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WO2006009260A1 - Procédé de détection du virus de l'hépatite e - Google Patents

Procédé de détection du virus de l'hépatite e Download PDF

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Publication number
WO2006009260A1
WO2006009260A1 PCT/JP2005/013513 JP2005013513W WO2006009260A1 WO 2006009260 A1 WO2006009260 A1 WO 2006009260A1 JP 2005013513 W JP2005013513 W JP 2005013513W WO 2006009260 A1 WO2006009260 A1 WO 2006009260A1
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Prior art keywords
nucleic acid
base sequence
seq
homology
virus
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PCT/JP2005/013513
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English (en)
Japanese (ja)
Inventor
Tsutomu Kageyama
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Bml, Inc.
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Priority to JP2006524555A priority Critical patent/JPWO2006009260A1/ja
Publication of WO2006009260A1 publication Critical patent/WO2006009260A1/fr

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    • 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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis
    • C12Q1/707—Specific hybridization probes for hepatitis non-A, non-B Hepatitis, excluding hepatitis D

Definitions

  • the present invention relates to a method for detecting a virus, and more particularly to a method for detecting a hepatitis E virus.
  • Hepatitis E virus is a virus that induces hepatitis E and is classified as family Hepeviridae, genus Hepevirus, and has a small (diameter) diameter of about 7.2kb plus-strand RNA genome. 27-30nm) virus.
  • HEV was first cloned by Reyes et al. In 1990 (Reyes, G. R "MA Purdy, JP Kim, KC Luk, LM Young, KE Fry, and DW Bradley. 1990. Isolation of a cDNA from tHEVirus res ponsible for enterically transmitted non— A, non— B hepatitis.
  • HEV HEV is classified into four types (genotypes I to IV) (Schlauder, GG, and I. K. Mushahwar. 2001. uenetic heterogeneity of hepatitis E virus. J Med irol, vol.6 5: 282- 92) Many cases of hepatitis E are caused by drinking water contaminated with viruses, and in developing countries, hepatitis E is often caused by drinking water contaminated with HEV. A. C, and G. b. Fout. 2002. Development of a molecular method to identify hepatitis E virus in water. J Virol Methods, vol. 101: 175-88).
  • This virus is transmitted by oral infection and develops after an incubation period of 15 days and 60 days. There are many cases of subclinical infection, and some people do not develop even if infected. Typical symptoms are acute hepatitis with gastrointestinal symptoms such as abdominal pain, fever, and vomiting, and strong jaundice with brown urine appears. After jaundice lasts for 12 to 15 days, it usually recovers one month after onset. HEV viremia appears prior to jaundice, and the virus is excreted in the stool. Like hepatitis A, hepatitis E is not chronic, but there are cases where viremia continues for a long time with excretion in the stool. The fatality rate is 0.5-4.0% in the case of onset. For pregnant women, the fatality rate with a high rate of fulminant hepatitis is as high as 17-33%.
  • HEV has also been found in sewage in non-indigenous areas, and there is concern about contamination of HEV to the ocean and shellfish (Pina, S., J. Jofre, SU Emerson, RH Purcell, and R. Girones.
  • the present inventor has examined practical means for detecting HEV, and in a specific region of the HEV gene, the mutant is highly conserved even between mutant strains. There are parts where the effects of mutations are very large, and by using the gene region as a HEV detection index, it was found that HEV can be detected efficiently in various aspects. completed.
  • the present invention detects a nucleic acid having a base sequence of 10 bases or more selected from a base sequence corresponding to the base sequence shown in SEQ ID NO: 1 with respect to the detection target, and detects the nucleic acid.
  • the present invention provides a virus detection method (hereinafter also referred to as the present detection method), wherein the detection target is positive for hepatitis E virus (HEV).
  • HEV hepatitis E virus
  • HEV genotypes are currently classified into four types, GI to GIV, and the base shown in SEQ ID NO: 1
  • the sequence is the nucleotide sequence of the 5253 to 5434 nt cDNA of HEV (GI) strain “M73218: Genbank No. J [hereinafter, this strain may be referred to as a reference strain (prototype).” This is a region that forms part of the HEV “region where ORF2 and ORF3 regions overlap”.
