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

A technology of real-time fluorescence quantification and detection kit, which is applied in the direction of fluorescence/phosphorescence, microbial measurement/inspection, biochemical equipment and methods, etc. It can solve the problems of PCR product contamination, heavy workload, false positives, etc., and shorten the reaction time Effects of time, reliable results, increased specificity and accuracy

CN101967525AActive Publication Date: 2011-02-09CHONGQING MEDICAL UNIVERSITY
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Patent Information

Authority / Receiving Office
CN · China
Current Assignee / Owner
Publication Date
2011-02-09

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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.
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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...

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Embodiment Construction

[0044] 1. Preparation of kits

[0045] 1. The design of fluorescent quantitative PCR primers and probes, the length of the amplified target fragment is 199bp, all of which were synthesized and labeled by Shanghai Ying (Invitrogen) Biotechnology Co., Ltd.

[0046] The upstream primer sequence is SEQ ID NO2;

[0047] The downstream primer sequence is SEQ ID NO3;

[0048] The sequence of the fluorescent probe is SEQ ID NO5.

[0049] 2. Preparation of standard products

[0050] The standard product is a recombinant plasmid inserted into the p24 fragment, constructed by our laboratory. According to the plasmid concentration can be converted into copy number, the plasmid was diluted to 1×10 8 copies / μl, stored at -20°C, and diluted 10-fold before use.

[0051] 3. Optimization of annealing temperature

[0052] The Tm values ​​of the upstream and downstream primers are 57.8°C and 57.3°C, respectively, and the annealing temperature decibels are set to 54.8°C, 55.5°C, 56.6°C,...