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WO2023030162A1 - Procédé de détection de qpcr de type sonde étendue pour snv - Google Patents

Procédé de détection de qpcr de type sonde étendue pour snv Download PDF

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WO2023030162A1
WO2023030162A1 PCT/CN2022/114842 CN2022114842W WO2023030162A1 WO 2023030162 A1 WO2023030162 A1 WO 2023030162A1 CN 2022114842 W CN2022114842 W CN 2022114842W WO 2023030162 A1 WO2023030162 A1 WO 2023030162A1
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primer
probe
primers
epitaxial
snv
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杨永臣
张泓
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Children's Hospital Of Shanghai
Daozhi Precision Medicine Technology Shanghai Co Ltd
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Children's Hospital Of Shanghai
Daozhi Precision Medicine Technology Shanghai Co Ltd
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    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the field of gene detection, in particular to an extension probe type qPCR detection method for single base variation (SNV).
  • SNV single base variation
  • Single nucleotide variation is a single nucleotide variation in the DNA sequence. Single nucleotide variation widely exists in the human genome or other biological genomes, and it plays a regulatory role in biological processes such as RNA transcription and protein expression, and is closely related to human diseases.
  • SNVs include SNPs, which are not distinguished or given different names according to the population frequency of single base variation, whether the variation is from germ cells or somatic cells.
  • SNV detection There are many methods for SNV detection, such as PCR-Sanger sequencing, next-generation sequencing, pyrosequencing, TaqMan probe qPCR method, PCR-RFLP enzyme digestion method, Multiplex SNaPshot method, Sequenom MassArray method, AS-PCR method , chip method and so on.
  • AS-PCR allele specific PCR
  • a typical AS-PCR consists of four primers: an outer upstream primer, an outer downstream primer, an inner upstream specific primer, and an inner downstream specific primer.
  • the internal upstream specific primer and the internal downstream specific primer are respectively combined with the positive strand and the reverse strand of DNA, and their 3' ends are respectively matched with different allele types of the same SNV on the DNA.
  • the specificity of the specific primer is increased (this is not required) by introducing an intentional mismatch at the second or third position at the 3' end.
  • the four primers have three kinds of amplification products: the outer upstream primer and the outer downstream primer are amplified, and the products are used as internal references for amplification and provide more templates for the amplification of specific primers; the outer upstream primer and the inner downstream specific The primers are used to amplify the SNV allele type corresponding to the internal test downstream specific primer; the inner upstream specific primer and the outer downstream primer are used to amplify the SNV allele type corresponding to the internal test upstream specific primer.
  • the amplification products of the outer upstream primer and the inner downstream specific primer, and the amplification products of the inner upstream specific primer and the outer downstream primer have different lengths. After amplification, electrophoresis is performed, and whether the SNV is wild-type, mutant or heterozygous is judged by the presence or absence of amplified bands with two specific primers.
  • the fourth variant has traditionally been used. This is because, no matter the amplification product of the outer upstream primer and the inner downstream specific primer, or the amplification product of the inner upstream specific primer and the outer downstream primer, it is covered by the amplification product of the outer upstream primer and the outer downstream primer, and then The latter is the inevitable product of the typical mode of AS-PCR.
  • the amplification product of the outer upstream primer and the inner downstream specific primer, or the amplification product of the inner upstream specific primer and the outer downstream primer cannot design specific probes to indicate the amplification product in which the specific primer participates.
  • the amplification curve can reflect the 3' end base binding situation of the specific primer without interference from other products. Therefore, this method has been widely used in various application scenarios such as clinical cancer SNV detection and virus mutation site detection.
  • the disadvantage of this variant is that the decrease in binding efficiency due to the introduction of deliberate mismatches on the specific primers cannot be compensated for by the amplification of the template provided by the two outer primers, resulting in most cases Reduced detection sensitivity and specificity.
  • the technical problem to be solved by the present invention is to design a qPCR detection scheme to obtain a more accurate and low-cost method for detecting nucleic acid variation sites.
  • the detection scheme used in the present invention is a modification based on the non-quantitative AS-PCR mode of the classic four primers or three primers, and the detection probe is designed outside the amplification region to indicate the amplification region.
  • a method for the rate of amplification of internal products Specifically, the middle mismatched primer is extended outward from the 5' end, and the accessory probe and accessory primer are designed on the extended fragment, so that the probe is located outside the amplification target region and can reflect the amplification region. effect of internal amplification.
