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CN-121992099-A - Digital PCR detection system, kit, method and application for detecting single nucleotide polymorphism and copy number variation of CYP2D6 gene

CN121992099ACN 121992099 ACN121992099 ACN 121992099ACN-121992099-A

Abstract

The application discloses a digital PCR detection system, a kit, a method and application for detecting single nucleotide polymorphism and copy number variation of CYP2D6 gene, and belongs to the technical field of molecular biology. The method comprises the steps of designing 3 pairs of CYP2D6 and reference gene RPP30 specific primers, matching 4 locked nucleic acid modified TaqMan fluorescent probes, constructing a single-tube multiplex digital PCR detection system, and synchronously and accurately detecting three single nucleotide polymorphisms of CYP2D6, CYP 6, and CYP2D6, and 5 copy number variation, wherein the RPP30 reference gene can synchronously realize sample amplification quality control and absolute quantitative calibration of target genes. The detection system relies on the advantages of high specificity of the LNA probe and high sensitivity of digital PCR, the detection sensitivity reaches 0.02 ng/. Mu.L, the detection period is short, and the result is stable and reliable.

Inventors

  • WANG HUIJUAN
  • YANG QIANQIAN
  • LI GANG
  • ZOU QIQI
  • LIU CHENYANG
  • LI LI
  • Xiao Qinli

Assignees

  • 陕西佰美基因股份有限公司

Dates

Publication Date
20260508
Application Date
20260320

Claims (10)