  • This detection method detects differences in HEV genotypes by detecting the base sequence of a specific gene region existing in all genotypes of HEV when detecting HEV in a detection target. It is a virus detection method characterized by being capable of both exceeding detection and detecting HEV for each genotype.
  • the numbers of individual bases in the base sequence of the HEV gene are in accordance with the Genbank No. shown in FIG.
  • guanine (G) the 5253rd base of the above-mentioned prototype M73218 strain, corresponds to guanine (G), the first base of SEQ ID NO: 1.
  • This "base sequence of a specific gene region existing in all genotypes of HEV” corresponds to "a base sequence corresponding to SEQ ID NO: 1" according to the present invention. That is, by detecting the presence of the base sequence corresponding to SEQ ID NO: 1 in the detection target and the Z or its contents, qualitative detection, quantitative detection, and further detection of HEV in the detection target are: By grasping the genotype of the causative virus, it is possible to establish a treatment policy for the detection target provider and specify a virus contamination source.
  • nucleic acid constituting the base sequence includes, but is not limited to, oligoribonucleotides and oligodeoxyribonucleotides. Peptide nucleic acids, methylphosphonate nucleic acids, S-oligonucleic acids, morpholyl nucleic acids, etc. Of artificially synthesized nucleic acids.
  • the nucleic acid to be detected must be an oligonucleotide selected from the base sequence corresponding to SEQ ID NO: 1 and continuous for at least 10 bases, and the maximum is about 180 bases. In general, it is preferable that the oligonucleotide is continuous for about 15 to 30 bases.
  • the base difference with respect to the base sequence represented by SEQ ID NO: 1 is 10% Must be within.
  • the difference in bases between all genotypes of the conserved region in the HEV “region where ORF2 region and ORF3 region overlap” is within 10%, and the quasi-conserved region (conserved between different genotypes, However, the difference in bases between HEVs of the same genotype in a gene region that is well conserved in the same genotype is also within 10%! /.
  • an organism individual for example, any mammal including humans, monkeys, pigs, ushi, and sheep, or oysters, clams, clams, shijimi, mussels, etc.
  • Biological samples such as body fluids, blood, serum, lymph, feces, and tissues collected from marine products, as well as environmental water including seawater, river water, lake water, sewage, drainage, tap water, well water, etc.
  • food and food production facilities food itself, deposits such as cooking utensils in food production facilities, deposits such as clothing of food producers, papers and cloths that wipe them, and the like can also be detected.
  • Each detection target can be subjected to appropriate pretreatments such as homogenization, concentration, and extraction as necessary, and the sample obtained in this way can also be used as a test target. is there.
  • the method of obtaining genes from these detection targets it is possible to select an appropriate method according to the type of detection target used.
  • the detection target to be used is generally immersed or suspended in water or the like.
  • viral RNA can be extracted by a conventional method such as the acid phenol method (AGPC method: Acid Guanidin Phenol Chroloform method).
  • AGPC method Acid Guanidin Phenol Chroloform method
  • Examples of gene amplification methods include PCR method, RT-PCR method, real-time PCR method, SDA ⁇ strand displacement amplification (NASDA) method, NASBA (Nucleic Acid Sequence Based Amplification) method, LAMP (Loop—mediated isothermal amplification) Law.
  • Nucleic acid amplification products can be detected by electrophoresis, electrochemical sensors such as nucleic acid detection chips, gene sensors using quartz crystal, or fluorescently labeled primers or probes.
  • nucleic acid amplification products can be detected as HEV-derived genomic fragments by measuring fluorescence intensity with a fluorescence sensor using a fluorescence intercalation agent.
  • HEV genomic fragments can be detected using the above-mentioned oligonucleotides even in methods that do not require amplification of the target gene, such as the invader method.
  • the detection of the target nucleic acid in this detection method is mainly performed by positively or negatively detecting nucleic acid nucleic acids and hybrids, and a specific method thereof is a known detection method.
  • Techniques can be used. For example, dot hybridization, slot hybridization, Southern hybridization, Northern hybridization method, etc., or in combination with various amplification methods, TaqMan probe, molecular beacon probe (
  • the target HEV-derived genomic fragment can be detected by analysis using a real-time quantification method using molecular beacon probes), noisy hybridization probes, LUX (light upon extension) primers, etc.