  • extension sequence can be designed almost arbitrarily, not only can it easily avoid the sequence identity with the outer amplification product, but more importantly, when we use the same pattern to detect another mutation site, we only need to replace the upstream primer, downstream The parts on the primers and specific primers that match the target region, while retaining the extended part of the 5' end of the accessory probes, accessory primers, and specific primers, thereby realizing the repeated use of accessory primers and accessory probes, saving expensive new Probe synthesis costs.
  • the unidirectional epitaxial probe qPCR detection system has a good ability to distinguish SNVs using a probe-based qPCR reagent that does not have 3’-5’ exonuclease activity.
  • the ability of the unidirectional epitaxial probe qPCR detection system to detect SNV has been further confirmed through amplification curve, sensitivity test, specificity analysis and other tests.
  • the unidirectional epitaxial probe qPCR detection system of the present invention has better performance in detecting SNVs, especially in detection scenarios with low proportion of mutations, such as the detection of gene mutation sites in cancer tissues, the detection of virus drug resistance sites, etc. application value.
  • AS-PCR technology with probe is a more accurate and efficient SNV detection technology.
  • the detection sensitivity and specificity of the AS-PCR technique with probes are slightly lower.
  • the invention firstly discloses a design mode of primers and probes. Based on the classic PCR principle, this mode includes a set of extension components and a downstream primer; wherein the extension components include extension primers, accessory primers, and accessory probes.
  • the feature of the extension primer is: except for the region where the 3' section of the primer binds to the template, an artificial sequence is extended outward at the 5' end of the primer, and the artificial sequence is divided into a 5' section and a 3' section according to the relative position.
  • the aforementioned so-called accessory primer is a primer that can be reversely complementary to the reverse complementary sequence of the 5' segment of the artificial sequence; the aforementioned so-called accessory probe is a band that can be reversely complementary to the reverse complementary sequence of the 3' segment of the artificial sequence Probes with luminescent and extractive groups. There is no overlap or partial overlap between the accessory primer and the accessory probe on the region corresponding to the extension primer.
  • the aforementioned so-called downstream primers refer to primers that form a pair of primers with the extension primers and can perform PCR amplification on the template in a normal PCR system.
  • the downstream primer binds and extends the single strand following the extension product of the upstream primer, which can serve as the binding object of the accessory primer and accessory probe, and initiate qPCR amplification.
  • This is a probe design mode where the probe is designed on the primer, especially when the probe is not suitable for designing in the product region between the two primers, it gives another option for probe design.
  • the epitaxial probe qPCR method we call this method the epitaxial probe qPCR method, and the schematic diagram is shown in Figure 1: epitaxial probe qPCR.
  • epitaxial probe qPCR Furthermore, combining the epitaxial probe qPCR method with the principle of AS-PCR, we designed an epitaxial probe qPCR detection system for the purpose of SNV detection.
  • the system modifies the 3' end of the extension primer.
  • Genotype matching can replace the second or third base at the 3' end to improve binding specificity.
  • the Taq enzyme used does not have 3' to 5' exonuclease activity
  • the primer is extended and qPCR can be started, and the primer cannot be used when it does not match.
  • Extension qPCR cannot be started or is extremely difficult to start. According to the amplification curve triggered by the fluorescent signal, the allelic type of SNV and the proportion of this type can be analyzed.
  • This epitaxial component modified for the purpose of SNV detection is called a specific epitaxial component, and the epitaxial primer contained in it is called a specific epitaxial primer.
  • This method a specific epitaxial probe-based qPCR method, and its schematic diagram is shown in Figure 2.
  • an outer upstream primer is designed upstream of the specific extension primer, and on the amplification product of the outer upstream primer and the downstream primer, an outer upstream primer and the extension primer are set in the middle position.
  • An internal reference probe bound to the template with a fluorescence different from that of the attached probe is used to indicate the amplification efficiency of the outer upstream primer and the downstream primer (expressed as a Ct value), by comparing the Ct value with that of the attached probe on the extension primer The Ct value can be used to calculate the relative proportion of the specific extension primer corresponding to the genotype.
  • the second mode is: for two allelic types of a certain SNV, two corresponding specific extension components are designed on the forward and reverse strands respectively, and outer amplification primers are designed on the outer side respectively.