  1. 1. The digital PCR detection system for detecting CYP2D6 gene single nucleotide polymorphism and copy number variation is characterized by synchronously detecting three single nucleotide polymorphisms of CYP2D6 x 10, CYP2D6 x 36 and CYP2D6 x 41 and CYP2D6 x 5 copy number variation types, wherein the detection system comprises 3 pairs of specific primers and 4 TaqMan specific fluorescent probes; The 3 pairs of specific primers are a CYP2D6 x 10 specific primer pair, a CYP2D6 x 41 specific primer pair and an internal reference gene RPP30 specific primer pair respectively; The 4 TaqMan specific fluorescent probes are modified by LNA, and specifically comprise CYP2D6 x 1 wild type probes, CYP2D6 x 10 mutant probes, CYP2D6 x 41 mutant probes and RPP30 internal reference probes.
  2. 2. The digital PCR detection system for detecting single nucleotide polymorphisms and copy number variations of the CYP2D6 gene according to claim 1, wherein said specific primer pair for CYP2D6 x 10 comprises a forward primer CYP2D6 x 10-F as shown in SEQ ID No. 1 and a reverse primer CYP2D6 x 10-R as shown in SEQ ID No. 2, wherein the sequence of the CYP2D6 x1 wild type probe is shown in SEQ ID No. 5 and the sequence of the CYP2D6 x 10 mutant probe is shown in SEQ ID No. 6; The specific primer pair of CYP2D6 x 41 comprises a forward primer CYP2D6 x 41-F shown by SEQ ID NO. 3 and a reverse primer CYP2D6 x 41-R shown by SEQ ID NO. 4, wherein the sequence of the mutant probe of the CYP2D6 x 41 is shown by SEQ ID NO. 7; The specific primer pair of the reference gene RPP30 comprises a forward primer RPP30-F shown in SEQ ID NO. 8 and a reverse primer RPP30-R shown in SEQ ID NO. 9, and the sequence of the reference probe of the RPP30 is shown in SEQ ID NO. 10.
  3. 3. The digital PCR assay for detecting single nucleotide polymorphisms and copy number variation of CYP2D6 gene of claim 1 wherein said assay is a 15 μl PCR reaction system comprising, in particular, 3 μl L Probe dPCR HiTapMix reagent a, 0.75 μl L Probe dPCR HiTapMix reagent B, 10 μΜ CYP2D6 x 10 forward primer 0.8 μl, 10 μΜ CYP2D6 x 10 reverse primer 0.8 μl, 10 μΜ CYP2D6 x 1 wild type probe 0.6 μl, 10 μΜ CYP2D6 x 10 mutant probe 0.6 μl, 10 μΜ CYP2D6 x 41 forward primer 0.6 μl, 10 μΜ CYP2D6 x 41 reverse primer 0.6 μl, 10 μΜ CYP2D6 x 41 mutant probe 0.4 μl, 10 μΜ RPP30 forward primer 0.4 μl, 10 μΜ RPP30 reverse primer 0.4 μl, and the remainder of 532 μl sample is 2 μl and the remainder is nucleic acid sample of 15 μl.
  4. 4. The digital PCR detection system for detecting single nucleotide polymorphisms and copy number variations of the CYP2D6 gene of claim 1, wherein the PCR reaction procedure of said detection system is: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 10s and extension at 62℃for 30s, 40 cycles.
  5. 5. A digital PCR detection kit for detecting single nucleotide polymorphisms and copy number variations of the CYP2D6 gene, said kit comprising the digital PCR detection system of any one of claims 1-4.
  6. 6. A method for detecting single nucleotide polymorphisms and copy number variations of the CYP2D6 gene based on the digital PCR detection system according to any one of claims 1 to 4, said method comprising the steps of: extracting genome DNA of a sample to be detected, and measuring the concentration and purity of the DNA for later use; Mixing a nucleic acid sample, a specific primer, a TaqMan specific fluorescent probe and a PCR reaction reagent according to a 15 mu L PCR reaction system proportion to obtain a PCR reaction solution; placing the PCR reaction liquid into a digital PCR instrument, amplifying by a PCR reaction program, and collecting fluorescent signals by FAM, VIC, ROX and CY5 channels; And taking an amplification signal of the VIC channel reference gene RPP30 as quality control, calculating the copy number ratio of the target gene to the reference gene under each fluorescent channel on the premise of qualified quality control, and judging the genotypes of CYP2D6 x 10, CYP2D6 x 36 and CYP2D6 x 41 and the copy number variation types of CYP2D6 x 5 according to the copy number ratio.
  7. 7. The method according to claim 6, wherein the sample to be tested is a blood sample, and the steps of extracting genomic DNA of the sample to be tested, and determining DNA concentration and purity for later use comprise: Extracting genome DNA by a column extraction method or an automatic extraction instrument, dissolving the extracted DNA in eluent, and measuring concentration and purity by using an ultraviolet spectrophotometer.
  8. 8. The method of claim 6, wherein the criteria for the determination are: CYP2D6 5/CYP2D 65. Only the VIC fluorescent channel has a signal, (FAM+ROX)/VIC=0; CYP2D6 5/CYP2D 61 ROX/FAM/VIC channel has fluorescent signal, (FAM+ROX)/VIC=0.5; CYP2D 6. Times.5 is not deleted, FAM, ROX, VIC and CY5 have fluorescent signals, (FAM+ROX)/VIC=1; Wherein, in the case of no deletion of CYP2D6 x 5, the criteria for interpretation of CYP2D6 x 10, CYP2D6 x 41 and CYP2D6 x 36 genotypes are: CYP2D6 x 1/CYP2D6 x1, wherein only FAM and VIC channels have fluorescence signals, and the copy numbers of the two fluorescence channels are the same; CYP2D6 x 10/CYP2D6 x 1: 10 mutation rate = ROX/(fam+rox) =0.5; CYP2D6 x 10/CYP2D6 x 10 mutation rate = ROX/VIC = 1; CYP2D6 x 41/CYP2D6 x 1. 41 mutation rate = CY5/VIC = 0.5; CYP2D6 x 41/CYP2D6 x 41. 41 mutation rate = CY5/VIC = 1; CYP2D6 x 1/x10-/36 mutation: ROX/fam=2, mutation rate of x 10=rox/(fam+rox) =0.66; CYP2D6*10-*36/*10-*36:ROX/VIC=2。
  9. 9. Use of the digital PCR detection system according to any one of claims 1-4 or the digital PCR detection kit according to claim 5 for the preparation of a clinical detection reagent related to CYP2D6 genotyping.
  10. 10. Use of the digital PCR detection system according to any one of claims 1-4 or the digital PCR detection kit according to claim 5 for guiding personalized precision dosing.