  • the target HEV genome fragment can also be detected by methods that do not require amplification of the target gene, such as the invader method.
  • nucleic acid according to the present invention can be added with necessary modifications, and these modified nucleic acids are also As long as the base sequence is within the scope of the present invention, it is similarly assumed to be within the scope of the present invention.
  • additional element include enzyme modification (peroxidase, etc.), fluorescent labeling (luciferin, etc.), chemical modification, protein modification (piotine, etc.), radioisotope labeling, and the like.
  • a specific base sequence in the gene amplification product is used. It is preferred to use a means capable of monitoring the presence of the row.
  • Representative examples of such methods include, for example, the detection method using the above-described molecular beacon probe, the detection method using the Taq-Man Probe, and the LUX (Light Upon Extension). Examples include detection methods using primers.
  • the detection method using a molecular beacon probe is a hairpin type that can be used to monitor the formation of gene amplification products by the PCR method or the like during or after the amplification process.
  • This is a method for detecting a gene using a hybridization probe (molecular beacon probe) (Nature Biotechnology 1998 16: 49-53).
  • the ends of the nucleic acid constituting the molecular beacon probe are complementary to each other. Usually, these ends are joined together to form a so-called stem structure, and the loop portion in the striking stem structure is a gene amplification. It is designed to be complementary to the target region of the product.
  • the phosphor and the non-fluorescent quencher are bound to both ends of the nucleic acid and released in solution, a hairpin structure is formed, so that the fluorophore and the quencher interact with each other. And the fluorescence disappears.
  • the loop portion binds to the complementary base sequence, and as a result, the structure of the entire probe changes. Since the phosphor and the quencher are separated from each other and the quenching effect of the quencher on the phosphor is eliminated, the phosphor emits the original fluorescence.
  • the increase in fluorescence intensity due to the elimination of this quenching effect is proportional to the increase in gene amplification products having a base sequence complementary to the nucleic acid.
  • the target nucleotide sequence can be detected not only after the gene amplification process but also during the gene amplification process. That is, the target nucleic acid in the detection sample can be detected using the increase in fluorescence intensity as an index.
  • the above-mentioned fluorescent label and quencher label are usually labeled with 6-carboxyfluorescein (6-FAM) or 6-carboxy-4,7,2 'at the 5th end of the nucleic acid.
  • TET 7'-tetrachlorofluorescein
  • TTAMARA 5-carboxytetrame thylrhodamine
  • DABC YL 4- (4′-dimethylaminophenylazo) benzoic acid
  • the detection method using the Taq-Man Probe is a hybridization probe that can be used to monitor the formation of a gene amplification product by PCR or the like during the amplification process with fluorescence ( This is a method for detecting genes using Taq-Man Probe (Experimental Medicine Vol.15 No.7 (Special Issue) p46-51, 1997, etc.).
  • the Tac Couman probe is a nucleic acid labeled with 5, a fluorescein fluorescent dye (reporter dye) at the end, and a rhodamine fluorescent dye (taentia dye) at the 3 'end.
  • the fluorescence of the reporter dye is suppressed by the quencher dye due to the Forster resonance energy.
  • the primer and the Tatooman probe anneal the nucleic acid complementary to the Taucman probe nucleic acid of the gene amplification product and the extension reaction proceeds, TaqDNA polymerase 5, ⁇ 3, due to the endonuclease activity, Hydrolysis occurs from the 5 'end of the probe, and when the 5' end reporter dye is released from the 3 'end quencher, the fluorescence intensity of the reporter dye increases.
  • the increase in fluorescence intensity by the reporter dye is proportional to the increase in gene amplification products having a base sequence complementary to the nucleic acid.
  • the target base sequence can be detected not only after the gene amplification process but also during the gene amplification process. That is, the target nucleic acid in the detection sample can be detected using the increase in fluorescence intensity as an index.
  • the above-described fluorescent labeling in the Tac Couman probe usually has a fluorescein-based fluorescent dye such as 6-FAM or TET at the 5 'end of the nucleic acid and a rhodamine such as TAM ARA at the 3' end.