  • the accessory probes of the two specific epitaxy components have different fluorescence. According to the amplification curves of the two fluorescences, the relative proportions of the two genotypes of the SNV can be judged.
  • this scheme a two-way epitaxial probe qPCR detection system, and its schematic diagram is shown in Figure 4.
  • the third mode is: for the two allelic types of a certain SNV, two specific epitaxial components are designed in the same direction on the forward or reverse strands, and they combine with each other at the same position in a competitive relationship. Design amplification primers. The accessory probe sequences of the two specific extension components are different, so that each corresponds to an extension primer and has different fluorescence. According to the amplification curves of the two fluorescences, the relative proportions of the two genotypes of the SNV can be judged.
  • This scheme a qPCR detection system of unidirectional paired epitaxial probes, and its schematic diagram is shown in Figure 5.
  • PCR reagents such as Taq enzymes that do not have 3'-5' exonuclease activity, can be used.
  • the detection scheme designed for the mutation site N501Y on the new coronavirus can determine the COVID-19 in the template.
  • COVID-19 commonly known as COVID-19, named by the World Health Organization as 2019-nCoV
  • 2019-nCoV the detection scheme designed for the mutation site N501Y on the new coronavirus
  • the presence or absence of -19, the presence and proportion of N501Y, and the proportion of the N501Y mutant plasmid in the wild-type plasmid can be judged in the simulation experiment, and the accuracy can reach less than 1%.
  • Primers and probes used include: N501Y-F1: 5'-ACTGAAATCTATCAGGCCGGT-3' (SEQ ID NO 1); N501Y-R1: 5'-ACAAACAGTTGCTGGTGCAT-3' (SEQ ID NO 2); N501Y-F2: 5' -ATCTCCGTTCCTAAGGTTGGACAAAGCTTCCGATTAGGGATTCTCCATGTCAATCATATGGTTTCCAACCCACCT-3' (SEQ ID NO 3); N501Y-F3:5'-ATCTCCGTTCCTAAGGTTGGA-3' (SEQ ID NO 4); N501Y-P1:5'-FAM-AGCTTCCGATTAGG3GATTCTCCATG-BHQ ); N501Y-P2: 5'-VIC-CTTGTAATGGTGTTGAAGGTT-MGB-3' (SEQ ID NO 6).
  • N501Y-F1 and N501Y-R1 are outer primers
  • F2 is an extension-specific primer
  • F3 and P1 are accessory primers and accessory probes, respectively
  • P2 is an internal reference probe.
  • Test according to the PCR program recommended by the probe qPCR kit and the recommended qPCR instrument. By comparing the fluorescence curve of FAM with that of VIC, the ratio of N501Y mutant plasmid to wild-type plasmid can be accurately judged, and the accuracy can reach less than 1%.
  • the experimental method and results are shown in the specific implementation method.
  • the results show that the AceQ Universal U+Probe Master Mix V2 reagent has a better ability to distinguish wild-type and mutant plasmids than the Premix Ex Taq TM reagent.
  • KRAS gene Exon 2 p.G12S (34G>A), p.G12D (35G>A), p.G12C (34G>T), p.
  • G12V 35G>T
  • p.G12A 35G>C 522
  • p.G13D 38G>A
  • KRAS gene Exon 3 p.Q61H (183A>C)
  • KRAS gene Exon 4 p.K117N (351A >C)
  • p.Al46T 436G>A
  • p.A146V 437C>T
  • NRAS gene Exon 2 p.G12D(35G >A)
  • NRAS gene Exon 3 p.Q61R (182A>G), p.Q61K (181C>A 580
  • PIK3CA gene Exon20 p.H1047R ( CAT>CGT), p.H1047L (CAT>CTT), p.E542K (GAA>
  • the designed detection scheme can determine whether the BRAF gene Exon 15 :p.V600E (1799T>A) with or without and the ratio, the results of simulation experiments show that it has better detection accuracy.
  • the above method is based on the general knowledge of PCR. If there is no special point, you can refer to the conventional method in this field or the materials and methods used in the "Molecular Cloning Experiment Guide” (Cold Spring Harbor).
  • the above-mentioned combination of primers and templates is based on the annealing rules of PCR, the 5' and 3' ends are cut according to the target of the probe, the composition of the PCR system, the base replacement rules in the primers, and the labeling position of the luminescent group in the probe , the type of luminescent group, etc., are common knowledge of PCR.
