Extraction-free direct amplification reagent for real-time fluorescent quantitation PCR and application thereof

Abstract

The invention provides an extraction-free direct amplification reagent for real-time fluorescent quantitation PCR and application thereof. The direct amplification reagent comprises the following components: sodium hydroxide (NaOH), Surfactin, dimethyl sulfoxide (DMSO), sucrose and chelex-100. When the reagent is used, there is no need to extract and purify nucleic acid from a sample, only the sample and the reagent need to be mixed according to the equal volume ratio of 1:1. The mixture can be directly used for fluorescent PCR detection. The extraction-free direct amplification reagent is applicable to nucleic acid extraction of human genome, bacteria and viruses in mouth swabs, nasopharyngeal swabs, serum, plasma, genital tract secretion and other samples. The reagent is suitable for both the fluorescence PCR of a probe method and the PCR of a dying method, is suitable for a variety of clinical applications and has the advantages of low cost, simple operation, accurate results and the like, and detection can be simple and quickly completed.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to an extraction-free direct amplification reagent for real-time fluorescent quantitative PCR and its application. Background technique [0002] The polymerase chain reaction (PCR) technology is a simple, fast, highly sensitive, and specific gene amplification method. The process usually includes 20-50 cycles of denaturation, annealing, and extension. The target nucleic acid sequence can be specifically amplified to several million times within 1.5-3 hours. In recent years, due to the emergence of real-time fluorescent PCR machines, ordinary PCR no longer needs gel electrophoresis analysis, which eliminates the need for post-PCR operations and eliminates PCR pollution. At present, real-time fluorescent PCR technology has been widely used in various fields such as infectious disease detection, genetic disease detection, tumor gene mutation detection and so on. [0003] The products...

AI Technical Summary

Problems solved by technology

At this stage, the nucleic acid extraction of samples mainly adopts the spin column method and the magnetic bead method. Both methods have to go through steps such as lysis, binding, washing, repeated washing, and elution. The operation is complicated and takes 1 to 2 hours. Large amounts of protein allosteric agents and organic solvents are required

At the same time, nucleic acid extraction also requires professional laboratory equipment and environment, and has special requirements for operators.

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