Epitaxial probe type qPCR detection method for SNV
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A detection method and probe-based technology, which can be used in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. It can solve the problems of decreased binding efficiency, decreased detection sensitivity and specificity, and achieve high sensitivity and specificity, cost-reducing effect
Pending Publication Date: 2021-12-28
SHANGHAI CHILDRENS HOSPITAL
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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
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Embodiment 1
[0057] Example 1 Detection of New Coronavirus N501Y
[0058] We intend to develop a new coronavirus N501Y site detection kit based on the unidirectional epitaxial probe qPCR detection system to prove the effectiveness of the unidirectional epitaxial probe qPCR detection system.
[0059] 1 Materials and methods
[0060] 1.1 Preparation of N501Y mutant and wild-type plasmids
[0061] According to the gene sequence of SARS-CoV-2 VOC 202012 / 01" or "B.1.1.7." entered by GenBank, select the 1398th to 1588th sites in the spike protein gene sequence (its position in the full-length sequence 22935 to 23125), the wild-type sequence and the N501Y mutant sequence were synthesized by General Biosystems (Anhui) Co., Ltd. by splicing primers, and inserted into the EcoRV blunt-end restriction site of the puc57 plasmid. The plasmid was transformed and extracted Finally, the wild-type plasmid and the mutant plasmid were diluted with double distilled water to 1×10 to the 10th power copies / uL, ...
Embodiment 2
[0099] The detection of embodiment two multiple SNPs
[0100] We plan to develop c.-3279T>G and c.1091C>T detection methods on UGT1A1 based on the bidirectional epitaxial probe qPCR detection system to prove the effectiveness of the bidirectional epitaxial probe qPCR detection system in SNP detection and the fluorescent probe Multiplexability in detecting different SNPs.
[0101] 1 Materials and methods
[0102] 1.1 Subjects under inspection
[0103] Peripheral blood samples from 22 neonates, aged 0-1 years, all came from the Neonatology Department of Shanghai Children's Hospital. The sample acquisition has obtained the informed consent of the legal representative or family members.
[0104] 1.2 Sanger sequencing
[0105] By amplifying the UGT1A1 exon, c.-3279T>G attachment region, and then performing Sanger sequencing, the variation of the subject's UGT1A1 gene was detected.
[0106] 1.3 Design of primers and probes for two-way epitaxial probe qPCR detection system for d...
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Abstract
The invention discloses an epitaxial probe type qPCR detection method for single nucleotide variation (SNV). Four primers are designed according to known single nucleotide variation sites, two of the four primers are outer side primers, the other two primers are specific primers, bases at the 3' ends of the specific primers are respectively complementary with bases of a mutation template and a normal template, mismatch can be introduced or not introduced into bases near the 3' ends, epitaxy of a segment of artificial sequence is carried out from the 5' end of each specific primer, a homodromous affiliated primer is designed on the 5' segment of each artificial sequence, an affiliated probe is designed on the 3' segment of each artificial sequence, and in the amplification of the specific primers and downstream primers, the affiliated probes on the specific primers are degraded and emits light in the extension of the affiliated primers so as to indicate the amplification efficiency of the specific primers. If only one genotype of the single nucleotide variation sites is detected, the two outer side primers, one specific primer, the affiliated primer of the specific primer and the probes can be used. The method still has a plurality of implementation modes, and compared with probe type AS-PCR, the method has higher sensitivity and lower cost.
Description
technical field [0001] The invention relates to the field of gene detection, in particular to an extension probe type qPCR detection method for single base variation (SNV). Background technique [0002] Single nucleotide variation (Single nucleotide variants, SNV) 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. In this paper, 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. [0003] 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...
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