  • the two accessory probes shared by the two sites are: General-Fam: 5'-FAM-agcttccgattagggattctccatg-MGB-3' (SEQ ID NO 15); General-Vic: 5'-VIC-gcacaccgttgactgcttatgac-MGB-3 '(SEQ ID NO 16); the accessory primer shared by the two sites is: General-F: 5'-ATCTCCGTTCCTAAGGTTGGA-3'(SEQ ID NO 17). Test according to the PCR program recommended by the probe qPCR kit and the recommended qPCR instrument. By comparing the fluorescence curve of FAM with that of VIC, the determined SNP genotype was consistent with the results of first-generation sequencing.
  • the primer and probe combination of the SNV extension probe type qPCR detection method includes a set of extension components and a downstream primer;
  • extension components include extension primers, accessory primers, and accessory probes
  • the extension primer extends an artificial sequence outward at the 5' end, and the artificial sequence is divided into a 5' segment and a 3' segment according to the relative position;
  • accessory primer is a primer that can be reverse complementary to the reverse complementary sequence of the 5' segment of the artificial sequence
  • the so-called accessory probe is a probe with a luminescent group and an extraction group that can be reverse complementary to the reverse complementary sequence of the 3' segment of the artificial sequence;
  • downstream primers refer to primers that form a pair of upstream and downstream primers with the extension primers, and can perform PCR amplification on the template in a normal PCR system.
  • the epitaxial probe type qPCR detection method of SNV described in the present invention includes but not limited to:
  • the primer and probe sequences designed based on the epitaxial probe qPCR design mode and derivation mode disclosed in the present invention are different from those disclosed in this patent, and are still covered by the claims.
  • the species source of the gene variation detection applied by the method of the present invention includes but not limited to human beings, various animals, various plants, microorganisms, viruses and artificially designed or modified genes, such as originating from humans, and the sample types include but not limited to : Blood, urine, throat swab, feces, alveolar lavage fluid, pus, drainage fluid, tissue blocks, etc.
  • the beneficial effect that the present invention obtains comprises:
  • the epitaxial probe type qPCR detection disclosed in the present invention, and the unidirectional epitaxial probe qPCR detection system developed accordingly, have higher sensitivity and specificity than the traditional probe type AS-PCR;
  • the probe qPCR detection system has higher sensitivity and specificity than the TaqMan probe qPCR method in SNV detection, and the two probes can be reused between different detection sites, which greatly reduces the cost.
  • the new coronavirus N501Y site detection kit developed based on the unidirectional epitaxial probe qPCR detection system can detect less than 1% of the mutant plasmids in the wild-type plasmid.
  • Figure 1 Schematic diagram of epitaxial probe qPCR.
  • FIG. 2 Schematic diagram of specific epitaxial probe-type qPCR method.
  • Fig. 3 Schematic diagram of the qPCR detection system of the unidirectional epitaxial probe.
  • Fig. 4 Schematic diagram of the qPCR detection system of the bidirectional epitaxy probe.
  • Fig. 5 Schematic diagram of qPCR detection system of unidirectional paired epitaxial probes.
  • Figure 6 N501Y, the sequencing map of the mutant plasmid and the wild-type plasmid.
  • Figure 9 N501Y the standard curve of the amplification curves of different concentrations of mutant plasmids.
  • Figure 11 N501Y the standard curve of the amplification curves of wild-type plasmids at different concentrations. Wherein, the closer the data points are to the fitting curve, the more reliable the method of the present invention is.
  • Figure 12 N501Y, amplification curves of mutant plasmids with different ratios under 10 to the 8th power copy plasmid.
  • Use 10 to the 8th power positive plasmid for dilution, and the amplification curves from top to bottom are 100%, 10%, 4&, 2%, 1%, 0.5%, 0.25%, and 0 dilution ratios. It can be seen from the figure that at a concentration of 10 to the 8th power, a mutation ratio of 0.25% can be distinguished.
  • Figure 13 N501Y the standard curve of the amplification curves of mutant plasmids with different ratios under 10 to the 8th power copy plasmid.
  • Figure 14 N501Y, under 10 to the 6th power copy plasmid, the amplification curves of mutant plasmids with different ratios.
  • Use 10 to the 6th power positive plasmid for dilution, and the amplification curves from top to bottom are 100%, 10%, 4&, 2%, 1%, 0.5%, 0.25%, and 0 dilution ratios. It can be seen from the figure that at a concentration of 10 to the 6th power, a mutation ratio of 2% can be distinguished.
