Variant porcine reproductive and respiratory syndrome virus (PRRSV) TaqMan fluorescence quantitative RT-PCR detecting kit and application thereof

Abstract

The invention discloses variant porcine reproductive and respiratory syndrome virus (PRRSV) TaqMan fluorescence quantitative RT-PCR detecting kit and application thereof. A primer and a TaqMan probe are designed and synthesized by referring to an NSP2 fragment gene sequence of the variant PRRSV and common PRRSV of a GenBank. By optimizing the reaction condition and constructing a standard plasmidproduct, a method for diagnosing the variant PRRSV by TaqMan fluorescence quantitative RT-PCR is established. A result indicates that the method has the advantages of strong specificity, high sensitivity, and the like and can detect the standard plasmid product with 264 copy numbers, and the virus quantity of 0.5623TICD50 is 10 times more sensitive than RT-PCR. By detecting 22 disease samples, 8 disease samples are positive, and the positive rate is 36.4 percent. Because the method has the advantages of quantification, high speed, accuracy, sensitivity, and the like, the invention is suitablefor the diagnosis on the swinery infected variant PRRSV in the early stage, the medium stage and the later stage and plays an important role in effectively diagnosing, preventing and treating the highly pathogenic PRRSV.

Description

technical field [0001] The invention relates to a mutant porcine reproductive and respiratory syndrome virus TaqMan fluorescent quantitative RT-PCR detection device, in particular to a mutant porcine reproductive and respiratory syndrome virus TaqMan fluorescent quantitative RT-PCR detection kit and its application. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS) is a new infectious disease that emerged in the middle and late 1980s, caused by porcine reproductive and respiratory syndrome virus (PRRSV). It is manifested as severe reproductive disorders such as abortion, premature birth, stillbirth, mummified fetus, and respiratory symptoms in piglets and fattening pigs. The disease first appeared in the United States in 1987, and then spread rapidly in Canada, European countries and Asia. Since the outbreak of the disease at the end of 1995 in my country, it has become one of the main diseases that endanger the pig industry in my country. S...

AI Technical Summary

Problems solved by technology

At present, there are many detection methods for reproductive and respiratory syndrome virus, such as virus isolation, in situ hybridization and RT-PCR technology, etc. Although virus isolation and in situ hybridization have high sensitivity, the operation is cumbersome and the cost is high. The cycle is long and the requirements for disease materials are high, so it is not suitable for the rapid diagnosis of clinical PRRSV; although the conventional RT-PCR detection method is convenient, fast and sensitive, RT-PCR requires electrophoresis and is prone to EB or molecular Contamination, and can not be carried out timing, quantitative monitoring, etc., and fluorescent quantitative RT-PCR method can overcome the above shortcomings, fluorescent quantitative PCR method is a new method introduced by the United States AppliedBiosystems in 1996, it by adding fluorescent groups in the PCR reaction, using Fluorescent signal accumulation monitors the entire PCR process in real time, and finally uses the standard curve to quantitatively analyze the unknown template. It not only realizes the leap from qualitative to quantitative PCR, but also has more timing, quantification and specificity than conventional PCR. Strong and effective solution to the PCR pollution problem, etc.

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