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Multiplex RT-PCR detection kit for porcine reproductive and respiratory syndrome virus and application thereof

A technology of RT-PCR and respiratory syndrome, which is applied in the field of multiple RT-PCR detection kits for porcine reproductive and respiratory syndrome virus, can solve the problems of low detection rate, long detection cycle, and low specificity, and achieve The effect of low cost, less reagents, and broad market application prospects

Active Publication Date: 2014-03-12
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to determine whether porcine reproductive and respiratory syndrome exists in a certain area, the method of detecting animal serum antibodies is not completely reliable. It is also necessary to pass etiological detection to confirm the existence of the pathogen. The general detection methods are biological experiments and serotype diagnostic techniques However, some of these methods have low sensitivity, some have low specificity, some have low detection rates, the detection cycle is too long, and the cost of these technologies is generally too high

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  • Multiplex RT-PCR detection kit for porcine reproductive and respiratory syndrome virus and application thereof
  • Multiplex RT-PCR detection kit for porcine reproductive and respiratory syndrome virus and application thereof
  • Multiplex RT-PCR detection kit for porcine reproductive and respiratory syndrome virus and application thereof

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Effect test

Embodiment 1

[0028] How to use the multiplex RT-PCR detection kit for European and American strains of porcine reproductive and respiratory syndrome virus:

[0029] 1. Nucleic acid extraction of European and American strains of porcine reproductive and respiratory syndrome virus:

[0030] Collect the blood of the animal to be tested or the tissue of the dead animal, make a 1:5 emulsion suspension, use the Trozol method to extract the whole genome RNA, use the RNA as a template, and use the specific European-type and American-type pig strains in the kit Multiplex RT-PCR was performed on the primers of reproductive and respiratory syndrome virus, and electrophoresis was performed on the obtained product. According to the electrophoretic pattern, it was analyzed whether the tested animals were infected with European-type and American-type strains of porcine reproductive and respiratory syndrome virus.

[0031] 2. Multiplex RT-PCR reaction:

[0032] This kit designs and synthesizes two pairs ...

Embodiment 2

[0036] Establishment of PRRSV multiplex RT-PCR method:

[0037] According to Example 1, the RNA of PRRSV was sequentially added to the Ep tube as the template amount was 0.5 μl-4 μl, the PRRSV-specific upstream and downstream primers were 0.2 μl-2 μl, and the annealing temperatures were 57 ° C, 58 ° C, 59 ° C and 60°C, and then carried out RT-PCR amplification respectively. Test results such as figure 1 As shown, when adding 2 μl of corresponding PRRSV RNA as a template to a 50 μl reaction system, 0.8 μl of PRRSV-specific upstream and downstream primers, and annealing temperatures of 58° C., the best results were obtained.

Embodiment 3

[0039] Specificity and sensitivity test of PRRSV multiplex RT-PCR method:

[0040] 1. PRRSV multiplex RT-PCR sensitivity test:

[0041] 1.1 Quantification of European and American PRRSV:

[0042] Viral RNA extracted from European and American PRRSV according to 4.0×10 5 Copies of RNA, 4.0×10 4 Copies of RNA, 4.0×10 3 Copies of RNA, 4.0×10 2 copies of RNA and 4.0 x 10 1 Copies of RNA were diluted separately.

[0043] 1.2 Result detection:

[0044] RT-PCR products were subjected to agarose gel electrophoresis. Then stained with ethidium bromide, photographed and analyzed in the BIO-RAD gel imager, the electrophoresis results are as follows figure 2 As shown, it can be seen from the figure that the multiplex RT-PCR of European and American PRRSV can specifically detect the virus target gene, and at least 40 copies of RNA can be detected.

[0045] 2. Established PRRSV multiplex RT-PCR specificity test:

[0046] 2.1 Extraction of porcine circovirus-type 2 virus, porcine ...

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Abstract

The invention discloses a multiplex RT-PCR detection kit for a porcine reproductive and respiratory syndrome virus. The kit comprises two pairs of primers used for amplification of genes of an American porcine reproductive and respiratory syndrome virus and a European porcine reproductive and respiratory syndrome virus; and the invention provides an application method for the detection kit. The invention has the following beneficial effects: the multiplex RT-PCR detection kit for the porcine reproductive and respiratory syndrome virus provided by the invention is fast compared with classical virus isolation, single RT-PCR and serum typing methods and can conveniently identify and distinguish any one belonging to American and European porcine reproductive and respiratory syndrome viruses in one shot so as to facilitate timely and fast responses to epidemic disease situations, regions and environments and implementation of correct treatment in shortest time; meanwhile, the Trizol extraction method for extraction of the whole genome RNA in the invention has the characteristics of rapidness, simpleness, low cost and usage of a small amount of desired reagents, is especially applicable to extraction of small samples and has wide market application prospects.

Description

technical field [0001] The invention relates to a livestock virus detection kit, in particular to a multiple RT-PCR detection kit for porcine reproductive and respiratory syndrome virus. Background technique [0002] Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS) is caused by porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV). And mummified fetuses, piglets and fattening pigs have respiratory diseases, reduced production performance, reduced feed remuneration, and increased mortality. [0003] Although a lot of research has been done on the etiology, serotyping and diagnosis of PRRSV at home and abroad, because it is almost all over the world, the threat to pigs and related products all over the world is well known. For tens of billions of dollars. In particular, it is difficult to distinguish it from other reproductive and respiratory syndromes clinically. At...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/686C12Q1/70C12Q2537/143C12Q2531/113
Inventor 刘永生丁耀忠张杰周建华陈豪泰马丽娜
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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