Real-time fluorescent quantitative PCR detection method for apple stem pitting virus

Abstract

The invention provides a real-time fluorescent quantitative PCR detection method for an apple stem pitting virus, and belongs to the field of virus molecule detection. The method comprises the following steps: with cDNA of an infected material as a template, and ASPV-cp-F2 and ASPV-cp-R2 as primers, carrying out PCR amplification, so as to obtain positive recombinant plasmid standards; with copy number concentration of the positive recombinant plasmid standards and positive recombinant plasmid standards with the concentration as templates, building a standard curve by a Ct value of real-time fluorescent quantitative PCR employing specific primers and probes; carrying out real-time fluorescent quantitative PCR on a tested material according to the same condition; and achieving quantitative detection of the apple stem pitting virus of the tested material by comparing the Ct value with the standard curve. According to the method, quantitative determination of the apple stem pitting virus is achieved; the detection result can be directly read out through computer software; and the problems of false positivity of the detection result and environmental pollution are overcome.

Description

technical field [0001] The invention relates to the field of virus molecular detection, in particular to a real-time fluorescent quantitative PCR detection method for apple stem pox virus. Background technique [0002] Apple stem pox virus is one of the latent viruses that commonly occur on apple and pear trees. The virus can cause various diseases such as apple stem pox, pear vein yellow disease, and pear stone pox, which can cause apple and pear yields to decline. , the quality deteriorates, seriously affecting the production of fruit trees in my country. The current method for detecting ASPV is mainly the conventional RT-PCR technique. [0003] The conventional RT-PCR detection technical scheme of ASPV includes the following steps: [0004] 1. Design routine RT-PCR detection primers; [0005] 2. Extract the RNA of the test sample from the fruit tree and reverse transcribe it into cDNA; [0006] 3. Using cDNA as a template, use specific amplification primers for convent...

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Problems solved by technology

[0010] (1), the operation steps are relatively cumbersome, time-consuming and labor-intensive;

[0011] (2) The PCR amplification product needs to be post-processed, and the detection result can only be obtained by electrophoresis of the amplification product through agarose gel, but the carcinogen EB needs to be added in the process of making the agarose gel, which is easy to pollute the laboratory environment. It will also pose a threat to the health of the experimenters. At the same time, cross-contamination between different samples is prone to occur during the post-processing process, resulting in false positives in the test results;

[0012] (3) Conventional RT-PCR technology can only detect viruses qualitatively, but not quantitatively

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