  • the dye of the system can be carried out according to a conventional method (for example, Nucleic Acids Research 1993 21 (16): 3761-3766).
  • the detection method using a LUX (Light Upon Extension) primer is a method of detecting the amplification of a target gene by monitoring the fluorescence during the amplification process for the formation of gene by-products by PCR or the like.
  • the LUX primer is a nucleic acid designed to label a single fluorophore near the 3 'end and take a hairpin structure between the 5' end. Length is through Usually it is 20 to 30 bases, and when the primer has a hairpin structure, it has a quenching ability and does not emit fluorescence, but when it is incorporated into a primer-stranded PCR product, the quenching is released and the fluorescence signal increases. By measuring this increase in signal, the amplified gene product of interest is quantitatively analyzed (Nazarenko, I. et al. (2002) Nucleic Acids Research 30: e37.).
  • detection nucleic acid probes or primers can be used as detection nucleic acid probes that can be used as components of the detection kit described later.
  • HEV detection in the present detection method can be performed by quantifying and detecting a target nucleic acid by the above-described means, such as detection of a gene amplification product having a target base sequence. For example, it can be detected as qualitative information of positive or negative without quantification. HEV can be detected by using detection information (quantitative values and qualitative information) such as gene amplification products as an index and correlating it with the presence / absence of HEV in the sample, and further with the abundance.
  • FIG. 1 is a drawing showing the results of examining the diversity of HEV genomes.
  • FIG. 2 is a drawing showing the strain lines in the HEV genome.
  • FIG. 3 shows a parallel comparison of the base sequences in each strain of the HEV gene region used as a detection region in the present invention.
  • the three base sequences in the second row are the base sequences of SEQ ID NOs: 49, 51 and 57 from the left.
  • Fig. 3 shows a parallel comparison of the base sequences in each strain of the HEV gene region used as a detection region in the present invention.
  • the three base sequences in the second row are the base sequences of SEQ ID NOs: 49, 51 and 57 from the left.
  • the base sequence from the 3rd row to the 30th row is also the upper force, SEQ ID NO: 1, 15, 13, 10, 12, 17, 16, 11, 7, 8, 6, 4, 3, 2, 5, 9, 14, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 and 58.
  • the base sequences of the 32nd and 33rd lines are respectively , SEQ ID NOS: 18 and 53, and the base sequences of the 35th to 45th rows are SEQ ID NOS: 21, 27, 26, 25, 24, 28, 23, 22, 19 from the top, respectively. , 56 and 54, and the base lines in the 47th to 57th lines, Ui, Ue, et al., Respectively, Umi 37, 31, 32, 36, 33, 34, 35 , 30, 29, 38 and 55.
  • M80581 (SEQ ID NO: 2) .L25595 (SEQ ID NO: 3), L25547 (SEQ ID NO: 4), M94177 (SEQ ID NO: 5), L08816 (SEQ ID NO: 6), D11092 (SEQ ID NO: 7), D11093 (SEQ ID NO: 8), X98292 (SEQ ID NO: 9), AF185822 (SEQ ID NO: 10), M73218 (SEQ ID NO: 1), D10330 (SEQ ID NO: 1 1), AF459438 (SEQ ID NO: 12), AF076239 (SEQ ID NO: 13), X99441 (SEQ ID NO: 14), A F051830 (SEQ ID NO: 15), AY230202 (SEQ ID NO: 16), AY204877 (SEQ ID NO: 17)
  • GTV Early transmission TV
  • FIG. 3 shows the alignment of each type.
  • the white box region is a region having a sequence common to all strains, and oligonucleotides serving as a common primer and a common probe were designed here.
  • the gray box region is a region having a sequence common to only each genotype, and an oligonucleotide that is a probe specific to each genotype was designed here.
  • a nucleic acid having a base sequence corresponding to SEQ ID NO: 1 has at least 11 base groups out of 14 consecutive bases stored for each genotype, and The region is a region that differs by at least 5 bases from the corresponding base sequence of other genotypes.
  • the designed oligonucleotide sequence is shown below.
