. When two or more selected from the criteria 12 to 15 are used in the “step C”, the criteria may be used in any order.
  • a nucleotide sequence consisting of 17 consecutive nucleotides in the probe is at least two sites in the nucleotide sequence of one or more genomic DNAs selected from the group consisting of non-human organisms.
  • the non-human organism is not particularly limited as long as it does not exist.
  • the “non-human organism” refers to the same as the “non-human organism” in the criterion 1 ′.
  • a method for selecting a probe that satisfies the above criteria 8 ′ specifically, a sequence database (eg, GenBank, DDBJ, EMBL, etc.) and homology search software (eg, BLAST (registered trademark), etc.), etc.
  • the nucleotide sequence consisting of 19 consecutive nucleotides in the probe is 2 in the nucleotide sequence of one or more genomic DNAs selected from the group consisting of non-human organisms.
  • the method for selecting a probe that satisfies the above criteria 8 is not particularly limited as long as it does not exist in more than one place.
  • a sequence database for example, GenBank, DDBJ, EMBL, etc.
  • homology search software for example, , BLAST (registered trademark), etc.
  • the “human Alu model sequence” included in the “human Alu model sequence” is continuously contained in the genome of the “non-human organism”.
  • nucleotide identical to nucleotide sequence consisting of 19 nucleotides It is possible to know de sequence number exists. Further, by mapping the result of the homology search to the “human Alu model sequence”, a region in the human Alu model sequence where a probe satisfying the criterion 8 can be designed is specified. The probe to be satisfied can be selected.
  • the probe satisfying the above criteria 9 ′ is not particularly limited as long as it has a nucleotide length of 18 or more.
  • it is 20 to 60 nucleotides long, more preferably 20 to 40 nucleotides long, and further preferably 20 to 30 nucleotides long.
  • a probe having a length of 20 to 25 nucleotides, more preferably 20 to 23 nucleotides, more preferably 20 nucleotides, still more preferably 19 nucleotides, and more preferably 18 nucleotides can be mentioned.
  • the probe satisfying the above criteria 9 is not particularly limited as long as it has a nucleotide length of 20 or more.
  • the probe has a length of 20 to 60 nucleotides, more preferably 20 to 40 nucleotides, and further preferably 20 to 30 nucleotides.
  • a probe having a length of 20 to 25 nucleotides, more preferably 20 to 23 nucleotides, and more preferably 20 nucleotides can be mentioned.
  • a nucleotide sequence from the 3 ′ end to the third position of any one molecule between two molecules of a combination of a forward primer and a probe and a combination of a reverse primer and a probe As a probe satisfying the above criteria 10 ′, a nucleotide sequence from the 3 ′ end to the third position of any one molecule between two molecules of a combination of a forward primer and a probe and a combination of a reverse primer and a probe.
  • the nucleotide sequence complementary to (the first to third nucleotide sequence from the 3 ′ end) is not particularly limited as long as the other molecule does not contain it.
  • a probe satisfying the above criteria 10 is a combination of probes.
  • the column is not particularly limited as long as it does not contain other molecules.
  • the probe satisfying the above criteria 11 ′ or 11 is included in any one of the two molecules of the combination of probes, the combination of forward primer and probe, and the combination of reverse primer and probe.
  • a method for selecting a probe that satisfies the above criteria 10 ′, 10, 11 ′, or 11 is not particularly limited.
  • the probes may be linked to each other, a forward primer and a probe, or a reverse primer by a known program such as RNAstructure or Mfold.
  • a method of predicting a secondary structure formed between two molecules of a probe and determining whether the probe satisfies the criteria 10 ′, 10, 11 ′, or 11 based on the result may be mentioned. it can.
  • a nucleotide sequence complementary to a nucleotide sequence of 4 nucleotides or more is not particularly limited as long as the other molecule does not contain it.
  • a method for selecting a probe that satisfies the above criteria 12 is not particularly limited.
  • the secondary structure formed between two molecules of the forward primer and the probe or the reverse primer and the probe is predicted by a known program such as Mfold, and whether or not the probe satisfies the criteria 12 based on the result is predicted. Preferred method of judging It can gel.
  • the probe satisfying the above criteria 13 is not particularly limited as long as the 5 ′ terminal nucleotide of the probe is not G. Specifically, the probe whose 5 ′ terminal nucleotide is A, T, or C Can be mentioned. A probe that satisfies this criterion can avoid quenching by G when its 5 'end is labeled with a fluorescent substance. Therefore, when selecting a probe that is assumed to be used as a hydrolysis probe or a molecular beacon probe, it is preferable to select a probe that satisfies the above criteria 13.
  • the most stable dimer is formed between two molecules of a combination of probes, a combination of a forward primer and a probe, and a combination of a reverse primer and a probe.
  • Change in free energy is -7 kcal / mol or more, preferably -6.8 kcal / mol or more, more preferably -6.5 kcal / mol or more, further preferably -6.3 kcal / mol or more, more preferably -6.0 kcal.
  • the “change in free energy” may be different between two molecules in each combination of probes, a combination of forward primer and probe, and a combination of reverse primer and probe.
  • a method for selecting a probe that satisfies the above criteria 14 is not particularly limited.
  • the probe is formed between two molecules of a probe, a forward primer and a probe, or a reverse primer and a probe by a known program such as RNAstructure or Mfold.
  • a method of determining whether or not the probe satisfies the criterion 14 by calculating a change in free energy ( ⁇ G °) when the secondary structure is predicted and the most stable dimer is formed can be preferably exemplified. .
  • the probe that satisfies the above criteria 15 is not particularly limited as long as it has the shortest nucleotide length when comparing the nucleotide lengths of a plurality of probes to be selected.
  • PCR probe for human Alu detection of the present invention
  • probe of the present invention is not particularly limited as long as it is designed by the probe design method of the present invention, but specifically, Is a probe comprising the nucleotide sequence shown in SEQ ID NO: 37, 39, 42, 47 or 149, or at least 16 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO: 37, 39, 42, 47 or 149, preferably 17 nucleotides More preferably 18 nucleotides, even more preferably 19 nucleotides, more preferably 20 nucleotides, and one or several nucleotide sequences shown in SEQ ID NO: 37, 39, 42, 47 or 149 (for example, 1-10 pieces are preferred 1 to 5, more preferably 1 to 3, more preferably 1 to 2, more preferably 1), a probe comprising a nucleotide sequence in which nucleotides have been
  • a probe consisting of the nucleotide sequence shown in SEQ ID NO: 37, 39, 42, 47 or 149 is preferable, and a probe consisting of the nucleotide sequence shown in SEQ ID NO: 42 or 149 is preferable. Further preferred.
  • the probe of the present invention is preferably labeled with a labeling substance in order to detect and / or quantify the amplification product of the primer pair of the present invention. From the viewpoint of detecting or quantifying more rapidly or with higher sensitivity, It is preferably labeled with a fluorescent substance.
  • the labeling substance other than the fluorescent substance include biotin, a complex of biotin and avidin, and an enzyme such as peroxidase.
  • fluorescent substance in addition to a fluorescent protein such as luciferase, FITC (fluorescein isothiocyanate), 6 -FAM (6-carboxyfluorescein), TET (6-carboxy-4,7,2 ', 7'-tetrachlorofluorescein), JOE (6-carboxy-4', 5'-dichloro2 ', 7'-dimethoxy) Fluorescent dyes such as fluorescein), Cy3, Cy5, HEX (4,7,2 ′, 4 ′, 5,5,7′-hexachloro-6-carboxyfluorescein) are preferred.
  • fluorescent protein such as luciferase, FITC (fluorescein isothiocyanate), 6 -FAM (6-carboxyfluorescein), TET (6-carboxy-4,7,2 ', 7'-tetrachlorofluorescein), JOE (6-carboxy-4', 5'-dichloro2 ', 7
  • the probe of the present invention is more preferably double-labeled with a fluorescent substance (reporter fluorescent dye) and a quencher substance (quencher fluorescent dye), and the 5 'end is It is particularly preferable that the 3 ′ end is labeled with a quencher substance (quencher fluorescent dye).