  • the wild-type sequence inserted in the plasmid is:
  • the mutant sequence inserted in the plasmid is:
  • the sequence diagram is shown in Figure 6, the upper sequence in the figure is the mutant type, and the lower sequence is the wild type.
  • Primers and probes used include:
  • N501Y-R1 5'-ACAAACAGTTGCTGGTGCAT-3';
  • N501Y-F3 5'-ATCTCCGTTCCTAAGGTTGGA-3';
  • N501Y-P1 5'-FAM-AGCTTCCGATTAGGGATTCTCCATG-BHQ1-3';
  • N501Y-P2 5'-VIC-CTTGTAATGGTGTTGAAGGTT-MGB-3'.
  • each group is divided into three PCR reactions, and each qPCR system (25 ⁇ L) contains: 2 ⁇ Premix 12.5uL, F2 primer 0.5uL, F3 primer 2uL, R1 primer 2uL, P1 probe 1uL, P2 probe 1uL, In addition to 1uL of plasmid DNA, 2uL, 1uL, and 0uL of F1 were added to the three PCR reactions, respectively, and made up to 25uL with water. According to the different amplification reagents, templates, and the amount of F1 added, each tube is named Vm2, Vm1, Vm0, Vw2, Vw1, Vw0, Tm2, Tm1, Tm0, Tw2, Tw1, Tw0.
  • mutant plasmids Add the mutant plasmids to 1 ⁇ 10 7copies/ ⁇ L and 1 ⁇ 105 copies/ ⁇ L wild-type plasmids, and configure the proportions of mutant plasmids to be 100%, 50%, 10%, and 5% respectively. , 1%, 0.5%, 0.1%, 0% of mixed plasmids.
  • AceQ Universal U+Probe Master Mix V2 Vazyme, Cat#Q513-02
  • the N501Y mutant (m) plasmid with different content was used as the detection object for qPCR detection. Each experiment was repeated for three tubes. The operating apparatus and procedures are the same as the above experiments.
  • AceQ Universal U+Probe Master Mix V2 reagent has a good ability to distinguish between wild-type and mutant plasmids. From the amplification curve triggered by the internal reference probe, adding 1uL or 2uL F1 probe, no matter for the wild-type plasmid or the mutant plasmid, there are similar amplification curves. If F1 is not added, none of the internal reference amplification curves exceeds the fluorescence detection threshold.
  • the mutant plasmid was amplified, and the amplification curve was as shown in FIG. 8 .
  • the amplification curve obtained after 10-fold dilution of the mutant plasmid was used as a standard curve.
  • the slope of the internal reference standard curve was -4.3594, the linear correlation coefficient was 0.9968 for R2, and the longitudinal intercept was 53.959.
  • the slope of the epitaxial component standard curve is -4.042, the linear correlation coefficient is R2 is 0.9964, and the longitudinal intercept is 53.745 (Fig. 9).
  • the wild-type plasmid was amplified, and its amplification curve is shown in FIG. 10 .
  • the amplification signal of the internal reference could be detected at 1 ⁇ 10 4 copies. It can be seen from the figure that when the number of wild-type plasmids added to the amplification system is 1 ⁇ 108 copies, the Ct value of the amplification curve of the specific component is greater than 37, and the curve is not smooth.
  • the amplification curve obtained after 10-fold dilution of the wild-type plasmid was used as a standard curve.
  • the mutation rate can be detected in the wild-type plasmid background of 1 ⁇ 108 copies of less than 0.25%.
  • the logarithm of the number of mutant plasmids contained in the wild-type plasmid was used to make a standard curve.
  • the mutation rate can be detected in the wild-type plasmid background of 1 ⁇ 106 copies of less than 2%.
  • the problem with this method is that, on the one hand, due to the mismatching of the primers, the amplification efficiency is reduced, and even the amplification fails;
  • the template still has a weak amplification ability, and the mismatched primers lose the ability to specifically recognize the amplified product, resulting in the rapid accumulation of non-specific amplified products and the rapid rise of the amplification curve, resulting in a decrease in the specificity of detection.
  • the detection scheme we use is a modification based on the non-quantitative AS-PCR mode of the classic four primers or three primers, and the detection probe is designed outside the amplified region to indicate within the amplified region
  • the method of product growth rate Specifically, the middle mismatched primer is extended outward from the 5' end, and the accessory probe and accessory primer are designed on the extended fragment, so that the probe is located outside the amplification target region and can reflect the amplification region. effect of internal amplification.
