Isothermal nucleic acid amplification reaction reagent, isothermal nucleic acid amplification method and application thereof

Abstract

The invention discloses an isothermal nucleic acid amplification reaction reagent, an isothermal nucleic acid amplification method and application thereof; the isothermal nucleic acid amplification reaction reagent provided by the invention selects surfactin, TX-100, Tween 20 and other mild surfactants to cooperate with DMSO, sucrose, trehalose, hot start Bst polymerase and high-temperature-resistant hot start reverse transcriptase; the reaction specificity is high; pathogens can be split; genome nucleic acid of the pathogens can be released; a highly inhibition-resistant buffer system is provided; direct amplification of blood samples, respiratory tract samples and the like can be realized; and extraction-free amplification is realized.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an isothermal nucleic acid amplification reaction reagent, an isothermal nucleic acid amplification method and applications thereof. Background technique [0002] In the field of molecular detection, the methods used for target detection include PCR-direct sequencing method, PCR-pyrosequencing method, fluorescent quantitative PCR method, PCR-gene chip method, PCR-electrophoresis analysis, in situ hybridization (ISH), isothermal amplification, etc. Increase and so on a variety of methods. [0003] The operation process of PCR-Sanger sequencing method mainly includes four main steps of PCR amplification and PCR product purification, sequencing reaction, sequencing and result analysis; the main disadvantages of PCR-Sanger are: low sensitivity, special requirements for reagents and instruments, It is not easy to popularize; the operation is complicated, the cost is relativel...

AI Technical Summary

Problems solved by technology

[0003] The operation process of PCR-Sanger sequencing method mainly includes four main steps of PCR amplification and PCR product purification, sequencing reaction, sequencing and result analysis; the main disadvantages of PCR-Sanger are: low sensitivity, special requirements for reagents and instruments, Not easy to popularize; complex operation, relatively high cost, slow speed, and low throughput; PCR-pyrosequencing method has high detection sensitivity, and the analysis of short fragments is mainly carried out through special reagents and instruments; long fragments cannot be analyzed

[0006] However, in vitro amplification technology is prone to inhibition in practical applications.

Sampling methods, sample types, extraction reagents, etc. affect the quality of the obtained DNA or RNA templates, resulting in different degrees of amplification inhibition, which needs further improvement

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