  • HEV co-sense primer 5, -CGGCGGTGGTTTCTGGRGTG-3 ,: SEQ ID NO: 49
  • HECOM-AS primer 5——GGGCGCTKGGMYTGRTCNCGCCAAGNGGA—3: SEQ ID NO: 50
  • HEV common probe 5——GGGCGCTKGGMYTGRTCNCGCCAAGNGGA—3: SEQ ID NO: 50
  • TP-HECOM probe (5,-(FAM) -CCCCYATATTCATCCAACCAACCCCTTYG C- (TAMRA) -3 ': SEQ ID NO: 51)
  • TP-HEG1 probe (5, — (FAM) — CGCCCCSRATGTCACCGCT— (TAMRA) — 3 ,: Sequence number 52)
  • TP-HEG2 probe (Corresponds to GLM73218 5329-5352 nt and corresponds to GILM74506 5229-5332 nt)
  • TP-HEG2 probe (5,-(FAM) -CTTTGCCCCAGACGTTGCCGCTGC- (TAMRA) -3 ': SEQ ID NO: 53)
  • TP-HEG3 probe (Corresponds to GLM73218 5343-5365 nt and corresponds to GIILAF082843 5367-5389 nt) TP-HEG3 probe (5, — (FAM) -TCGTTTCACAAYCCGGGGCTGGA- (TAMRA) -3 ': SEQ ID NO: 54)
  • TP-HEG4 probe 5, — (FAM) -TTCGCATCTGACATWCCARCCGC- (TAMRA) -3 ′: SEQ ID NO: 55
  • GI: M 73218 corresponds to 5253-5432 nt
  • GII: M74506 corresponds to 5223-5402 nt
  • GIII: AF08284 3 corresponds to 5277-5456 nt
  • GVI: AB097811 corresponds to 5294-5473 nt (180nt)
  • a plasmid (pT7Blue vector) having ⁇ was prepared and used as a standard for each genotype of HEV.
  • the oligonucleotides of the nucleotide sequences shown in SEQ ID NOs: 49 to 55 are an oligonucleotide within the scope of the present invention. Based on this premise, the usage of SEQ ID NOS: 49 to 55 in this detection method will be described.
  • the HEV common probe SEQ ID NO: 51
  • the oligonucleotide of SEQ ID NO: 51 is used as a probe for the nucleic acid sample derived from the detection target. ⁇ If the hybridizing reaction is positive, it is possible to make the detection target HEV positive (genotype is not yet determined).
  • the oligonucleotide probe having the base sequence of SEQ ID NO: 51 can be labeled appropriately depending on the detection mode.
  • an amplification product of a nucleic acid in a nucleic acid sample derived from the detection target is detected using the molecular weight of the gene fragment directly appearing on electrophoresis as an index without performing a nucleic acid hybridization reaction,
  • an amplification product of the target gene region is detected, it is possible to make the detection target HEV positive (genotype is not determined).
  • a primer used for the amplification reaction of nucleic acid it is possible to use the primer of SEQ ID NO: 49 or 50.
  • the sequence described as the probe of SEQ ID NO: 51 is used as a primer. Is also possible.
  • the primer oligonucleotide sets that can be used in this embodiment are the oligonucleotide set of SEQ ID NOs: 49 and 50, the set of base sequences of SEQ ID NOs: 49 and 51, and the set of base sequences of SEQ ID NOs: 50 and 51.
  • a group is applicable.
  • the oligonucleotide primers having the nucleotide sequences of SEQ ID NOs: 49 to 51 can be appropriately labeled depending on the detection mode.
  • a nucleic acid hybridization reaction is performed on a nucleic acid amplification product in a nucleic acid sample derived from a detection target, and HEV is detected using the hybridization reaction as an index
  • the oligonucleotide of SEQ ID NO: 51 as a probe is subjected to a hybridization reaction with a nucleic acid sample subjected to an amplification reaction derived from the detection target. If the hybridization reaction is positive, HEV is detected in the detection target. It can be positive (genotype is undetermined).
  • the probe that can be used in this embodiment is an oligonucleotide having the nucleotide sequence of SEQ ID NO: 51
  • the set of primer nucleic acids is the set of oligonucleotides having the nucleotide sequences of SEQ ID NO: 49 and 50, SEQ ID NO: 49 and 51 This corresponds to the base sequence set of SEQ ID NOs: 50 and 51.