  • the fluorescent substance (reporter fluorescent dye) include the fluorescent dyes described above, and examples of the quencher substance (quencher fluorescent dye) include 6-carboxytetramethylrhodamine (TAMRA), 6-carboxy-X-rhodamine.
  • Rhodamine fluorescent dyes such as (ROX) and BHQ-1 ([(4- (2-nitro-4-methyl-phenyl) -azo) -yl-((2-methoxy-5-methyl-phenyl) -azo )]-Aniline), BHQ-2 ([(4- (1-nitro-phenyl) -azo) -yl-((2,5-dimethoxy-phenyl) -azo)]-aniline)
  • 6-FAM (6-carboxyfluorescein) is used as a fluorescent substance (reporter fluorescent dye), and a quencher substance (quencher fluorescence).
  • BHQ-1 [(4- (2-nitro-4-methyl-phenyl) -azo) -yl-((2-methoxy-5-methyl-phenyl) -azo)]-aniline) is particularly preferred Can be exemplified.
  • PCR primer / probe set for detecting human Alu of the present invention (PCR primer / probe set for detecting human Alu of the present invention)
  • the “PCR primer / probe set for detecting human Alu” of the present invention (hereinafter also simply referred to as “primer / probe set of the present invention”) comprises the primer pair of the present invention and the probe of the present invention.
  • the primer pair of the present invention composed of a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 18 and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 19, and SEQ ID NO: 37, Equipped with the probe of the present invention consisting of the nucleotide sequence shown in 39, 42 or 47; (2) a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 123 and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 148 Of the present invention constituted by And a probe comprising the nucleotide sequence shown in SEQ ID NO: 149; (3) a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 18 and reverse consisting of the nucleotide sequence shown in SEQ ID NO: 148
  • a probe pair of the present invention composed of a primer and a probe of the present invention consisting of the nucleotide sequence shown in SEQ ID NO: 37, 39,
  • the primer / probe set of the present invention may comprise the primer pair of the present invention and two or more probes of the present invention.
  • a primer pair of the present invention comprising a forward primer comprising: a reverse primer comprising the nucleotide sequence represented by SEQ ID NO: 19; and a probe group comprising the nucleotide sequence represented by SEQ ID NOs: 37, 39, 42 and 47
  • a primer / probe set provided with two or more of them can be mentioned.
  • the “method for detecting and / or quantifying the human genomic DNA of the present invention” of the present invention uses the DNA extracted from the test sample as a template and the above-mentioned book.
  • the method is not particularly limited as long as it is a method for detecting and / or quantifying human genomic DNA in the test sample, which includes the step of performing PCR using the primer pair of the invention.
  • the primer / probe set of the present invention is used.
  • a method for detecting and / or quantifying human genomic DNA in a test sample which preferably includes the following steps (I) to (III).
  • test sample is not particularly limited as long as it is a sample that can contain human genomic DNA, and may include DNA derived from non-human organisms in addition to human genomic DNA.
  • test sample include biological samples derived from non-human organisms, old samples, forensic samples, paleontological samples, etc., among others, prepared by transplanting human cells into non-human animals
  • Preferred examples include biological samples derived from xenograft model animals and old samples derived from ancient human bones.
  • non-human animals include non-human mammals such as mice, rats, guinea pigs, dogs, rabbits, pigs, goats and cows, and rodents such as mice, rats and guinea pigs. An animal can be mentioned more preferably.
  • the “human cell” may be any kind of cell as long as it is a human-derived cell such as a human stem cell, human cancer cell, human somatic cell, or human germ cell, but human mesenchyme. Particularly preferred examples include human stem cells such as lineage stem cells, human hematopoietic stem cells, human neural stem cells, human iPS cells, human ES cells, and human somatic cell-derived embryonic stem cells.
  • the “old sample” refers to a “non-fresh sample” that has passed several days to several million years in various environments, and the lower limit of the elapsed time is, for example, 2 days, 1 week, 2 weeks, 1 month, 3 months, 1 year, 3 years, etc.
  • the “old sample derived from old human bone” means a sample derived from human bones several years ago from the time of detecting and / or quantifying human DNA, and this old sample detects human DNA.
  • RNA degradation treatment is performed using RNase A and RNase T1 before the proteinase K treatment step in the “proteinase K / phenol extraction method” is preferable.
  • RNA degradation treatment is performed using RNase A and RNase T1 before the proteinase K treatment step in the “proteinase K / phenol extraction method” is preferable.
  • the “PCR using the primer pair of the present invention” is not particularly limited as long as it is a PCR using the primer pair of the present invention, and is an end point PCR method for detecting a PCR amplification product at the end of a cycle.
  • a real-time PCR method for monitoring an increase in PCR amplification products in real time may be used.
  • the reaction solution after completion of PCR is directly subjected to gel electrophoresis using agarose or the like, and the DNA fragment after electrophoresis is subjected to ethidium bromide staining, fluorescent reagent, etc.
  • a method of detecting can be exemplified.
  • SYBR registered trademark
  • SYBR registered trademark
  • Green II Green II
  • SYBR registered trademark
  • Gold Gold
  • BEBO YO-PRO-1
  • LCGreen registered trademark
  • SYTO-9 SYTO-13
  • SYTO-16 SYTO-60
  • SYTO-62 SYTO-64
  • SYTO-82 POPO-3
  • TOTO-3 BOBO-3
  • TO-PRO-3 YO-PRO-1
  • PicoGreen registered trademark
  • Nonspecific DNA intercalating dyes such as SYTOX® Orange and EvaGreen (registered trademark) can be used, and among them, SYBR (registered trademark) GreenI is preferably used.
  • the “real-time PCR using the primer / probe set of the present invention” is preferably a probe method using a fluorescently labeled probe of the present invention.
  • the type of the probe of the present invention used here is not particularly limited, and examples thereof include hydrolysis probes, molecular beacon probes, cycling probes, Eprobe (registered trademark), Qprobe (registered trademark), scorpion probes, and hybridization-probe.
  • a hydrolysis probe can be preferably mentioned.
  • the hydrolysis probe is usually a linear oligonucleotide in which the 5 'end of the nucleic acid probe is modified with a fluorescent substance (reporter fluorescent dye) and the 3' end is modified with a quenching substance (quencher).
  • a molecular beacon probe is usually an oligonucleotide that can take a stem-loop structure, in which the 5 ′ end of a nucleic acid probe is modified with a fluorescent substance (reporter fluorescent dye) and the 3 ′ end is modified with a quencher (quencher).
  • a cycling probe is usually a chimeric oligonucleotide composed of RNA and DNA, one end of which is modified with a fluorescent substance (reporter fluorescent dye) and the other end is modified with a quencher (quencher).
  • Eprobe is usually an artificial nucleic acid having two fluorescent dyes in thymine nucleotides, and fluorescence emission is suppressed in a single-stranded state not bound to the target, and emits fluorescence when bound to the target.
  • Qprobe is usually an oligonucleotide terminated with cytosine, and the cytosine at the end is labeled with a fluorescent substance, and is also called a guanine quenching probe.
  • the scorpion probe is usually an oligonucleotide that can take a hairpin loop structure in which one end of a nucleic acid probe is modified with a fluorescent substance (reporter fluorescent dye) and the 3 'end is modified with a quencher (quencher).
  • the hybridization-probe consists of a donor probe consisting of an oligonucleotide modified at the 3 'end with a fluorescent dye and an acceptor probe consisting of an oligonucleotide modified at the 5' end with a fluorescent dye. These probes are targeted. When the fluorescent dye is bound, both fluorescent dyes are close to each other, and the fluorescent dye of the acceptor probe is excited by the fluorescence of the fluorescent dye of the donor probe to emit light.
