PRRS rt-lamp detection method and detection kit

Abstract

The invention discloses a simple, rapid and specific RT-PCR detection method and detection kit for PRRS. The present invention uses reverse transcription loop-mediated isothermal amplification technology (RT-LAMP), designs 2 pairs of specific primers in 6 regions of the ORF7 gene conservative sequence of PRRSV nucleocapsid protein (N protein), and optimizes the reaction system and reaction conditions As well as sensitivity and specificity tests, a RT-LAMP detection method for PRRSV was established. The research results show that this detection method does not require complicated instruments, and the nucleic acid amplification reaction can be completed by only using an ordinary constant temperature water bath and incubating at isothermal conditions (63°C) for 1 hour. The accurate judgment of the test results provides a cheap, simple, fast, high sensitivity and good specificity new method for the detection of PRRS in the field, field and grassroots departments.

Description

technical field [0001] The invention relates to a RT-LAMP detection method and a detection kit for PRRS. Background technique [0002] Pig PRRS is mainly a disease caused by porcine reproductive and respiratory syndrome virus (Porcine Reproductive and Respiratory Syndrome virus, PRRSV) infection. From 1990 to 1991, PRRS broke out in Europe, causing more than 1 million pigs to die. The Dutchman Wenswort and others isolated the virus from sick pigs for the first time. The mortality rate of PRRS pigs is generally 50%, or even 90% to 95%, mainly causing abortion, premature birth, stillbirth and mummification in pregnant sows, while piglets mainly have symptoms such as coughing and dyspnea. PRRS is a Class B animal infectious disease stipulated by OIE. Once infected in intensive farms, it is difficult to purify. Since the first report in the United States in 1987, PRRS has spread to the world, causing huge economic losses every year. The pig industry poses a serious threat, rap...

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Problems solved by technology

Immunological detection methods such as indirect immunofluorescence test and immunoperoxidase monolayer test have long quarantine period, cumbersome operation and difficult diagnosis, which are not conducive to accurate, rapid and efficient monitoring of the PRRSV epidemic situation

With the development of modern molecular biology, a variety of molecular diagnostic techniques have been applied to the detection of PRRS, such as RT-PCR, gene chip technology, nucleic acid sequence-dependent amplification (NASBA), real-time fluorescent RT-PCR, error Amplification mutation assay (MAMA), etc., but these methods require expensive special instruments and reagents, and are not suitable for popularization and application in grassroots laboratories. The new nucleic acid amplification technology - loop-mediated isothermal amplification (LAMP) and other Compared with the detection method, it has the advantages of simplicity, rapidity, and specificity. It does not need a PCR instrument and can be amplified in a water bath. It is especially suitable for the promotion and use of grassroots laboratories.

At present, a simple, rapid and specific RT-PCR detection method that is convenient for the detection of PRRS in the field and in grass-roots laboratories has not been established.

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