Detection primer group, kits and detection method for porcine reproductive and respiratory syndrome virus fluorescent EMA

Abstract

The invention relates to a detection primer group, kits and a detection method for porcine reproductive and respiratory syndrome virus fluorescent EMA. The detection primer group of the porcine reproductive and respiratory syndrome virus fluorescent EMA contains an upstream primer, a downstream primer and a probe for detecting the genome Nsp2 gene sequence of the porcine reproductive and respiratory syndrome virus; the EMA detection kits comprise a kit A and a kit B. The detection method comprises the following steps: carrying out normal temperature nucleic acid amplification technique by using the detection kits; carrying out multiple enzymatic reactions by using M-MLV reverse transcriptase, an Rnase inhibitor, DNA helicase, single-stranded DNA binding protein, DNA polymerase and the like; under the constant condition of 37 to 45 DEG C, carrying out reverse transcription on the to-be-tested RNA to obtain cDNA within 10 to 30 minutes; meanwhile, amplifying the cDNA for millions of times; realizing rapid detection of the to-be-tested RNA by matching a fluorescence detection technology. The detection method for the porcine reproductive and respiratory syndrome virus fluorescent EMA,disclosed by the invention has the advantages of simple operation, short time, low requirement on instrument and suitability for rapid diagnosis of porcine communicable diseases.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a primer set, kit and detection method for porcine PRRS virus fluorescent EMA detection. Background technique [0002] Pig blue-ear disease, also known as reproductive and respiratory syndrome (Porcine Reproductiv and Respiratory Syndrome, PRRS). The main clinical manifestations are reproductive disorders in pregnant sows, abortion, weak fetuses, stillbirths, and mummified fetuses in pregnant sows; interstitial pneumonia in piglets and other symptoms. The causative agent is porcine blue ear disease virus (PRRS Virus, PRRSV), which is a positive-sense small RNA enveloped virus with a genome length of about 15kb. It is divided into American type and European type. my country's porcine PRRSV belongs to the American type; but since 2006, there have been 25 provinces in my country where the highly pathogenic porcine blue-ear disease virus (HP-PRRSV) was the main pathogen. High fever". T...

AI Technical Summary

Problems solved by technology

Once the pig is infected, it will show the characteristics of high morbidity and high mortality, causing more serious economic losses to the pig industry

[0003] Nowadays, the detection of porcine PRRS virus is mainly based on PCR method and immune method. Among them, the immune method has problems such as low specificity and low sensitivity, and the use of PCR method is becoming more and more popular, including electrophoresis and fluorescence methods. It is well known that electrophoresis Compared with the fluorescence method, there is one more step of electrophoresis, and the amplified product needs to be opened, which increases the risk of contamination, so the acceptance is not as high as that of the fluorescence method. The traditional PCR method needs to be equipped with an expensive PCR machine or a real-time fluorescent quantitative PCR machine, so it cannot For grassroots and field testing

[0004] For example, Chinese patent CN 105648119 A discloses a primer, a kit and a method for detecting porcine reproductive and respiratory syndrome virus. It takes a long time and requires a PCR instrument with high temperature control accuracy, which is relatively expensive. After amplification, electrophoresis detection is required, which adds a cumbersome step. At the same time, the product needs to be opened, which increases the risk of contamination.

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