Method for detecting nucleic acid of porcine reproductive and respiratory syndrome virus in one step

Abstract

The invention relates to a method for detecting nucleic acid of a porcine reproductive and respiratory syndrome (PRRS) virus by one step, which comprises the following steps of: collecting, processing and detecting samples, wherein in the collecting step, an animal blood sample is dripped into a full type approval (FTA) card sample area; and the detecting steps comprises the: (1) designing specific primers and a probe for general type PRRS virus, and marking carboxyfluorescein (FAM) and tetramethyl rhodamine (TAMARA) fluorescent groups on the probe; and (2) preparing and optimizing a detection system and a reaction condition, wherein a reaction system comprises 25 or 50 microliters of trihydroxymethyl aminomethane-hydrogen chloride (HCl) (the pH value is between 7.8 and 9.0), 0.1 to 0.5 micro mol of upstream primer and 0.1 to 0.5 micro mol of downstream primer, 100 to 400 micro mols of deoxynucleotide mixture, 0.1 to 0.5 micro mol of probe, 100 to 300 U of Moloney murine leukemia virus (M-MLV) reverse transcriptase, 1 to 5 U of thermostable deoxyribonucleic acid (DNA) polymerase, 4 to 8 mols of Mg<2+->, 300 to 500 nano mol of homogenized reference dyes ROX, and 1 to 15 microliters of sample which is re-suspended in trihydroxymethyl aminomethane-ethylene diamine tetraacetic acid (EDTA) buffer solution and is added before each time of reaction. In the method, the sample collection is easy and convenient, so that an FTA card containing s ribonucleic acid (RNA) sample can be posted to any one central laboratory to be detected according to a form of regular mails; and the pollution risks are reduced, and the detection sensitivity is improved.

Description

technical field [0001] The invention relates to a method for detecting porcine PRRS virus nucleic acid in one step. Background technique [0002] Existing detection methods for porcine PRRS virus nucleic acid include antibody detection and antigen detection. Antibody detection often lags behind antigen detection, so that the disease cannot be detected in a timely and accurate manner, leading to the outbreak of the disease and causing huge economic losses; the existing antigen detection method takes a long time to separate the virus, requires high environmental facilities, and requires high technical level. In rural and remote areas, small and medium-sized farmers and even large and medium-sized farms have inadequate environmental facilities and personnel skills, so early-warning or suspicious samples can only be sent to large-scale testing centers for testing; and their sensitivity is not high; and how to preserve RNA from degradation, It has always been a difficult problem...

AI Technical Summary

Problems solved by technology

Antibody detection often lags behind antigen detection, so that the disease cannot be detected in a timely and accurate manner, leading to the outbreak of the disease and causing huge economic losses; the existing antigen detection method takes a long time to separate the virus, requires high environmental facilities, and requires high technical level. In rural and remote areas, small and medium-sized farmers and even large and medium-sized farms have inadequate environmental facilities and personnel skills, so early-warning or suspicious samples can only be sent to large-scale testing centers for testing; and their sensitivity is not high; and how to preserve RNA from degradation, It has always been a difficult problem in the field of molecular biology. So far, there is no easy and feasible preservation method; the traditional method of transporting and storing these virus specimens requires professional freezing and transportation technology. This method is costly and requires certain professional knowledge. It is not easy to promote. Therefore, the disease cannot be detected in time and accurately

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