  • extension sequence can be designed almost arbitrarily, not only can it easily avoid the sequence identity with the outer amplification product, but more importantly, when we use the same pattern to detect another mutation site, we only need to replace the upstream primer, downstream The part of the primer and specific primer that matches the target region, while retaining the extended part of the 5' end of the accessory probe, accessory primer and specific primer, thereby realizing the repeated use of accessory primers and accessory probes, saving expensive new Probe synthesis costs.
  • this unidirectional extension probe qPCR detection system containing F1 primers is better than the previous scheme that does not contain F1 primers but directly uses specific primers and downstream primers for amplification. , with better specificity.
  • the unidirectional epiprobe qPCR assay provides good discrimination of point mutations at relatively high concentrations of F1 primers using probe-based qPCR reagents without 3'-5' exonuclease activity ability.
  • the ability of the unidirectional epitaxial probe qPCR detection system to detect SNV has been further confirmed through experiments such as concentration amplification curve, mutation sensitivity test, and mutation specificity analysis. Therefore, we believe that the unidirectional epitaxial probe qPCR detection system has great potential in the detection of SNV, especially in detection scenarios with a low mutation ratio, such as the detection of gene mutation sites in cancer tissues and the detection of viral drug resistance sites. better application value.
  • Embodiment 2 Detection of Multiple SNPs
  • the specific primers and outer primers used for c.-3279T>G site detection on UGT1A1 are:
  • UGT1A1 The specific primers and outer primers used for c.1091C>T site detection are:
  • the two accessory probes shared by the two sites are:
  • the accessory primers shared by both loci are:
  • a 20uL amplification system including: AceQ Universal U+Probe Master Mix V2 (Vazyme, Cat#Q513-02) 10 microliters, Ug3279-Fn 0.5uL, Ug3279-Rn 0.08uL, Ug3279-Fo 2uL, Ug3279-Ro 2uL, General-Fam 1uL, General-Vic 1uL, General-F 2uL, Human Genomic DNA 1uL, add water to make up 20uL.
  • qPCR is run on the ABI 7500Fast Dx qPCR instrument, and the set fluorescent signal type is consistent with the fluorescent signal type on the probe.
  • the operating program is: denaturation at 95°C for 3 minutes, and then 40 cycles include: 95°C for 15 seconds, 60°C for 45 seconds And collect fluorescence at this stage.
  • a 20uL amplification system including: AceQ Universal U+Probe Master Mix V2 (Vazyme, Cat#Q513-02) 10 microliters, Ug3279-Fn 0.5 uL, Ug3279-Rn 0.5uL, Ug3279-Fo 2uL, Ug3279-Ro 2uL, General-Fam 1uL, General-Vic 1uL, General-F 2uL, Human Genomic DNA 1uL.
  • the qPCR is run on the ABI 7500 Fast Dx qPCR instrument.
  • the set fluorescent signal type is consistent with the fluorescent signal type on the probe.
  • the operating program is: denaturation at 95°C for 3 minutes, and then 40 cycles including: 95°C for 15 seconds, 60°C for 45 sec and collect fluorescence at this stage.
  • the c.-3279T>G epitaxial qPCR amplification curve on UGT1A1 the three selected samples are TT, TG, GG types from left to right.
  • the qPCR results were consistent with the sequencing results (Figure 18).
  • the 15 samples were divided into three groups according to the c.-3279T>G genotype, and the amplification curves of each group were superimposed on each other.
  • the results are shown in FIG. 19 .
  • c.1091C>T qPCR amplification curve on UGT1A1 the three selected samples are TT, CT, CC types from left to right.
  • the qPCR results were consistent with the sequencing results (Figure 20).
  • the 7 samples were divided into three groups according to the c.1091C>T genotype, and the amplification curves of each group were superimposed on each other, and the results are shown in Figure 21.
  • the bidirectional epitaxial probe qPCR detection system Based on the bidirectional epitaxial probe qPCR detection system, the c.-3279T>G and c.1091C>T detection methods on UGT1A1 were developed, and the detection results were consistent with the sequencing results, proving that the bidirectional epitaxial probe qPCR detection system is effective in SNP detection of.