  • the PCR can be performed according to methods described in literature such as molecular cloning, A laboratory manual, KOD-Plus-Neo (Toyobo Co., Ltd.), TaKaRa PCR Amplification
  • Kit manufactured by TaKaRa
  • TaqMan registered trademark
  • Real-Time PCR Master Mixes manufactured by Thermo Fisher Scientific
  • each reagent necessary for the PCR reaction can be used for the PCR.
  • reagents examples include enzymes that polymerize nucleic acids (for example, polymerases), substances that serve as nucleic acid materials (for example, dNTPs), nucleic acid amplification buffers (for example, components having a buffering action such as Tris-Cl, Tween 20 and the like. Surfactant, etc.) and one or more selected from the group consisting of Mg 2+ , and necessary reagents can be additionally used depending on the type of PCR.
  • enzymes that polymerize nucleic acids for example, polymerases
  • substances that serve as nucleic acid materials for example, dNTPs
  • nucleic acid amplification buffers for example, components having a buffering action such as Tris-Cl, Tween 20 and the like.
  • Surfactant, etc. and one or more selected from the group consisting of Mg 2+ , and necessary reagents can be additionally used depending on the type of PCR.
  • a commercially available nucleic acid amplification device can be used depending on the type of nucleic acid amplification reaction to be used, and in particular, a device capable of measuring a probe signal (preferably a fluorescent signal) is also provided.
  • a preferred example is a nucleic acid amplification device.
  • real-time PCR devices that can both amplify nucleic acids and measure fluorescence signals include 7500 real-time PCR instruments from Applied Biosystems, CFX96 from Bio-RAD, and Light cycler 480 from Roche. Can do.
  • Steps (I) and (II) may be in the order of step (II) following step (I), the order of step (I) after step (II), or step (I) ) And step (II) may be carried out simultaneously, but it is preferred that step (I) and step (II) are carried out simultaneously.
  • the step (III) is carried out after carrying out the steps (I) and (II).
  • the “standard sample” used in the above step (II) is not particularly limited as long as it is a standard sample prepared by serial dilution of a known amount of human genomic DNA, but the human genomic DNA added to each PCR reaction tube is not limited. The amount is adjusted to be in the range of, for example, 1 fg to 10 ng, preferably 0.1 fg to 10 ng, more preferably 0.01 fg to 10 ng, still more preferably 0.001 fg to 10 ng, more preferably 0.0001 fg to 100 ng. Standard samples.
  • the “standard sample” may further contain genomic DNA derived from a non-human organism that can be included in the test sample, in addition to human genomic DNA. Further, the human genomic DNA contained in the “standard sample” may be fragmented according to the type of the “test sample”, for example, fragmented to an appropriate size in the range of 20 kbp to 100 bp. Human genomic DNA can be used.
  • step (II) As a method of “creating a calibration curve” in the above step (II), for example, it is created by plotting the intensity level of the fluorescent signal obtained by real-time PCR using the standard sample as a template against the amount of human genomic DNA. And a method of creating a Ct value of a fluorescent signal obtained by real-time PCR using the standard sample as a template against the amount of human genomic DNA.
  • Ct value means the number of cycles in which the amplification curve and the threshold value (Threshold) intersect in real-time PCR, and the amount of fluorescence resulting from the generation of the PCR amplification product reaches a certain amount (threshold value). Represents the number of cycles.
  • the Ct value decreases as the amount of target DNA initially contained in the sample increases, and conversely increases as the amount of target DNA included in the sample decreases.
  • the amount of human genomic DNA contained in the test sample can be calculated by comparing the intensity level or Ct value of the fluorescent signal obtained by real-time PCR of the test sample with the calibration curve.
  • the “human genomic DNA detection and / or quantification kit” of the present invention is a human genomic DNA comprising the primer pair of the present invention and / or the probe of the present invention.
  • the kit of the present invention may contain a package insert such as an instruction manual in addition to components (for example, a carrier, a pH buffer, a stabilizer, etc.) generally used in this type of detection kit.
  • the kit of the present invention further includes human genomic DNA for use as a standard sample and non-human organism-derived DNA (for example, mouse genomic DNA, rat genomic DNA, guinea pig genomic DNA, etc.) for use as a negative control. It may be.
  • Alu sequence contained in extracted DNA fragment As described in (1) above, it is considered that the Alu sequence is contained in one or more copies of 3000 nucleotide pairs of human genomic DNA.
  • the extracted human genomic DNA fragment has a length of 20,000 to 50,000 nucleotide pairs. Can be assumed. Based on such calculation, the present inventors have determined that it can be assumed that at least one copy of the Alu sequence is included in one fragment even in consideration of the possibility that the distribution of the Alu sequence is biased.
  • RNA-RNA binding is more stable than DNA-DNA binding, so the primer / probe may be trapped in RNA and may not be able to hybridize to the original target DNA. . Therefore, when quantifying a small amount of human genomic DNA as in the Alu-qPCR of the present invention, it is necessary to minimize the contamination of the RNA with the template. Therefore, the present inventors examined genomic DNA extraction conditions for minimizing RNA contamination. Specifically, cultured human fibroblasts (NHDF cells) were detached by trypsin, collected, and centrifuged (400 ⁇ g, room temperature, 5 minutes). The obtained cell pellet was suspended in TBS, and the number of cells was counted.
  • NHDF cells cultured human fibroblasts
  • PCI Nacalai Tesque, Inc .; hereinafter sometimes referred to as “PCI”) was added and mixed by vortexing. Centrifugation (maximum speed, 4 ° C., 15 minutes) was performed, and the aqueous layer (upper layer) was collected. Then, PCI was added again, mixed by vortexing, and centrifuged (maximum speed, 4 ° C., 15 minutes).
  • the aqueous layer was recovered, 0.2 times the amount of 10M ammonium acetate (Nippon Gene) and 2.5 times the amount of 100% EtOH (Wako Pure Chemical Industries) were added and mixed by inversion, followed by centrifugation. (Maximum speed, 4 ° C., 15 minutes). The supernatant was completely removed and washed with 70% EtOH. The pellet was dried for 10 minutes, and then an appropriate amount of TE buffer (pH 8.0) was added to dissolve the pellet. The concentration of nucleic acid contained in the obtained sample was measured by an ultraviolet absorbance method and a PicoGreen DNA quantification method. In the ultraviolet absorbance method, the absorbance of A260 was measured using NanoDrop.
  • RNA is mixed in each extraction sample at a certain ratio. Investigate whether they are doing. The results are shown in FIG. When neither RNase A nor RNase T1 was used, the proportion of RNA in the total nucleic acid in the sample was 76.1%.
  • RNA in the total nucleic acid in the sample was 65.2 to 43.3%.
  • RNase A (20 ⁇ g / mL) and RNase T1 (100 U) are used in combination, RNA accounts for 58.6% of the total nucleic acid in the sample, and RNase A (200 ⁇ g / mL) and RNase T1 (1000 U) were used in combination, the proportion of RNA was 32.0%.
  • RNA contamination can be greatly suppressed by using a combination of 200 ⁇ g / mL RNase A and 1000 U RNase T1 during genomic DNA extraction.
  • RNA digestion with 200 ⁇ g / mL RNase A and 1000 U / mL RNase T1 was performed during the extraction of genomic DNA from cells or tissues, and the DNA in the sample was also extracted. All concentrations were measured by the PicoGreen DNA quantification method.
  • Template 1 Buffer only (TE buffer or EASY Dilution buffer)
  • Template 2 50 ng / ⁇ L mouse genomic DNA
  • Template 3 Standard sample containing only 11 levels of human genomic DNA from 0.0005 fg / ⁇ L to 5 ng / ⁇ L
  • Template 4 8 levels of human genomic DNA from 0.5 fg / ⁇ L to 5 ng / ⁇ L and mouse Mixed standard sample prepared with a total concentration of 50 ng / ⁇ L of genomic DNA
  • a threshold (Threshold) is set at an appropriate position in the obtained amplification curve, and Ct A standard curve is created by calculating a value (Threshold Cycle) and plotting the Ct value for each amount of human genomic DNA.
