Sequencing method for clinical detection

Abstract

The invention discloses a method for sequencing a gene by combing a fluorescence quantitative polymerase chain reaction (PCR). The method comprises the following steps of: performing the fluorescence quantitative PCR, performing enzymolysis, performing a sequencing reaction, purifying a sequencing product, detecting with a sequencing instrument, and the like. The invention also discloses application of the method in the preparation of a gene detection reagent kit. In the method, the fluorescence quantitative PCR mode replaces the conventional qualitative PCR and electrophoresis mode to amplify target nucleic acid and detect amplification effects, so that the detection time is shortened, the problem that PCR aerosol is polluted when a cover is opened in the electrophoresis process is solved, the false positive is reduced, and the method is extremely suitable for medical fields such as clinical detection and the like. Meanwhile, according to the quantitative results of the fluorescent quantitative PCR on target fragments, the initial concentration of the target fragments can be acquired, the concentration of PCR products can be approximately judged, subsequent gene sequencing can be directly guided, and detection sensitivity and gene sequencing efficiency are improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a gene sequencing method combined with fluorescent quantitative PCR. Background technique [0002] Sequencing technology is an epoch-making invention in the field of biotechnology. Based on gene amplification (polymerase chain reaction, PCR), this technology can intuitively obtain the nucleic acid sequence of the target fragment, which provides the possibility for mutation discovery and disease analysis. Sequencing technology is recognized as the "gold standard" for sequence analysis and mutation site detection. Using sequencing technology, not only known mutation sites can be detected, but also unknown mutation sites can be discovered. At the same time, sequencing technology is also the theoretical basis for the typing of pathogenic microorganisms. The principle of subtyping of pathogenic microorganisms such as hepatitis B virus and hepatitis C virus is based on the difference in th...

AI Technical Summary

Problems solved by technology

[0005] (1) The reaction time is long, and qualitative PCR combined with electrophoresis detection generally takes 2 to 3 hours;

[0006] (2) During electrophoresis, the PCR reaction tube needs to be opened, and the gel needs to be cut during the PCR product purification process. These operations take a long time and are likely to generate a large amount of PCR product aerosol, causing false positives;

[0007] (3) The sensitivity of electrophoretic detection is low, resulting in missed detection of low-concentration templates;

[0008] (4) A large amount of PCR products will be lost during gel cutting, which will affect the sequencing effect of low-concentration templates;

[0009] (5) The yield of PCR products can only be roughly estimated by the electrophoresis pattern, and in the sequencing reaction, too much PCR product will consume the sequencing primers too quickly, resulting in abnormal sequencing peaks

However, fluorescent quantitative PCR is to design the detection target fragment and probe in the conserved region of the DNA or RNA of the target object. A single fluorescent quantitative PCR technology can only detect the load of the target nucleic acid but cannot detect the type of the target object and whether it has related mutation

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