  • the probe of SEQ ID NO: 51 and the oligonucleotide primer of the base sequence of SEQ ID NOs: 49 to 51 can be appropriately labeled depending on the detection mode.
  • HEV is the probe of SEQ ID NO: 52
  • HEV is the probe of SEQ ID NO: 53
  • HEV is the probe of SEQ ID NO: 54
  • HEV (GIV ) Is the probe of SEQ ID NO: 55.
  • the oligonucleotides of SEQ ID NOs: 52 to 55 are selected according to the genotype (GI to GIV) of the HEV to be detected.
  • a nucleotide individually as a probe a hybridization reaction is performed on a nucleic acid sample derived from the detection target, and if the hybridization reaction force is positive for any of the genotype probes, the detection target is not detected.
  • HEV can be positive in the genotype.
  • the oligonucleotide probes having the nucleotide sequences of SEQ ID NOs: 52 to 55 can be appropriately labeled depending on the detection mode.
  • an amplified product of a nucleic acid in a nucleic acid sample derived from a detection target is converted into a nucleic acid hybrid. If the amplified product of the target gene region is detected using the molecular weight of the gene fragment that appears directly on electrophoresis as an index It is possible to make the target genotype HEV positive.
  • a primer used for the nucleic acid amplification reaction it is preferable to use the oligonucleotides described as probes of SEQ ID NO: 49 or 50 and SEQ ID NOs: 52 to 55 as primers.
  • the primer oligonucleotide pair that can be used in this embodiment is the oligonucleotide pair (GI) having the nucleotide sequence of SEQ ID NO: 49 or 50 and SEQ ID NO: 52, or the oligonucleotide having the nucleotide sequence of SEQ ID NO: 49 or 50 and SEQ ID NO: 53 Group (GII), SEQ ID NO: 49 or 50 and oligonucleotide group (GIII) with the nucleotide sequence of SEQ ID NO: 54, and SEQ ID NO: 49 or 50 and oligonucleotide group (GVI) with the nucleotide sequence of SEQ ID NO: 55 .
  • the oligonucleotide primers having the nucleotide sequences of SEQ ID NOs: 49 to 50 and 52 to 55 can be appropriately labeled depending on the detection mode.
  • nucleic acid hybridization is performed on a nucleic acid amplification product in a nucleic acid sample derived from the detection target, and HEV is detected by genotype using the hybridization reaction as an indicator
  • the detection purpose is used.
  • a hybridization reaction is performed on a nucleic acid sample subjected to an amplification reaction derived from a detection target using an oligonucleotide of SEQ ID NO: 52 to 55 as a probe according to the genotype of HEV, and the hybridization reaction is positive
  • a primer used for the nucleic acid amplification reaction it is possible to use the primer of SEQ ID NO: 49 or 50 as well as the oligonucleotide of SEQ ID NO: 52 to 55 as a primer. is there.
  • the probe that can be used in this embodiment is an oligonucleotide having the nucleotide sequence of SEQ ID NO: 52 when the detection genotype is GI
  • the primer nucleic acid pair is an oligonucleotide having the nucleotide sequences of SEQ ID NOs: 49 and 50
  • a pair of oligonucleotides having the nucleotide sequences of SEQ ID NOs: 49 and 52 and a pair of oligonucleotides having the nucleotide sequences of SEQ ID NOs: 50 and 52.
  • the oligonucleotide having the nucleotide sequence of SEQ ID NO: 53 is the probe, and the primer nucleic acid pair is the oligonucleotide pair of SEQ ID NOS: 49 and 50, the sequence number 49
  • the sequence number 49 A set of oligonucleotides with base sequences of 53 and 53, base sequences of SEQ ID NOs: 50 and 53
  • the set of oligonucleotides is applicable.
  • the oligonucleotide having the nucleotide sequence of SEQ ID NO: 54 is the probe, and the primer nucleic acid pair is the oligonucleotide pair of SEQ ID NOS: 49 and 50, SEQ ID NO: 49 And oligonucleotide pairs having the nucleotide sequences of 54 and 54, and oligonucleotide pairs having the nucleotide sequences of SEQ ID NOS: 50 and 54 are applicable.