Description

Digital PCR detection system, kit, method and application for detecting single nucleotide polymorphism and copy number variation of CYP2D6 gene Technical Field The application relates to the technical field of molecular biology, in particular to a digital PCR detection system, a kit, a method and application for detecting Single Nucleotide Polymorphism (SNP) and Copy Number Variation (CNV) of CYP2D6 genes. Background Cytochrome P450 2D6 (CYP 2D 6) is an important drug metabolizing enzyme, can participate in metabolism of about 25% of common drugs in clinic, and has gene polymorphism (such as single nucleotide polymorphism, copy number variation and the like) which can cause enzyme activity difference so as to cause individual difference of drug curative effect or enhancement of toxic and side effects, so that accurate prediction of enzyme activity through CYP2D6 genotyping has important significance in guiding clinical individual administration and reducing administration risk. However, the CYP2D6, the CYP2D7 and the CYP2D8 form a homologous gene cluster, the nucleotide sequence similarity is more than 97%, and the existence of the homologous pseudogene brings significant technical challenges for the accurate detection of the CYP2D6 gene. Currently, main detection technologies of CYP2D6 single nucleotide polymorphism and copy number variation comprise QPCR, multiple Ligation Probe Amplification (MLPA), sequencing technology, conventional digital PCR and the like, but the technologies have the common defects that firstly, a detection target is single and is difficult to cover various variation types common in clinic, secondly, the quantitative capability is insufficient, such as the QPCR depends on standard curve indirect quantification, absolute and accurate analysis of the copy number variation cannot be realized, thirdly, the detection cost is high, the system is complex, such as the MLPA needs special enzyme and quality control reagent, the sequencing technology needs library construction, high-depth sequencing and complex biological analysis, the conventional digital PCR is not integrated with the high-specificity technology, and is easy to be interfered by homologous sequences, and fourthly, the detection mode of multiple detection tubes further increases the detection cost and the operation complexity, and the requirements of clinical quick and accurate detection are difficult to meet. Therefore, there is a need to develop a detection technology that can detect multiple key variations of CYP2D6 simultaneously, has high specificity, high sensitivity and absolute quantification capability, and is cost-controllable and simple to operate. Disclosure of Invention The application aims to provide a digital PCR detection system, a kit, a method and application for detecting single nucleotide polymorphism and copy number variation of CYP2D6 genes, which have the characteristics and advantages of comprehensive target coverage (synchronous detection of 3 SNP+1 CNV variations), high specificity (elimination of homologous gene interference by LNA probes), high sensitivity (0.02 ng/. Mu.L) and absolute quantification. In order to achieve the above objective, the present application provides a digital PCR detection system for detecting single nucleotide polymorphism and copy number variation of a CYP2D6 gene, wherein the detection system can synchronously detect three SNPs of CYP2D6 x 10, CYP2D6 x 36 and CYP2D6 x 41 and copy number variation of CYP2D6 x 5, and the detection system comprises 3 pairs of specific primers and 4 TaqMan specific fluorescent probes, wherein the 3 pairs of specific primers are respectively a CYP2D6 x 10 specific primer pair, a CYP2D6 x 41 specific primer pair and an internal reference gene RPP30 specific primer pair, and the 4 TaqMan specific fluorescent probes are all modified by LNA, and specifically comprise a CYP2D6 x 1 wild type probe (FAM label), a CYP2D6 x 10 mutant probe (ROX label), a CYP2D6 x 41 mutant probe (CY 5 label) and an RPP30 internal reference probe (VIC label). Further, the specific primer pair of CYP2D6 x 10 comprises a forward primer CYP2D6 x 10-F shown as SEQ ID NO. 1 and a reverse primer CYP2D6 x 10-R shown as SEQ ID NO. 2, the sequence of the CYP2D6 x1 wild-type probe is shown as SEQ ID NO. 5, the sequence of the CYP2D6 x 10 mutant probe is shown as SEQ ID NO. 6, the specific primer pair of CYP2D6 x 41 comprises a forward primer CYP2D6 x 41-F shown as SEQ ID NO. 3 and a reverse primer CYP2D6 x 41-R shown as SEQ ID NO. 4, the sequence of the CYP2D6 x 41 mutant probe is shown as SEQ ID NO. 7, and the specific primer pair of the internal reference gene RPP30 comprises a forward primer RPP30-F shown as SEQ ID NO. 8 and a reverse primer RPP30-R shown as SEQ ID NO.9, and the sequence of the internal reference gene RPP30 is shown as SEQ ID NO. 10. Further, the detection system is a 15 μL PCR reaction system, and specifically comprises 3 μ L Probe dPCR HiTapMix reagent A, 0