  • the range where the linearity of the calibration curve is recognized is the amount of human genomic DNA that can be detected by PCR. That is, by using templates 3 and 4, it is possible to determine a detection limit value for a sample containing only human genomic DNA or a mixture sample containing human and mouse genomic DNA.
  • the templates 1 and 2 are negative control samples, and the signals detected by PCR using the templates 1 and 2 are nonspecific. That is, the lower the Ct value in templates 1 and 2, the lower the specificity of PCR.
  • the detection limit value of the McBride primer / probe set is 1 fg when the template contains only human genomic DNA, but decreases to 1 pg when the template contains human genomic DNA and mouse genomic DNA. It became clear that. These results indicate that the McBride primer / probe set has low specificity and cannot detect and quantify trace amounts of human genomic DNA.
  • Example 3 [Identification of human Alu model sequence] As described above, the results of Example 3 indicate that the sensitivity and specificity of the McBride primer / probe set are low. The reasons for this are that a) the McBride primer / probe set may not be designed to sufficiently cover the Alu sequence in the human genome, and b) primers and / or probes. The background was considered to be increased by nonspecific binding between each other and nonspecific binding to mouse genomic DNA. Therefore, in order to solve the problem a), the present inventors based on the latest data on Alu sequences (Wheeler et al., Nucleic Acids Res, 41, D70-82. 2013), 46 Alu subs.
  • One “human Alu model sequence” representing a family consensus sequence was identified, and an attempt was made to design a primer / probe set that covers the most Alu sequences in the human genome using this human Alu model sequence.
  • multiple alignments were created using the ClustalW2 program based on all the consensus sequences of 46 Alu subfamilies registered in the Dfam1.3 database.
  • the frequency of occurrence of A, T, G, and C at each position of the multiple alignment is determined by the number of constituent factors for each of the 46 subfamilies.
  • a position-specific score matrix was created by calculating (copy number in the human genome) as a weight ratio (FIGS. 4-1 to 4-3). We used this position-specific score matrix to attempt to identify “human Alu model sequences” that cover the maximum number of Alu sequences in the human genome.
  • the prototype of the human Alu model sequence was determined as follows.
  • Y represents C or T
  • S represents C or G
  • R represents A or G, respectively.
  • Prototype of human Alu model sequence SEQ ID NO: 4: GGCCGGGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGG Y GGATCAC Y TGAGG Y CAGGAGTTCGAGACCAGCCTGGCCAACATGGTGAAACCCCGTCTCTACTAAAAATACAAAAATTAGCCGGGCGTGGTGGCG S G Y GCCTGTA R TCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATCGCTTGAACCCGGGAGGCGGAGGTTGCAGTGAGCCGAGATCGCGCCACTGCACTCCAGCCTGGG Y GACAGAG Y GAGACTC Y GTCTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
  • Human Alu model sequence (SEQ ID NO: 5): GGCCGGGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGCGGATCACTTGAGGTCAGGAGTTCGAGACCAGCCTGGCCAACATGGTGAAACCCCGTCTCTACTAAAAATACAAAAATTAGCCGGGCGTGGTGGCGCGTGCCTGTAATCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATCGCTTGAACCCGGGAGGCGGAGGTTGCAGTGAGCCGAGATCGCGCCACTGCACTCCAGCCTGGGCGACAGAGCGAGACTCTGTCTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
  • Primer3 (Primer3web version 4.0.0)
  • the present inventors comprise a forward primer, a reverse primer consisting of a partial sequence of the human Alu model sequence or its complementary sequence, And a combination of probes was selected. Specifically, in Primer 3, only the following parameters i) and ii) were changed, and the rest were all set to default settings, and the human Alu model sequence (positions # 1 to # 300) was input.
  • Mispriming Library (repeat library): RODENT_AND_SIMPLE ii) Pick hybridization probe (internal oligo), or use oligo below: Check and select the combination of primer and probe with the highest “quality” (ie, the lowest “objective function value”) from the output. did.
  • the selected combinations are: forward primer: CGGATCACTTGAGGTCAGAGA (SEQ ID NO: 6; positions # 57 to # 76 of the human Alu model sequence), reverse primer: GGTTCAAGCGATTCTCCCTGC (SEQ ID NO: 7; complement of positions # 206 to # 187 of the human Alu model sequence Sequence), probe: TGGTGAAACCCCGTCTCTAC (SEQ ID NO: 8; positions # 100 to # 119 of the human Alu model sequence) (FIG. 6).
  • These primers and probes were synthesized, and the 5 ′ end of the probe was labeled with FAM and the 3 ′ end was labeled with BHQ1 (hereinafter, this primer and probe combination is referred to as “Primer3 primer / probe set”). There).
  • PCR samples were prepared as follows. PCR sample preparation: Template 2 ⁇ L TaqMan Universal Master Mix II, no UNG (Applied Biosystems) 10 ⁇ L Forward primer (10 ⁇ M) 0.4 ⁇ L Reverse primer (10 ⁇ M) 0.4 ⁇ L Probe (12.5 ⁇ M) 0.5 ⁇ L 6.7 ⁇ L of water Total 20 ⁇ L
  • the inventors of the present invention selected non-specific binding between the primer and the probe and the primer / probe and mouse genomic DNA in the primer and probe sequence selection process. We thought that it was necessary to avoid sequences that would cause non-specific binding, so we set our own primer and probe selection criteria. Specifically, the following criteria 1 to 7 were set for selecting primer pairs, and the following criteria 8 to 15 were set for selecting probes.
  • the criteria 1 to 7 may be referred to as “primer criteria”, the criteria 8 to 11 as “essential probe criteria”, and the criteria 12 to 15 as “probe supplement criteria”.
  • the second nucleotide from the 3 ′ end is G or C;
  • At least one of the nucleotides from the 3 'end to the 3rd end of the primer is A or T;
  • [Criteria 6] A nucleotide sequence complementary to a continuous nucleotide sequence of 5 or more nucleotides contained in any one molecule between the two primers, between the forward primers, between the reverse primers, and between the forward primer and the reverse primer.
  • Two nucleotide sequences in the nucleotide sequence of one or more genomic DNAs selected from the group consisting of non-human organisms are nucleotide sequences consisting of 19 consecutive nucleotides in the probe.
  • the probe has a nucleotide length of 20 or more;
  • mice, rat, and nucleotide sequences at positions # 1 to # 282 of the human Alu model sequence are used as input sequences.
  • BLAST search http://blast.ncbi.nlm.nih.gov/Blast.cgi
  • the search database used is as follows. Mouse genome: all assemblies top-level, Annotation Release 105 Rat Genome: all assemblies, Annotation Release 105 Guinea Pig Genome: Cavpor3.0 reference Annotation Release 102
  • the search parameters were the initial parameters except for the following.
  • Program selection Megablast Short queries: off Word size: 16; Match / Mismatch Scores: 1, -4 Gap Costs: 5, 2 Filters and Masking: off
  • the above parameters are set so that the region that matches 100% or more with the nucleotide sequence of 19 nucleotides or more contained in positions # 1 to # 282 of the human Alu model sequence in the genome of mouse, rat and guinea pig can be extracted. It is.
  • As a result of the BLAST search it was found that there are 6055 nucleotide sequences that match 100% or more consecutive nucleotide sequences in human Alu model sequences in mice, 2951 in rats, and 13298 in Guinea pigs. It was.
  • FIG. 8 shows how many 19 nucleotide contiguous sequences starting from each position exist in the mouse, rat, and Guinea pig genomes for positions # 1 to # 264 of the human Alu model sequence. For example, in the position # 1 of FIG. 8, all the columns of mouse, rat, and guinea pig are described as “0”.
