Pyrophosphoric acid sequencing method combined with fluorogenic quantitative PCR technology

Abstract

The invention relates to a pyrophosphoric acid sequencing method combined with a fluorogenic quantitative PCR technology. The pyrophosphoric acid sequencing method comprises 1, fluorogenic quantitative PCR, 2, PCR product single-chain template preparation, and 3, sequencing reaction and detection. The pyrophosphoric acid sequencing method utilizes the fluorogenic quantitative PCR technology to replace the common PCR-electrophoresis combined mode for target amplification and amplification effect detection, shortens detection time, reduces a false positive risk and is very suitable for medical fields such as clinical detection. According to the fluorogenic quantitative PCR result, a sequencing template amount can be determined accurately, follow-up gene sequencing is guided, and detection sensitivity and gene sequencing efficiency are improved.

Description

technical field [0001] The invention relates to a pyrosequencing method combined with fluorescent quantitative PCR technology, which belongs to the field of biotechnology. Background technique [0002] Sequencing technology is the "gold standard" for sequence analysis and mutation site detection. Through sequencing, the nucleic acid sequence of the target can be intuitively obtained, which provides convenience for the discovery of genetic disease-related sites and the analysis of drug resistance sites. Nucleic acid sequence is also the basis for typing of pathogenic microorganisms. The subtype distinction of pathogenic microorganisms such as hepatitis B virus (HBV) and hepatitis C virus (HCV) is based on the difference in the whole genome sequence of the virus or the sequence difference in some regions. [0003] In recent years, some sequencing products and solutions have been used in the field of clinical diagnosis, including virus subtype analysis, drug site screening, gen...

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Problems solved by technology

[0005] (2) The sensitivity of pyrosequencing can reach 5%, and if combined with some mutation enrichment techniques, it can reach 1% or even lower, while the sensitivity of traditional Sanger sequencing is only about 15-20%;

[0006] (3) Pyrosequencing can be quantified, that is, the proportion of different genotypes in heterozygotes can be given, while traditional Sanger sequencing cannot do this;

[0007] (4) The results of pyrosequencing are automatically judged by software, while traditional Sanger sequencing requires manual analysis of mutation sites

[0009] (1) Time-consuming: PCR-electrophoresis detection of PCR products generally takes 2-3 hours;

[0010] (2) Easy to pollute: processes such as electrophoresis and PCR product purification (generally gel cutting and recovery) are time-consuming, and a large amount of PCR product aerosols are likely to be generated, causing false positives;

[0011] (3) Easy to missed detection: The sensitivity of electrophoresis detection is low, which is easy to cause missed detection;

[0012] (4) Easy to lose: part of the PCR product will be lost during the purification process, which will affect the detection effect of low-concentration templates;

[0013] (5) Difficult to quantify: The amount of PCR products can only be roughly estimated by electrophoresis. Too many products will consume sequencing primers too quickly and affect the detection effect. At the same time, some clinical items have quantitative requirements, such as hepatitis B Quantitative virus detection

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