Real-time fluorescence quantitative PCR (Polymerase Chain Reaction) detection kit for BDV (Borna Disease Virus) p24 segment

Abstract

The invention provides a fluorescence quantitative PCR (Polymerase Chain Reaction) kit with simple and convenient usage and high sensitivity and specificity, which can optimize and improve the amplification efficiency of the PCR, reduce the contamination rate of the PCR, exactly and fast detect the BDV nucleic acid of a sample. In the invention, the real-time fluorescence quantitative PCR detection kit for a BDV p24 segment comprises 10* reverse transcription reaction liquid, an AMV (Avian Myeloblastosis Virus) reverse transcriptase, 2* fluorescence quantitative PCR reaction liquid, a Taq polymerase, a standard substance and the negative control, wherein the fluorescence quantitative PCR reaction liquid is optimized reaction liquid. The invention can shorten the reaction time and reduce the possibility of the PCR contamination, and can also remarkably improve the quantitative accuracy and the amplification efficiency.

Description

technical field [0001] The invention belongs to the field of biological technology, and is a method for obtaining cDNA by reverse transcription of an mRNA sample, combined with real-time fluorescence quantitative PCR technology to detect the Borna disease virus nucleic acid in the quantitative sample, and specifically relates to a Borna disease virus real-time fluorescence quantitative PCR Detection kit. Background technique [0002] Borna disease virus (BDV) is a neurotropic single-stranded negative-sense RNA virus. The disease caused by BDV as a pathogenic factor is called Borna disease (BD). zoonoses. In recent years, indirect immunofluorescence (IFA), enzyme-linked immunosorbent assay (ELISA), immunoblotting (IB), RT-PCR and other methods have frequently detected BDV antigens, antibodies and Nucleic acid, suggesting that chronic infection of BDV may be related to human mental illness. However, different detection methods have great differences in the detection result...

AI Technical Summary

Problems solved by technology

We have previously developed a BDV nested fluorescent quantitative RT-PCR kit (Xu Ping, Xie Peng, Zou Dezhi, et al. Establishment of a fluorescent quantitative nested RT-PCR detection method for Borna disease virus[J]. Journal of Chongqing Medical University , 2003, 28(6): 688-691), although the specificity of nested fluorescent quantitative PCR is high, but there are many steps and the workload is heavy, and the intermediate steps need to process PCR products, which is prone to PCR product contamination when detecting a large number of specimens. False positive, we redesigned the primers when developing the new BDV fluorescent quantitative PCR kit, and optimized the fluorescent quantitative PCR reaction buffer, annealing temperature, primer probe concentration, dNTPs concentration, magnesium ion concentration, and developed a post-reverse transcription Fluorescence quantitative one-step detection kit, no need to process the second-step PCR product, reducing the possibility of PCR product contamination

Images