  • the present inventors also considered the condition 2 that the lengths of the forward primer and the reverse primer are each 20 nucleotides or more, out of positions # 1 to # 282 of the human Alu model sequence.
  • the positions that can meet the criteria 1 are shown in white, and the positions that cannot meet the criteria 1 are shown in gray.
  • Primer3's forward primer is It was found that three complete sequences were present, seven McBride forward primers, 79 McBride reverse primers, and 186 McBride probes.
  • the McBride reverse primer since the McBride reverse primer has a total length of 19 nucleotides, the entire primer perfectly matches the rodent genomic sequence. As mentioned above, the sensitivity of the McBride primer / probe set is significantly reduced when template 4 (mixed sample of human and mouse genome) is used, which means that the McBride primer recognizes rodent genome sequences. Is considered to be the cause.
  • Criteria 2 From our experience so far, it has been found that the length is preferably 20 nucleotides or more in order to give the primer sufficient specificity. For this reason, Criteria 2 with a primer length of 20 nucleotides or more was set.
  • Criteria 1 and 2 remove most of the primers with 19 or more nucleotide complementarity with the rodent genome, but still have primers with 18 or less nucleotide complementarity with the rodent genome. For this reason, when the primer full length (20 nucleotides or more) matches the nucleotide sequence used as a template (that is, when it specifically binds to the Alu sequence), the present inventors only have 18 nucleotides in the primer. By designing the primer so that the difference in Tm value between the case where it does not match the template nucleotide sequence (that is, nonspecific binding to a sequence other than the Alu sequence) is maximized, I wanted to prevent specific amplification.
  • the two nucleotides at both ends of the primer may not form a complementary bond with the rodent genome.
  • the Tm value decreases.
  • At least one end of the 5 ′ or 3 ′ ends of the primer is defined as G
  • Criteria 3 is provided, in which, when the terminal nucleotide is not G or C, the second nucleotide from the terminal is G or C.
  • the primer specificity should be designed so that when the primer and the target sequence are complementarily bound, the binding stability at the 3 'end of the primer is low (the Gibbs free energy is high).
  • the criterion 3 is set so that the second nucleotide from the 3 ′ end or the end of the primer is G or C. Since the free energy of G or C is lower than that of A or T, there is a possibility that the stability in the vicinity of the 3 ′ end of the primer is extremely increased by the criterion 3. In order to prevent such a possibility, criterion 4 having at least one of the first to third nucleotides from the 3 ′ end as A or T was set.
  • Criteria 9 From our experience so far, it has been found that the length is preferably 20 nucleotides or more in order to give the probe sufficient specificity. Therefore, Criteria 9 was set in order to select a probe of 20 nucleotides or more.
  • probes that can be used as Alu-qPCR primers can be selected.
  • a probe having more desirable properties can be selected using one or more of the criteria 12 to 15.
  • criteria 12 As described above, the inventors of the present invention have one of the causes for the decrease in the sensitivity of the McBride primer / probe set. It was thought that a complementary strand of 4 nucleotides was formed (FIG. 10). Therefore, in order to exclude a nucleotide sequence that tends to cause complementary strand formation (homodimer and / or heterodimer formation) between the primer and the probe, criteria 12 were set.
  • Criteria 13 Various fluorescent molecules are known to be quenched by proximity to a guanine base. For this reason, when the 5 ′ end of the probe is labeled with a fluorescent substance, it is desirable that the 5 ′ end nucleotide is not G. For this reason, criteria 13 were set.
  • the criterion 14 was set in order to select a probe having a change in free energy of ⁇ 7 kcal / mol or more when the most stable dimer was formed between the primer and the probe.
  • [Selection of primer pair of the present invention] Selection of candidate primers A plurality of candidate primer sequences were selected from human Alu model sequences using the primer design program Primer3. Specifically, in Primer 3, only the following parameters i) to iii) were changed, and the rest were all set to default settings, and the human Alu model sequence (positions # 1 to # 300) was input. i) Task value: pick_primer_list ii) Numbers To Return value: 150 iii) Mispriming Library (repeat library) value: RODENT_AND_SIMPLE The settings i) and ii) are for obtaining all the primer sequences that can be designed, and the setting iii) is for avoiding crossover with rodent sequences. As a result of this selection, 106 forward primer candidates and 102 reverse primer candidates were obtained.
  • the forward primer candidate is judged to satisfy the criteria 1 and is selected.
  • the positions of the 3 ′ ends of the forward primer candidates shown in gray in FIG. 8 (positions # 1 to 35, 37 to 41, 57 to 62, 68 to 84, 86, 87, 96 to 98, 112 to 120, 128 to 136, 140-186, 239-243, 250, 251 and 261-264), the forward primer Candidate was determined not to meet the criteria 1. As a result, 49 candidates were selected from the 106 forward primer candidates as satisfying the criteria 1, and 48 candidates were selected as satisfying the criteria 1 from the 102 reverse primer candidates. .
  • RNAstructure version 5.6 a secondary structure prediction program provided by Mathews Lab at Rochester University (http://rna.urmc.rochester.edu/index.html)
  • Mathews Lab at Rochester University http://rna.urmc.rochester.edu/index.html
  • RNAstructure a secondary structure formed between two molecules consisting of the same nucleotide sequence is predicted by RNAstructure, and i) either one of them
  • the other molecule does not contain a nucleotide sequence complementary to the nucleotide sequence from the 3 'end to the 3rd end of the molecule (the 1st to 3rd nucleotide sequence from the 3'end); ii) contained in either molecule Nucleotide sequence complementary to a continuous nucleotide sequence of 5 or more nucleotides that is not included in the other molecule; iii) complementary to a nucleotide sequence of 4 or more nucleotides that consists of G or C included in one of the molecules Primer candidates that satisfy the conditions i) to iii) were selected.
  • the secondary structure of each was predicted by RNAstructure, and a combination satisfying all the above conditions i) to iii) was searched. As a result, it was found that only combinations of the forward primer candidate F10 and the reverse primer candidate R1 satisfy all the above i) to iii). From the above results, the forward primer F10 (GGTGAAACCCCGTCTCTACT; SEQ ID NO: 18) and the reverse primer R1 (GGTTCAAGCGATTTCCCTGC; SEQ ID NO: 19) were selected as primers satisfying the criteria 1 to 7.
  • [Selection of probe of the present invention] Necessity of probe As a method for detecting fluorescence in real-time PCR, an intercalator method using a reagent that non-specifically binds to an amplification product and a reagent that binds to a nucleotide sequence specific to the amplification product are used. There are two types of probe methods. Since the intercalator method also detects non-specific nucleotide sequences, it is generally considered that the probe method is superior from the viewpoint of specificity. In the present invention, the aim is to measure the number of copies of Alu contained in a sample by real-time PCR.
  • probe candidates in the region amplified by the forward primer F10 and the reverse primer R1 were selected. Specifically, in Primer3, change only the parameters of i) to vii) below, and use the default settings for all others, and enter the nucleotide sequence of positions # 120 to # 186 of the human Alu model sequence. did.
  • the setting of vii) is based on the report that the Tm value of the probe is preferably about 10 ° C. higher than the Tm of the primer (Holland, PM, Abramson, RD, Watson, R. and Gelfand, DH). (1991) Detection of specific polymerase chain reaction product by utilizing the 5 '---- 3' exonuclease activity of Thermus aquaticus DNA polymerase.Proc Natl Acad Sci USA, 88, 7276-7280., Bustin, SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. Journal of molecular endocrinology, 25, 169-193.). As a result of this selection, 76 probe candidates were obtained.