  • the detection of two sites uses different amplification products and the same fluorescent probe, which proves that the bidirectional epitaxial probe qPCR detection system can realize the reusability of fluorescent probes when detecting different SNP sites.
  • the amplification curves of different samples are very similar, indicating that using different concentrations of DNA (when performing quantitative PCR, the consistency adjustment of sample DNA concentration was not performed), similar amplification curves can be obtained when the genotype is the same, and are not affected by other factors. The effect of is small, so this method is suitable for detecting SNPs.
  • the Tm values of the two probes may be different.
  • the amplification curve will deviate from the preset result: when a certain allele is completely missing, there is still an overly strong amplification curve signal.
  • adjust the concentration of the primers of the two alleles to obtain the desired effect.
  • the bidirectional epitaxial probe qPCR detection system is a cheaper and more accurate detection method than the TaqMan probe qPCR method.
  • ATCTCCGTTCCTAAGGTTGGAcaagcacaccgttgactgcttatgactACCCACTCCATCGAGATTCCT (SEQ ID NO 22);
  • Upstream outer primer 5'-92AATGCTTGCTCTGATAGGAAAATGA-3' (SEQ ID NO 23);
  • Downstream outer primer 5'-332AGTAACTCAGCAGCATCTCAGGG-3' (SEQ ID NO 24);
  • Upstream medial probe General-Vic: 5'-VIC-gcacaccgttgactgcttatgac-MGB-3';
  • Downstream medial probe General-Fam: 5'-FAM-agcttccgattagggattctccatg-MGB-3';
  • the sequence of the accessory primer is: General-F: 5'-ATCTCCGTTCCTAAGGTTGGA-3'.
  • Example 2 Using the qPCR reagents and methods described in Example 1, the amplification curve test and mutation sensitivity test were carried out, and the BRAF gene V600E standard product (1799T>A) (article number-specification: CW2767S-1 ⁇ g Hangzhou Nuoyang Biotechnology Co., Ltd.) was used as the template.
  • test results show that the detection effect of the reagent on the BRAF gene Exon 15:p.V600E (1799T>A) site is consistent with the detection effect on the new crown mutation site in Example 1.

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Abstract

La présente invention concerne un procédé de détection qPCR de type sonde étendue pour un variant nucléotidique simple (SNV). Quatre amorces sont conçues selon des sites connus de variants nucléotidiques simples, deux des amorces étant des amorces externes et les deux autres étant des amorces spécifiques, les bases aux extrémités 3' des amorces spécifiques étant respectivement complémentaires des bases d'une matrice de mutation et d'une matrice normale ; des mésappariements peuvent être introduits ou non dans les bases proches des extrémités 3' ; et un segment de séquence artificielle est étendu à l'extrémité 5' de chaque amorce spécifique, une amorce accessoire dans la même direction étant conçue sur le segment 5' de la séquence artificielle et une sonde accessoire étant conçue sur son segment 3'. Dans l'amplification des amorces spécifiques et des amorces aval, les sondes accessoires sur les amorces spécifiques sont dégradées et émettent de la lumière dans l'extension des amorces accessoires de façon à indiquer l'efficacité d'amplification des amorces spécifiques. Si un seul génotype des sites de variant nucléotidique simple est détecté, on peut utiliser deux amorces externes, une amorce spécifique, ainsi que l'amorce accessoire et la sonde de l'amorce spécifique. Le procédé possède également de nombreux modes de mise en œuvre, et présente une sensibilité plus élevée et un coût plus faible qu'une AS-PCR de type sonde.
PCT/CN2022/114842 2021-09-01 2022-08-25 Procédé de détection de qpcr de type sonde étendue pour snv Ceased WO2023030162A1 (fr)

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CN113846147A (zh) * 2021-09-01 2021-12-28 上海市儿童医院 一种SNV的外延探针式qPCR检测方法
CN114410848B (zh) * 2022-03-30 2022-07-05 深圳联合医学科技有限公司 检测SARS-CoV-2的组合物、试剂盒、方法及其用途
CN116121453B (zh) * 2022-11-11 2024-08-20 重庆浦济生命科技有限公司 一种高灵敏荧光pcr检测引物和探针改进结构及其应用
CN120138137B (zh) * 2025-04-09 2025-09-02 四川大家医学检测有限公司 一种基于数字pcr的人braf基因v600e突变定量检测的引物探针组、试剂盒及装置

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