  • the oligonucleotide having the nucleotide sequence of SEQ ID NO: 55 is the probe, and the primer nucleic acid pair is the oligonucleotide pair of SEQ ID NOS: 49 and 50, SEQ ID NO: 49
  • SEQ ID NO: 49 This includes a pair of oligonucleotides having a base sequence of 55, and a pair of oligonucleotides having the base sequences of SEQ ID NOS: 50 and 55.
  • the present invention is also an invention that provides a detection kit (this detection kit) for carrying out this detection method.
  • an amplification primer for amplifying the whole or a part of the nucleotide sequence corresponding to the nucleotide sequence of SEQ ID NO: 1 of HEV typically, Oligonucleotides having the nucleotide sequences of SEQ ID NOs: 49 to 50, provided that the oligonucleotides shown as common probes of SEQ ID NO: 51 (when performing detection exceeding genotypes) or specific probes of SEQ ID NOs: 52 to 55 As described above, the oligonucleotide (when detecting for each genotype) can also be used as a primer.] Or 2) Hybridize to the nucleic acid of a specific HEV to be detected. A probe for detecting the nucleic acid (a common probe of SEQ ID NO: 51 for detection exceeding genotype, or a specific sequence of SEQ ID NO: 52 to 55 for detection of each genotype) Professional B) is included depending on the specific detection method.
  • kits may be formed by combining necessary reagents such as a reaction vessel and a reaction buffer in combination with substances, enzymes, and the like.
  • the present detection kit is not limited to the above combinations, and can be provided in combination with various types as necessary.
  • This detection kit which is considered to be the most typical, has the following minimum configuration: It is.
  • HEV gene amplification primer set A nucleic acid having a nucleotide sequence having 90% or more homology with the nucleotide sequence shown in SEQ ID NO: 49, and a nucleic acid having a nucleotide sequence having 90% or more homology with the nucleotide sequence shown in SEQ ID NO: 50.
  • Probe nucleic acid having a base sequence having 90% or more homology with the base sequence shown in SEQ ID NO: 51, and Z or having 90% or more homology with the base sequence shown in SEQ ID NOs: 52 to 55 A probe nucleic acid set having a base sequence.
  • a detection method using a common probe was examined. First, using the HEV common sense primer of SEQ ID NO: 49, the set of HEV common antisense primer of SEQ ID NO: 50, and the HEV common probe of SEQ ID NO: 51, the detection sensitivity for each gene type standard was determined.
  • reaction solution Buffer 25 ⁇ 1, HECOM-S primer 500nM, HE COM-AS primer 500nM, TP-HECOM probe 5-20pmol
  • each standard DNA Prepare 5-5 x 10 7 copies, adjusted with sterile distilled water to a total volume of 50 ⁇ l), and perform PCR reaction at ⁇ 7900 ( ⁇ , USA) (PCR cycle is 50 ° C for 2 minutes ⁇ 95 ° C)
  • the fluorescence intensity of 10 minutes ⁇ 95 ° C. for 15 seconds ⁇ 56 ° C. for 1 minute
  • Standard DNA was prepared separately for each genotype.
  • all genotype standards DNA can be detected from 5 copies / reaction, and by referring to the Ct value (threshold cycle number), HEV standards of all genotypes are quantitative regardless of genotype. It was apparent that it was detectable.
  • the detection method using each genotype-specific probe was examined using each genotype standard.
  • the HEV common sense primer of SEQ ID NO: 49, the set of HEV common antisense primer of SEQ ID NO: 50, and each HEV of SEQ ID NO: 52 to 55 Detection sensitivity for each genotype standard was determined using a gene-type specific probe individually.
  • reaction solution (Buf fer 25 ⁇ 1, HECOM—S primer 500nM ⁇ HECOM— AS primer 500nM ⁇ TP— HEG KTP-HEG2, TP-HEG3 or TP -HEG4)
  • infectious HEV was prepared from the stool of a patient suffering from hepatitis E in India and injected into a Japanese macaque. Fecal stool 10 days after infection, serum before HEV infection and Serum serum from 10 days to 60 days after HEV infection (total of 6 points every 5 days for the first 10-30 days after the first infection and 4 points every 8 days for the remaining 31-60 days) The HEV was detected using this method.