  • the present inventors include a nucleotide sequence identical to the nucleotide sequence consisting of 19 nucleotides in the mouse, rat, and guinea pig genome in the region of positions # 121 to # 146 of the human Alu model sequence. I checked again. Specifically, using the nucleotide sequence of positions # 121 to # 146 of the human Alu model sequence as an input sequence, a BLAST search of the entire genome of mouse, rat, and Guinea pig (http://blast.ncbi.nlm.nih.gov /Blast.cgi). The search database used is as follows.
  • the number of probe candidates that satisfy both of the criteria 8 and 9 was limited to 13 shown in Table 3 below. Further, since the probe may be either a sense strand or an antisense strand, in addition to the 13 probe sequences shown in Table 3, their complementary sequences shown in Table 4 were also selected as probe candidates.
  • RNAstructure version 5.6 Secondary structure prediction program RNAstructure (RNAstructure version 5.6) provided by Mathews Lab of Rochester University (http://rna.urmc.rochester.edu/index.html) Thus, those satisfying the criteria 10 and 11 were selected from the probe candidates shown in Tables 3 and 4.
  • the present inventors predicted that the 26 probes described above would have the same level of function as probes.
  • supplementary criteria for probes (criteria 12 to 15) were used. Further selection was made using. Specifically, first, 13 candidates (probes P14 to P26) satisfying the criteria 12 were selected from the 26 candidates. Next, 11 candidates (P16 to P26) satisfying the criteria 13 were selected from these 13 candidates. Furthermore, 7 candidates (P14 to P20) satisfying the criteria 14 were selected from these 11 candidates. Finally, P16 satisfying the criteria 15 was selected from these seven candidates.
  • FIG. 11 shows the positions of the selected forward primer F10, reverse primer R1, and probe P16 in the human Alu model sequence.
  • F10 (GGTGAAACCCCGTCTCTACT; SEQ ID NO: 18) is used as the forward primer used in the Alu-qPCR of the present invention
  • R1 (GGTTCAAGCGATTCTCCCTGC; SEQ ID NO: 19) is used as the reverse primer
  • P11 ATACAAAAATAGTAGCGGGCG; SEQ ID NO: 37
  • P13 TACAAAAATTTAGCCGGGCGT; SEQ ID NO: 39
  • P16 CGCCCCGGCTAATTTTTTTAT; SEQ ID NO: 42
  • P21 ACGCCCCGCTAATTTTTGTA; SEQ ID NO: 47).
  • the combination of the probe P16, the forward primer F10, and the reverse primer R1 is “the primer / probe set A of the present invention”, and the combination of the probe P11, the forward primer F10, and the reverse primer R1 is “the primer / probe set of the present invention”.
  • B the combination of probe P13, forward primer F10 and reverse primer R1 is“ primer / probe set C of the present invention ”, and the combination of probe P21, forward primer F10 and reverse primer R1 is“ primer / probe of the present invention ”
  • Each may be referred to as “set D”.
  • the primer / probe sets A to D of the present invention may be collectively referred to as “the 20 nt primer / probe set of the present invention”.
  • Template 5 50 ng / ⁇ L rat genomic DNA
  • Template 6 Mixed standard sample prepared from human genomic DNA at 8 levels from 0.5 fg / ⁇ L to 5 ng / ⁇ L and rat genomic DNA to a total concentration of 50 ng / ⁇ L Primer / probe of the present invention Using the set A and the above templates 1 to 6, PCR samples were prepared as follows.
  • FIG. 16 shows the results.
  • the Ct value was about 50 (“Buf.” In FIG. 16). This result suggests that almost no non-specific binding between the primer and the probe is formed in the primer / probe set C of the present invention.
  • the Ct value was 40 or more (“Rat” in FIG. 16).
  • FIG. 17 shows the results.
  • no Ct value could be detected (“Buf.” In FIG. 17).
  • the Ct value was 40 or more (“Rat” in FIG. 17).
  • mice [Detection of human cells xenografted in mice]
  • xenograft model mouse and DNA extraction Xenograft model by injection administration of 500,000 human mesenchymal stem cells (human MSC) into the tail vein of NOD-scid mice (7 weeks old, male) Mice were prepared (18 cases in total). One day, three days, and one week after administration, the mice were euthanized, and the lungs, kidneys, and livers were excised (6 cases each), immediately frozen in liquid nitrogen, and stored at ⁇ 80 ° C. Each organ was freeze-ground and added with Lysis buffer containing 200 ⁇ g / mL RNase A and 1000 U / mL RNase T1, and incubated at 37 ° C. for 1 hour.
  • Lysis buffer containing 200 ⁇ g / mL RNase A and 1000 U / mL RNase T1
  • proteinase K solution manufactured by Wako Pure Chemical Industries, Ltd.
  • PCI manufactured by Nacalai Tesque
  • the aqueous layer was recovered, 0.2 times the amount of 10M ammonium acetate (Nippon Gene) and 2.5 times the amount of 100% EtOH (Wako Pure Chemical Industries) were added and mixed by inversion, followed by centrifugation. (Maximum speed, 4 ° C., 15 minutes). The supernatant was completely removed and washed with 70% EtOH. The pellet was air-dried for 10 minutes, and then an appropriate amount of TE buffer (pH 8.0) was added to dissolve the pellet. The DNA concentration contained in the obtained sample was measured by the PicoGreen DNA quantification method. In addition, lungs, kidneys, and livers were also collected from control mice (total 3 cases) not administered with human MSC, and genomic DNA was extracted and quantified in the same manner as described above.
  • Template Templates 7 to 15 were prepared as follows using the genomic DNA extracted in (1) above. Templates 9, 12, and 15 are samples to be quantified, and were prepared by diluting genomic DNA extracted from each organ of a xenograft model mouse to 50 ng / ⁇ L. Templates 7, 10, and 13 are negative control samples corresponding to templates 9, 12, and 15, respectively. Templates 8, 11, and 14 are standard sample samples for creating calibration curves for quantification of templates 9, 12, and 15, respectively. Note that TE buffer was used for the preparation of templates 7 to 15.
  • Template 7 50 ng / ⁇ L genomic DNA derived from the lungs of control mice
  • Template 8 Mixed standard sample template 9 prepared by adjusting human genomic DNA at 8 levels from 0.5 fg / ⁇ L to 5 ng / ⁇ L and genomic DNA from lungs of control mice to a total concentration of 50 ng / ⁇ L: Sample template 10 containing genomic DNA derived from the lungs of xenograft model mice 1 day, 3 days, or 1 week after human MSC transplantation at a concentration of 50 ng / ⁇ L: 50 ng / ⁇ L genomic DNA from kidney of control mouse Template 11: Mixed standard sample template 12 prepared by adjusting human genomic DNA at 8 levels from 0.5 fg / ⁇ L to 5 ng / ⁇ L and genomic DNA from kidney of control mouse to a total concentration of 50 ng / ⁇ L: Sample template 13 containing genomic DNA from kidneys of xenograft model mice 1 day, 3 days, or 1 week after human MSC transplantation at a concentration of 50 ng
  • the amount of human genomic DNA contained in the whole lung of the xenograft model mouse was calculated by multiplying this value by the dilution factor at the time of preparing template 9 and the total volume ( ⁇ L) of genomic DNA extracted from the lung. .
  • the whole lungs 1 day, 3 days and 1 week after MSC transplantation contained 79345.527 pg, 8851.071 pg, and 674.765 pg of human genomic DNA, respectively.
  • the number of human MSCs contained in the whole lung of a xenograft model mouse was calculated with the weight of genomic DNA contained in one human cell being 6 pg.
  • the median value in the box plot shows that there are 10,000 or more MSCs in the lung 1 day after the MSC transplantation, but it decreases to 1316 after 3 days and 74 after 1 week ( FIG. 18B).
  • the amount of human genomic DNA contained in the whole kidney of the xenograft model mouse was calculated by multiplying this value by the dilution factor at the time of preparing the template 12 and the total volume ( ⁇ L) of genomic DNA extracted from the kidney. .
