RT-RPA (reverse transcription recombinase polymerase amplification) detection kit for fast detecting high-pathogenicity porcine reproductive and respiratory syndrome virus and application thereof

A technology for respiratory syndrome and high pathogenicity, applied in the direction of microorganism-based methods, microorganism measurement/testing, biochemical equipment and methods, etc., to achieve good specificity, increase specificity, and simple methods

Active Publication Date: 2016-05-11
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

At present, there is no RT-RPA test established at home and abroad to detect HP-PRRSV

Method used

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  • RT-RPA (reverse transcription recombinase polymerase amplification) detection kit for fast detecting high-pathogenicity porcine reproductive and respiratory syndrome virus and application thereof
  • RT-RPA (reverse transcription recombinase polymerase amplification) detection kit for fast detecting high-pathogenicity porcine reproductive and respiratory syndrome virus and application thereof
  • RT-RPA (reverse transcription recombinase polymerase amplification) detection kit for fast detecting high-pathogenicity porcine reproductive and respiratory syndrome virus and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The establishment of the RT-RPA detection kit and detection method of embodiment 1 rapid detection HP-PRRSV

[0038] 1. Design and synthesis of primers and probe sequences

[0039] Studies have shown that compared with C-PRRSV, H-PRRSV lacks about 30 amino acids in the NSP2 region, so the present invention selects highly pathogenic porcine reproductive and respiratory syndrome virus NSP2 deletion region to design specific primers and probes, and The homologous sequences of NSP2 genes from EF112445, EF517962, EF635006, EF641008, EU109503, EU144079, EU187484, EU200961, EU880431 and EU880435 in GenBank were compared in order to detect as many HP-PRRSV as possible. Both probes and probes were synthesized by Sangon Biotech (Shanghai, China).

[0040] 2. Virus strains and cells

[0041] All strains used in this study are preserved by our laboratory: HP-PRRSV / SD0907 (GenBank: KF562320.1), HP-PRRSV / JS0912 (GenBank: KF562318.1), classicalPRRSV strain CH-1R (GenBank: EU807840),...

Embodiment 2

[0064] The purposes of the RT-RPA detection kit of embodiment 2HP-PRRSV in field detection

[0065] 1. Sample

[0066] Sixty-eight field tissue samples were collected from eight pig farms suspected to have HP-PRRS in Shandong Province. Twelve serum samples were collected from healthy pigs. Viral genome extraction is the same as in Example 1.

[0067] 2. Detection method

[0068] (1) real-timeRT-PRA method

[0069] The experimental system was as follows: 13.75 μL hydrolysis buffer, 1.05 μL upstream primer (10 μM), 1.05 μL downstream primer (10 μM), 0.075 μL RPAexo probe (10 μM), 2 μL RNA template, 4.825 μL ddHO and 1.25 μL acetic acid Magnesium (magnesium acetate, 280mM).

[0070] The sequences of the primers and probes are as follows:

[0071] Upstream primer: 5'-AGCTGATGACACCTTTGAGTGGGTCGGCACCAGTT-3' (shown in SEQ ID NO.1);

[0072] Downstream primer: 5'-CGTCTGTGAGGACGCAGACAAATCCAGAGGCTCAT-3' (shown in SEQ ID NO.2);

[0073] Probe: 5'-GTCGGCACCAGTTCCTGCACCGCGTAGAAC

...

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Abstract

The invention discloses an RT-RPA (reverse transcription recombinase polymerase amplification) detection kit for fast detecting a high-pathogenicity porcine reproductive and respiratory syndrome virus and application thereof. The kit comprises a pair of primers and a probe, the sequences of the primers are shown as SEQ ID NO.1 and SEQ ID NO.2, and the sequence of the probe is shown as SEQ ID NO.3. It is proved through experiments that the kit can detect adverse effects of the high-pathogenicity porcine reproductive and respiratory syndrome virus (HP-PRRSV), a hog cholera virus, a C-type porcine reproductive and respiratory syndrome virus, a porcine circovirus type II, a porcine pseudorabies virus and a foot and mouth disease virus in a specificity mode. It is proved through experiments that the kit can detect out templates of at least 70 copies at the temperature of 40 DEG C on the condition of 20 min amplification, and the conformity between the kit and RT-qPCR is high. This shows that the kit can detect HP-PRRSV fast, efficiently and sensitively and provides an effective technological means for differential diagnosis of HP-PRRSV.

Description

technical field [0001] The invention relates to a kit for detecting highly pathogenic porcine reproductive and respiratory syndrome virus and its application, in particular to an RT-RPA detection kit for rapidly detecting highly pathogenic porcine reproductive and respiratory syndrome virus and its application. purposes, the invention belongs to the field of preventive veterinary medicine inspection. Background technique [0002] In the laboratories of developing countries, due to the limitation of basic equipment and technology required for PCR implementation, most developing countries still focus on using traditional test methods, such as serological methods, microscopy techniques, or culturing and identifying infectivity and non-communicable diseases. In most cases, despite the lack of experimental equipment to use these methods, and the lack of routine practice of comprehensive management of these diseases. As a result, these countries have long been plagued by disease...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/686C12Q1/70C12Q2561/113C12Q2563/107C12Q2545/114C12Q2521/507C12Q2521/107C12Q2521/319
Inventor 杨洋张志东秦晓东
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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