  • the stool specimen was prepared with 10 mM Phos phate buffered saline (PBS) so as to be 10% (w / v).
  • RNA extraction from stool samples and serum samples was performed using the QIA Viral RNA Kit (QIAGEN, USA). Next, reverse transcription reaction was performed for each RNA sample.
  • Reverse transcription reaction was performed with each RNA sample (81), reverse transcription reaction solution 12 1 (1 ⁇ l of 10 mM dNTP solution, 75 pmol random hexamer, 3 Ounits RNAsin (Promega, USA), 200 units Superscript II RNAseH ( ⁇ ) Reverse- transcriptase (Invitrogen, USA), lOOmM DTT 1 ⁇ 1 and 5x reverse transcription buffer (250 mM Tris-Hcl (pH 8.3), 375 mM KC1, 15 mM MgC12) After dilution with sterile distilled water and reaction at 42 ° C for 1 hour or longer, the enzyme was inactivated at 99 ° C for 5 minutes to prepare cDNA (RT Products) for each RNA sample.
  • the reaction mixture (Buffer 25 1, HECOM-S primer 500 nM ⁇ HECOM-AS primer 500 nM ⁇ (TP- HEG1, TP- HEG2, TP- HEG3 or TP- HEG4 ) Probe 5 to 20 pmol, RT Products 2 ⁇ 1 adjusted with sterile distilled water to a total volume of 50 ⁇ 1, and measured the fluorescence intensity during the PCR reaction over time under the conditions described above. All did not react forcefully with the TP-HEG1 probe. That is, all of these HEV genotypes were determined to be GI.
  • a comprehensive or selective HEV high-sensitivity detection system can be established.
  • the HEV detection method of the present invention can be used for blood for blood transfusion, blood products, foods, environmental tests, etc., and the genotype-specific detection system can be used for elucidation of infection routes, epidemiological studies, and the like.

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Abstract

L'invention concerne un procédé de détection d'un virus qui consiste à détecter, chez un sujet soumis à cette détection, un acide nucléique ayant une séquence de bases constituée de 10 ou plus de 10 bases consécutives sélectionnées dans une séquence correspondant à la séquence de bases représentée par la SEQ ID n° : 1 qui chevauche la région ORF2 et la région ORF3 dans un gène du virus de l'hépatite E et à considérer le sujet comme positif au virus de l'hépatite E dans le cas où l'acide nucléique ci-dessus est détecté ; et un kit de détection pour la réalisation du procédé. Selon ce procédé, on peut établir un système exhaustif ou sélectif de détection du VHE avec une sensibilité élevée. Ce procédé de détection est applicable à du sang pour transfusion, à des préparations à base de sang, à des aliments, à des examens environnementaux et ainsi de suite. De plus, un système de détection spécifique à un génotype est utilisable pour mettre en évidence une voie d'infection et effectuer des études épidémiologiques.
PCT/JP2005/013513 2004-07-23 2005-07-22 Procédé de détection du virus de l'hépatite e WO2006009260A1 (fr)

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JP2015517810A (ja) * 2012-04-18 2015-06-25 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ Ｈｅｖアッセイ
US10047406B2 (en) 2013-08-14 2018-08-14 Gen-Probe Incorporated Compositions and methods for detecting HEV nucleic acid
WO2023234486A1 (fr) * 2022-06-03 2023-12-07 주식회사 세니젠 Kit pour pcr en temps réel pouvant détecter le virus de l'hépatite e

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2015517810A (ja) * 2012-04-18 2015-06-25 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ Ｈｅｖアッセイ
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US10689718B2 (en) 2012-04-18 2020-06-23 Roche Molecular Systems, Inc. HEV Assay
US10047406B2 (en) 2013-08-14 2018-08-14 Gen-Probe Incorporated Compositions and methods for detecting HEV nucleic acid
EP4299767A3 (fr) * 2013-08-14 2024-03-27 Gen-Probe Incorporated Compositions et procédés de détection de l'acide nucléique du hev
WO2023234486A1 (fr) * 2022-06-03 2023-12-07 주식회사 세니젠 Kit pour pcr en temps réel pouvant détecter le virus de l'hépatite e

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