Real-time fluorescent quantitative RT-PCR detection primer, probe and kit for Palyamserogroup virus

Abstract

The invention relates to a real-time fluorescent quantitative RT-PCR detection primers, probe and kit for Palyamserogroup virus, and belongs to the technical field of animal virus molecular biologicaldetection. The kit comprises an upstream primer, a downstream primer, the probe matched with the primers for use, a negative control template, a positive control template, a standard template and a PCR amplification reagent. The kit is adopted for detection, the reaction speed is high, and the whole amplification process is less than 1 hour; only virus RNA needs to be extracted, reverse transcription is not needed, operation steps are few, easy and convenient, and RNA degradation and pollution can be effectively avoided; meanwhile, after qRT-PCR amplification is completed, whether PALV RNA exists in a sample to be detected or not can be directly judged without agarose gel electrophoresis; and in addition, a standard curve established with the help of the standard template can be used forquantitatively detecting the PALV RNA in the clinical sample, so that the working efficiency is greatly improved, and the detection cost is reduced.

Description

technical field [0001] The invention belongs to the technical field of animal virus molecular biology detection, in particular to amplification primers, TaqMan probes and assembled detection reagents for rapid detection of Paliam serogroup viruses popular in China by using TaqMan real-time fluorescent quantitative RT-PCR technology box. Background technique [0002] Palyamserogroup virus (PALV) is a member of the Orbivirus genus of the Reoviridae family and is widely prevalent in tropical and subtropical regions between 49°N and 35°S. PALV is mainly transmitted through the blood-sucking bites of female blood-sucking insects Culicoides to animals, infecting cattle and sheep and other ruminants, resulting in abnormal production of pregnant animals, mainly manifested as abortion, premature birth, stillbirth or anencephaly. From 1985 to 1986, the Paliam virus epidemic broke out in Japan. Its clinical symptoms were congenital abnormalities in newborn cattle, accompanied by hydro...

AI Technical Summary

Problems solved by technology

[0006] (1) Disadvantages of nested RT-PCR: the first amplification product needs to be used as the template for the second amplification, which is easy to introduce contamination and lead to false positives; it needs to be detected by agarose gel electrophoresis, which is time-consuming longer

[0007] (2) Insufficiency of C-ELISA: It mainly detects antibodies in animal blood, and it generally takes 2 to 3 weeks from animal infection with PALV to antibody production. Therefore, C-ELISA method cannot be used for early detection before antibody production. clinical diagnosis

[0009] Fluorescent quantitative PCR technology is a new type of nucleic acid qualitative and quantitative technology introduced by ABI Company in the United States in 1996. Since its invention, this technology has been widely used in the detection of bacteria, viruses and other pathogens. It has strong characteristics, high sensitivity and fast detection speed. and high-throughput detection, and more importantly, this technology can be used for clinical sample detection, but there is no one-step real-time fluorescent quantitative RT-PCR (one step quantitative real timeRT-PCR, qRT-PCR) for PALV at present. PCR) detection method available

Images