LAMP (Loop-Mediated Isothermal Amplification) detection method of PRV (Porcine Rotavirus) inverse transcription and application

Abstract

The invention discloses an LAMP (Loop-Mediated Isothermal Amplification) detection method of PRV (Porcine Rotavirus) inverse transcription and an application. The detection method comprises the following steps: (1) building a RT-LAMP reaction system, and setting a negative control at the same time, wherein the RT-LAMP reaction system is 25 [mu] L and comprises 2 [mu] L of template, 0.5 [mu]L of 0.8 [mu]M FIP primer, 0.5 [mu]L of 0.8 [mu]M BIP primer, 0.25 [mu]L of 0.2 [mu]M of F3 primer, 0.25 [mu]L of 0.2 [mu]M of B3primer,2[mu]L of dNTP (Diethyl-Nitrophenyl Thiophosphate), 5 [mu]M of MgSO4 (Magnesium Sulfate), 8UBst of DNA polymerase and 1*ThermoPol Buffer, and 1[mu]L of MLV (Murine Leukemia Virus) reverse transcriptase, and supplementing the volume to 25 [mu]L by sterile water; and the negative control template is sterile water; (2) after reacting for 20-60min at 61-65 DEG C in a constant temperature water bath boiler, terminating for 20 min at 80 DEG C, carrying out AGE (Agarose Gel Electrophoresis) identification on an amplification product or adding 2 [mu]L of SYBR Green dye, and observing a result in an UV lamp (Ultraviolet Lamp) or by naked eyes. According to the LAMP detection method provided by the invention, a convenient, fast and accurate molecular biological diagnosis method can be provided for the clinical diagnosis and epidemiological investigation of the PRV.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a rapid detection method for porcine rotavirus and the application of the method in differential detection of PRV and PrV, PPV, PEDV, TGEV and PRRSV. Background technique [0002] Rotavirus (rotavirus, RV) is a member of the genus Rotavirus in the Reoviridae family and is the main pathogen that causes viral diarrhea in infants and young animals. Porcine rotavirus (porcine rotavirus, PRV) is one of the main pathogens causing viral diarrhea in piglets, which has caused serious economic losses to the swine industry all over the world. Porcine rotavirus infection is also very common in my country, and the positive rate of weaned piglets in some areas is as high as 72%. The rotavirus genome consists of 11 dsRNA fragments, which encode 6 structural proteins and 6 non-structural proteins (NSPs) of the virus respectively. The structure of the virus is very stable. VP6 protein is an important...

AI Technical Summary

Problems solved by technology

Traditional serological methods, such as enzyme-linked immunosorbent assay (ELISA) and colloidal gold technology, are difficult to detect early PRV infection and have low sensitivity

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