PCR (polymerase chain reaction) product melting point analysis method for quantitatively detecting species composition of mixed-type sample

Abstract

The invention discloses a PCR (polymerase chain reaction) product melting point analysis method for quantitatively detecting species composition of a mixed-type sample. The method comprises the following steps: carrying out primer design by using genes with moderate evolutionary rate of each species in the mixed sample; optimizing multiplex PCR conditions according to single PCR of the corresponding template and primers; carrying out amplification on different sample DNAs (deoxyribonucleic acids) with known component content under the optimized conditions; determining the melting curve of the multiplex PCR product to obtain the melting peak-to-peak height value of the corresponding component, and drawing a standard curve according to the peak height ratio and corresponding sample component content ratio; and for the sample with unknown component content ratio, carrying out amplification and melting curve determination under the same conditions to obtain the peak height ratio, and calculating the corresponding component content ratio according to the standard curve. The method is simple and quick, is low in cost, does not need to detect all the templates to be detected on the basis of the high-quality standard curve, and implements quantitative analysis on the species or components in the mixed-type sample.

Description

technical field [0001] The invention relates to a detection method for various species in a mixed sample, in particular to a PCR product melting point analysis method for quantitatively detecting the species composition of a mixed sample. Background technique [0002] Many biological species have great differences in the degree of protection, potential allergen types, quality and commodity value; due to the insignificant difference in morphological characteristics between similar species or the difficulty in identifying species due to post-processing damage to morphological characteristics, these are all The differential application of its grades and the implementation of special protection measures cause serious obstacles, and it is also a great test for the detection level of the detection method. In particular, the detection of closely related species poses great challenges to the specificity and sensitivity of molecular detection methods. At present, the identification ...

AI Technical Summary

Problems solved by technology

If we can invent a method that can improve sensitivity and specificity at the same time, and overcome the key problems of poor reproducibility, false positives and false negatives, etc. Nucleic acid detection provides theoretical basis and technical support, and will have broad application prospects and huge market potential in various biological monitoring fields

[0004] In recent years, many PCR quantitative detection methods have made varying degrees of progress in both sensitivity and specificity, but it is still difficult to achieve both sensitivity and specificity

For the detection and analysis of mixed species, the existence of non-target templates increases the interference of specific primers, especially the mutual interference of genes of closely related species exacerbates this negative effect

At present, the combination of multiplex PCR and product melting point determination can qualitatively detect some pathogenic bacteria at the same time, but the quantitative analysis method for mixed samples has not yet been established, and it is even more difficult in the application of rapid high-throughput detection

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