Multiplex RT-PCR (Reverse Transcription-Polymerase Chain Reaction) quick detection kit and primer for SIV (Swine Influenza Virus) and PRRSV (Porcine Reproductive and Respiratory Syndrome Virus)

A technology of RT-PCR and detection kit, which is applied in the field of porcine respiratory virus nucleic acid detection

Active Publication Date: 2020-01-31
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Generally speaking, compared with commonly used diagnostic methods such as electron microscope observation, virus isolation, immunohistochemistry, ELISA serological detection, and fluorescent antibody technology, the multipl...

Method used

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  • Multiplex RT-PCR (Reverse Transcription-Polymerase Chain Reaction) quick detection kit and primer for SIV (Swine Influenza Virus) and PRRSV (Porcine Reproductive and Respiratory Syndrome Virus)
  • Multiplex RT-PCR (Reverse Transcription-Polymerase Chain Reaction) quick detection kit and primer for SIV (Swine Influenza Virus) and PRRSV (Porcine Reproductive and Respiratory Syndrome Virus)
  • Multiplex RT-PCR (Reverse Transcription-Polymerase Chain Reaction) quick detection kit and primer for SIV (Swine Influenza Virus) and PRRSV (Porcine Reproductive and Respiratory Syndrome Virus)

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1, kit assembly and use of the present invention

[0033] For ease of use, the reagents used in the present invention are assembled into a four-type virus multiplex RT-PCR rapid detection kit, which is mainly composed of PCR standard products, positive control products, negative control products, primers and boxes. Corresponding container holes are provided in the box body for placing reagents respectively.

[0034] As shown in Table 1, the primers include four pairs of PCR primers (primers synthesized by Shanghai Jieli Biotechnology Co., Ltd., and the amount of synthesis is 1OD per tube of primers), which are respectively aimed at H1-SIV, H3-SIV, NA-PRRSV and EU- PRRSV four types of virus. All upstream primers of the four pairs of PCR primers are packaged in the same tube, and all downstream primers are packaged in the same tube. In the 50 μL total reaction system, dilute the primers according to the molar ratio of primers 3:3:3:3:5:5:3:3, where the initia...

Embodiment 2

[0049] Embodiment 2, the present invention detects the specificity experiment of four types of viruses

[0050] According to the multiplex PCR reaction system, DreamTaq Hot Start Green PCR Master Mix (ThermoScientific) 25 μL, upstream primer mix 8 μL, downstream primer mix 8 μL, ddH 2 O 8 μL, add 1 μL of sample cDNA. Templates include 1:NA-PRRSV; 2:EU-PRRSV; 3:H1-SIV; 4:H3-SIV; 5:ASFV; 6:PRV; 7:PCV; 8:PRoV; 9:PEVG; 10:PAstV; 11: CSFV; 12: ultrapure water negative control. The reaction was carried out on a Life ProFlex PCR amplification instrument. The reaction program was as follows: pre-denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 54.5°C for 30 s, extension at 72°C for 1 min, and finally extension at 72°C for 5 min. Take 10 μL of the PCR product, spot it in the well of a 2% agar gel electrophoresis plate, and run the electrophoresis at 140V for about 20 minutes. After UV imaging on the gel imager, take pictures and jud...

Embodiment 3

[0051] Embodiment 3, the sensitivity experiment that the present invention detects four types of viruses

[0052] Multiplex PCR sensitivity detection reaction system: DreamTaq Hot Start Green PCR Master Mix (Thermo Scientific) 25 μL, upstream primer mix 8 μL, downstream primer mix 8 μL, ddH 2O 5 μL, add 4 μL 10-fold serially diluted H1-SIV, H3-SIV, NA-PRRSV and EU-PRRSV positive plasmid mixture (9.86×10 9 ~9.86×10 0 copies / μL) template. The reaction was carried out in a Life ProFlex PCR amplification instrument. The reaction program was as follows: pre-denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 54.5°C for 30 s, extension at 72°C for 30 s, and finally extension at 72°C for 5 min. Take 10 μL of the PCR product and detect it by 2% agarose gel electrophoresis.

[0053] Conventional single-plex PCR sensitivity detection reaction system: DreamTaq Hot Start Green PCR Master Mix (Thermo Scientific) 25 μL, upstream primer 2 μ...

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Abstract

The invention discloses a multiplex RT-PCR (Reverse Transcription-Polymerase Chain Reaction) quick detection primer for H1 and H3 subtype SIVs (Swine Influenza Virus) and North America and Europe PRRSVs (Porcine Reproductive and Respiratory Syndrome Virus). The primer comprises four pairs of PCR primers which independently aim at four types of viruses, wherein four pairs of PCR primers are primers1 to 8 and independently have base sequences of sequence tables SEQ. ID. NO.1 to SEQ. ID. NO.8. Hereby, the inventor also researches a corresponding quick detection method and a kit thereof, and a detectable quantity for a recombinant plasmid standard substance can be lowered to 9.86*10<0>copies/[mu]L. The invention has the characteristics of high specificity, good sensitivity, short time consumption and low cost, provides a quick and convenient tool for detecting swinery mixed infection, lays a solid foundation for clinic quick authentication and detection and laboratory epidemiological investigation, is favorable for pig industry to formulate a disease prevention and control scheme in time, and reduces a swinery death rate and economic loss.

Description

technical field [0001] The invention belongs to the technical field of porcine respiratory virus nucleic acid detection, in particular to multiple RT-PCR rapid detection kits and primers for H1 and H3 subtype SIV (swine influenza virus) and American and European PRRSV (porcine reproductive and respiratory syndrome virus) . Background technique [0002] Pig farming has become a relatively important pillar industry in my country's agricultural field. In recent years, with the transformation of feeding methods towards intensification and scale, the occurrence of multiple pathogenic mixed infections in pig herds has intensified. In particular, the pathological changes and clinical symptoms caused by certain types of pig diseases are not typical, and there are even similarities in epidemiology, which makes it difficult for us to distinguish such diseases based on subjective feelings in our daily work. It is even more impossible to visually confirm that the diseased pigs are infe...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2537/143C12Q2521/107
Inventor 陈樱余良政韦祖樟王豪林霜任同伟
Owner GUANGXI UNIV
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