  • the whole kidneys 1 day, 3 days, and 1 week after MSC transplantation contained 1260.5989 pg, 165.4567 pg, and 25.8333 pg of human genomic DNA, respectively.
  • the weight of genomic DNA contained in one human cell was 6 pg
  • the number of human MSCs contained in the whole kidney of the xenograft model mouse was calculated.
  • the median value in the box plot showed that 191 MSCs were present in the kidney 1 day after MSC transplantation, and decreased to 22 after 3 days and 4 after 1 week (Fig. 19 (b)). From the above, it was revealed that MSC was present in the kidney even after one week after transplantation in the xenograft model mice. According to a conventional report, in the xenograft model mice in which human cells were transplanted by tail vein injection, it was considered that no engraftment of human cells was observed except in the lungs.
  • the results shown in FIG. 19 are the first results showing that transplanted human MSCs are engrafted in the mouse kidney.
  • the amount of human genomic DNA contained in the whole liver of the xenograft model mouse was calculated by multiplying this value by the dilution factor at the time of preparing the template 15 and the total volume ( ⁇ L) of genomic DNA extracted from the liver. .
  • 715.940 pg of human genomic DNA was contained in the entire liver one day after MSC transplantation.
  • the number of human MSCs contained in the whole liver of a xenograft model mouse was calculated with the weight of genomic DNA contained in one human cell being 6 pg. As a result, it was found that 107 MSCs were present in the liver one day after MSC transplantation as the median value in the box plot, but became below the detection limit after 3 days and 1 week (FIG. 20 (b)). )).
  • Template preparation and real-time PCR Templates 16 and 17 were prepared as follows using the genomic DNA extracted in (1) above.
  • the template 17 is a sample to be quantified, and the template 16 is a standard sample sample for creating a calibration curve.
  • PCR was carried out under the same conditions as in Example 14. Note that a TE buffer was used for preparing the templates 16 and 17.
  • Template 16 Sample template 17 containing genomic DNA derived from kidney of subrenal graft model mouse at a concentration of 50 ng / ⁇ L: A mixed standard sample prepared with human genomic DNA at 8 levels from 0.5 fg / ⁇ L to 5 ng / ⁇ L and genomic DNA from kidneys of control mice to a total concentration of 50 ng / ⁇ L
  • Template 18 Sample template 19 containing human genomic DNA fragmented to 20 kbp in 10 concentrations from 0.05 fg / ⁇ L to 50 ng: Sample template 20 containing human genomic DNA fragmented at 1000 bp in 10 concentrations from 0.05 fg / ⁇ L to 50 ng: Sample containing human genomic DNA fragmented at 250 bp at 10 concentrations from 0.05 fg / ⁇ L to 50 ng (3) Real-time PCR PCR samples were prepared in the same manner as in Example 11 using the primer / probe set A of the present invention and the templates 18 to 20, and real-time PCR was performed using the LightCycler 480 real-time PCR system (Roche Life Sciences).
  • the PCR reaction was performed by heat denaturation at 95 ° C. for 10 minutes, followed by 5 cycles of 95 ° C. for 30 seconds, 56 ° C. for 4 minutes 30 seconds, and 72 ° C. for 30 seconds, and then 95 ° C. for 30 seconds. The cycle of 56 ° C. for 30 seconds and 72 ° C. for 30 seconds was repeated 45 times.
  • the template and sample were prepared using a low adsorption tube and a chip so that a trace amount of DNA was not lost. (4) Sensitivity to fragmented DNA The results of real-time PCR in (3) above are shown in FIGS.
  • a calibration curve was created based on Ct values obtained by real-time PCR of templates 18 to 20 (standard samples using fragmented DNAs of 20 kbp, 1000 bp, and 250 bp in length). In any case, 10 fg to 10 ng The linearity of the calibration curve was observed between (Figs. 26-28). These results indicate that Alu-qPCR using the primer / probe set A of the present invention can maintain its sensitivity even when the DNA contained in the sample to be quantified is highly fragmented. It was.
  • Template Templates 21 to 28 were prepared as follows using the chicken, porcine, bovine, and canine genomic DNA extracted in (1) above. Templates 21, 23, 25, and 27 are negative control samples corresponding to templates 22, 24, 26, and 28, respectively.
  • Template 21 50 ng / ⁇ L chicken genomic DNA
  • Template 22 Chicken genome mixed standard sample template 23 prepared by adjusting human genomic DNA at 10 levels from 0.05 fg / ⁇ L to 50 ng and chicken genomic DNA to a total concentration of 50 ng / ⁇ L: 50 ng / ⁇ L porcine genomic DNA
  • Template 24 Porcine genome mixed standard sample template 25 prepared by adjusting human genomic DNA at 10 levels from 0.05 fg / ⁇ L to 50 ng and porcine genomic DNA to a total concentration of 50 ng / ⁇ L: 50 ng / ⁇ L of bovine genomic DNA
  • Template 26 Bovine genome mixed standard sample template 27 prepared by preparing human genomic DNA of 10 levels from 0.05 fg / ⁇ L to 50 ng and bovine genomic DNA to a total concentration of 50
  • Templates 30 to 32 are samples to be quantified, and 1 ⁇ L of genomic DNA sample extracted from ancient human bones was used.
  • the template 29 is a standard sample sample for creating a calibration curve for quantification of the templates 30 to 32.
  • Template 29 Standard sample template 30 containing human genomic DNA fragmented at 1 kbp in 10 concentrations from 0.05 fg / ⁇ L to 50 ng: Sample template 31 from Nouchi 17: Sample template 32 from Hatauchi No. 26: Sample from Hatauchi 45
  • FIG. 33 shows the amplification curve obtained by real-time PCR of templates 29 to 32
  • FIG. 34 shows the calibration curve created based on the Ct value obtained by real-time PCR of template 29. Show.
  • the amplification curves of the ancient bone-derived samples (templates 30 to 32) all have a calibration curve within the linear range (10 fg to 10 ng), and can be accurately quantified by the Alu-qPCR of the present invention. Became clear.
  • the samples derived from No. 17 in the field, No. 26 in the field, and No. 45 in the field are each 2 .19 pg / ⁇ L, 0.11 pg / ⁇ L, and 0.61 pg / ⁇ L of human genomic DNA were found to be contained (FIG. 35).
  • the Tm value of the above known primer / probe set was calculated, and BLAST search for human and rodent genomes was performed for each sequence.
  • the primer / probe sets of the above documents 1, 3 to 5 were designed such that the Tm value of the probe was about 5 to 10 degrees higher than that of the primer.
  • any of the primer sets of the following documents 6 to 13 the same sequence as the sequence consisting of 18 consecutive bases contained therein must be present in two or more places in the genome of mouse, rat and / or guinea pig. Became clear.
  • Reference 6 Mira, E., Lacalle, RA, Gomez-Mouton, C., Leonardo, E. & Manes, S. Quantitative determination of tumor cell intravasation in a real-time polymerase chain reaction-based assay. Clin. Exp. Metastasis 19, 313-318 (2002).
  • Reference 7 Nehmann, N., Wicklein, D., Schumacher, U. & Muller, R.
  • Reference 10 Schneider, T., Osl, F., Friess, T., Stockinger, H. & Scheuer, WV Quantification of human Alu sequences by real-time PCR--an improved method to measure therapeutic efficacy of anti-metastatic drugs in human xenotransplants. Clin. Exp. Metastasis 19, 571-582 (2002).
  • Reference 11 Umetani, N. et al. Increased integrity of free circulating DNA in sera of patients with colorectal or periampullary cancer: direct quantitative PCR for ALU repeats. Clin. Chem. 52, 1062-1069 (2006).
  • Reference 12 Walker, J. A. et al.
  • the modified criteria 1, 2, 5, and 7 to 10 and the criteria 3, 4, 6, and 11 are used in combination (in the present specification, a criterion for a primer combining the modified criteria 1, 2, 5, and 7 and the criteria 3, 4, and 6 is referred to as "Criteria 1". And may be referred to as “criteria 8 ′ to 11 ′”. Also, the criteria for the probe in which the modified criteria 8 to 10 and the criteria 11 are combined may be referred to as “criteria 8 ′ to 11 ′”).
  • the nucleotide sequence consisting of 18 consecutive nucleotides in the forward primer and the reverse primer is selected from the group consisting of non-human organisms.
  • the nucleotide sequence of one or more genomic DNAs and the above-mentioned modified criteria 8“ a nucleotide sequence comprising 18 consecutive nucleotides in a probe is a non-human organism.
  • nucleotide sequence consisting of 17 consecutive nucleotides in the reverse primer and the reverse primer is a sequence that does not exist in two or more nucleotide sequences of one or more genomic DNAs selected from the group consisting of non-human organisms ”
  • the nucleotide sequence consisting of 17 consecutive nucleotides in the probe is present in two or more places in the nucleotide sequence of one or more genomic DNAs selected from the group consisting of non-human organisms” Select the "no" sequence.
  • FIG. 39 is selected in order to select a sequence which does not exist in two or more places in the sequence.
  • the nucleotide sequence consisting of 17 consecutive nucleotides in the forward primer and the reverse primer is selected from the group consisting of non-human organisms.
  • FIG. 40 was prepared in order to select a sequence which does not exist in two or more nucleotide sequences of one or more genomic DNAs.
  • primer candidates were selected from human Alu model sequences using the primer design program Primer3web4.1.0. Specifically, “Old Secondary Structure Alignments” of Primer 3 was used, parameters were set as follows, and human Alu model sequences (positions # 1 to # 300) were input. i) Max Self Complementarity: 5.0 ii) Max 3 'Self Complementarity: 4.0 iii) Max Pair Complementarity: 5.0 iv) Max 3 'Pair Complementarity: 5.0 Next, from the candidates selected by Primer, candidates that satisfy the criteria 1 ′ were selected using FIG. 40, and then primer pairs that satisfy the criteria 2 ′, 5 ′, and 7 ′ were further selected.
  • the 39 forward primer candidates (F1 ′ to F39 ′) and 20 reverse primer candidates (R1 ′ to R20 ′) thus selected are shown in Tables 5 and 6, respectively.
  • “Position #” in Tables 5 and 6 indicates the position # of the human Alu model sequence that is the starting point of each primer.
  • FIG. 41 shows the secondary structure of primer dimers (forward primers F34 'and reverse primers R20') predicted using RNAstructure.
  • a sequence in the sense direction (TGCAGTGGCGCGATCTCG; SEQ ID NO: 149) was selected.
  • probe P1 ′ such a probe is referred to as “probe P1 ′”.
  • the combination of forward primer F34 ', reverse primer R20', and probe P1 ' may be referred to as "primer / probe set E of the present invention".
  • Template 33 Sample containing human genomic DNA fragmented at 100 bp at 10 concentrations from 0.05 fg / ⁇ L to 50 ng (3)
  • Real-time PCR PCR samples were prepared using the primer / probe set E of the present invention and the template 33 in the same manner as in Example 11, and real-time PCR was performed using the LightCycler 480 real-time PCR system (Roche Life Sciences). The PCR reaction was performed by heat denaturation at 95 ° C. for 10 minutes, followed by 5 cycles of 95 ° C. for 30 seconds, 56 ° C. for 4 minutes 30 seconds, and 72 ° C. for 30 seconds, and then 95 ° C. for 30 seconds. The cycle of 56 ° C. for 30 seconds and 72 ° C. for 30 seconds was repeated 45 times.
  • FIG. 43 shows the results of real-time PCR in (3) above.
  • a calibration curve was created based on the Ct value obtained by real-time PCR of template 33 (standard sample using fragmented DNA of 100 bp length)
  • linearity of the calibration curve was recognized between 10 fg and 10 ng. (FIG. 43). From this result, it was revealed that Alu-qPCR using the primer / probe set E of the present invention can detect and quantify human genomic DNA highly fragmented to about 100 bp with high sensitivity.
  • human genomic DNA can be obtained from samples with poor conditions that could not be quantified by the prior art (for example, samples containing a large amount of other organism genomes or old samples in which DNA is highly fragmented). Can be detected and quantified with high sensitivity. Therefore, by such a method, engraftment / proliferation of human cells in a xenograft model animal can be confirmed accurately. Such a method can also be used to detect a small amount of human genomic DNA contained in a forensic sample or paleontological sample.

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Abstract

L'invention concerne un procédé de détection et/ou de dosage d'ADN génomique humain dans un échantillon d'essai à une sensibilité élevée. Une " séquence modèle d'Alu humaine " représentant la séquence consensus de 46 sous-familles d'Alu humaines est identifiée, et des amorces-sondes de PCR candidates pour la détection d'Alu sont sélectionnées à l'aide de cette séquence modèle d'Alu humaine. Ensuite, un ensemble amorce-sonde qui est une séquence oligonucléotidique qui s'hybride spécifiquement avec la séquence d'Alu humaine et qui comprend une séquence oligonucléotidique apte à minimiser la formation de dimère est sélectionné à l'aide de critères propriétaires. L'ADN génomique humain dans l'échantillon d'essai peut être détecté et/ou analysé à une sensibilité élevée par la réalisation d'une PCR quantitative en temps réel à l'aide de l'amorce-sonde de PCR pour la détection d'Alu ainsi obtenue.
PCT/JP2017/042943 2016-11-30 2017-11-30 Procédé de détection d'adn génomique humain Ceased WO2018101375A1 (fr)

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WO2021136559A1 (fr) * 2019-12-30 2021-07-08 江苏艾尔康生物医药科技有限公司 Procédé de distinction d'adn humain sur la base d'une technologie pcr quantitative fluorescente
WO2024005105A1 (fr) * 2022-06-30 2024-01-04 積水メディカル株式会社 Procédé de mesure de cellules humaines
WO2024048659A1 (fr) * 2022-08-30 2024-03-07 国立大学法人秋田大学 PAIRE D'AMORCES DE PCR POUR LA DÉTECTION DE L'ALu HUMAIN, SONDE DE PCR POUR LA DÉTECTION DE L'ALu HUMAIN, ENSEMBLE D'AMORCES ET DE SONDES DE PCR POUR LA DÉTECTION DE L'ALu HUMAIN, PROCÉDÉ DE DÉTECTION ET/OU DE QUANTIFICATION DE L'ADN GÉNOMIQUE HUMAIN, ET PROCÉDÉ D'AIDE À LA PRÉDICTION DE LA PRÉSENCE OU DE L'ABSENCE D'UN CARCINOME DES CELLULES RÉNALES OU D'UN CANCER DE LA PROSTATE

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* Cited by examiner, † Cited by third party
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WO2024005105A1 (fr) * 2022-06-30 2024-01-04 積水メディカル株式会社 Procédé de mesure de cellules humaines
WO2024048659A1 (fr) * 2022-08-30 2024-03-07 国立大学法人秋田大学 PAIRE D'AMORCES DE PCR POUR LA DÉTECTION DE L'ALu HUMAIN, SONDE DE PCR POUR LA DÉTECTION DE L'ALu HUMAIN, ENSEMBLE D'AMORCES ET DE SONDES DE PCR POUR LA DÉTECTION DE L'ALu HUMAIN, PROCÉDÉ DE DÉTECTION ET/OU DE QUANTIFICATION DE L'ADN GÉNOMIQUE HUMAIN, ET PROCÉDÉ D'AIDE À LA PRÉDICTION DE LA PRÉSENCE OU DE L'ABSENCE D'UN CARCINOME DES CELLULES RÉNALES OU D'UN CANCER DE LA PROSTATE

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