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Multiple RT-PCR quick detection kit for five porcine enteroviruses and application thereof

A RT-PCR, detection kit technology, applied in recombinant DNA technology, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of long time, difficulty, low accuracy of serological methods, etc.

Inactive Publication Date: 2018-10-30
GUANGXI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] Studies have shown that the mixed infection of multiple viruses can cause diarrhea and disease in pigs, which seriously endangers the production of pigs, and it is difficult to distinguish based on clinical symptoms and epidemiology, so it is necessary to rely on laboratory testing techniques for differential diagnosis
Among them, electron microscope observation, virus isolation, immunohistochemistry and fluorescent antibody techniques are complex and difficult to operate; ELISA pathogen rapid detection kits and conventional RT-PCR methods are widely used, but the accuracy of serological methods is low , the commonly used single PCR method takes a long time

Method used

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  • Multiple RT-PCR quick detection kit for five porcine enteroviruses and application thereof
  • Multiple RT-PCR quick detection kit for five porcine enteroviruses and application thereof
  • Multiple RT-PCR quick detection kit for five porcine enteroviruses and application thereof

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Embodiment 1

[0034] Embodiment 1, kit assembly and use of the present invention

[0035] For the convenience of use, the reagents used in the present invention are assembled into five kinds of porcine enterovirus multiple RT-PCR rapid detection kits, which are 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.

[0036] The primers include five pairs of PCR primers (Table 1, primers synthesized by Shanghai Jieli Biotechnology Co., Ltd., the synthetic amount is 1OD per tube of primers), which are respectively aimed at five porcine enteroviruses: PEDV, TGEV, PDCoV, PRoV, and PKV. It has the base sequence of SEQ.ID.NO.1 to SEQ.ID.NO.10 in the sequence listing. All upstream primers among the five pairs of PCR primers are packaged in the same tube, and all downstream primers are packaged in the same tube.

[0037] Table 1, multiple PCR pr...

Embodiment 2

[0048] Embodiment 2, the present invention detects the specificity experiment of five kinds of viruses

[0049] According to the multiplex PCR reaction system, Green Taq Mix (Vazyme) 12.5 μL, upstream primer mix 0.5 μL, downstream primer mix 0.5 μL, ddH 2 O 8.5 μL, add 3 μL positive sample cDNA template respectively 1: PEDV; 2: TGEV; 3: PDCoV; 4: PRoV; 5: PKV; 6: PEDV, TGEV, PDCoV, PRoV and PKV; and PKV; 8: TGEV, PDCoV, PRoV and PKV; 9: PDCoV and PKV; 10: TGEV and PRoV; 11: PEDV and PDCoV; 12: PEDV and PKV; 13: PEDV, TGEV and PKV; 14: PDCoV, PRoV and PKV; 15: PEDV, PDCoV and PKV; 16: non-targeting template; 17: ultrapure water negative control. The reaction was carried out in a life ProFlex PCR amplification instrument. The reaction program was as follows: pre-denaturation at 94°C for 5 minutes, followed by 34 cycles of denaturation at 94°C for 40 seconds, annealing at 53°C for 40 seconds, extension at 72°C for 50 seconds, and finally extension at 72°C for 10 minutes. Take 1...

Embodiment 3

[0050] Embodiment 3, the present invention detects the sensitivity experiment of five kinds of viruses

[0051] Multiplex PCR sensitivity detection reaction system: Green Taq Mix (Vazyme) 12.5 μL, upstream primer mix 0.5 μL, downstream primer mix 0.5 μL, ddH 2 O 8.5 μL, add 3 μL 10-fold serially diluted PEDV, TGEV, PDCoV, PRoV and PKV positive plasmid mixture (7.56×10 7 ~7.56×10 0 copies / μL) template. The reaction was carried out in a lifeProFlex PCR amplification instrument. The reaction program was as follows: pre-denaturation at 94°C for 5 min, followed by 34 cycles of denaturation at 94°C for 40 s, annealing at 53°C for 40 s, extension at 72°C for 50 s, and finally extension at 72°C for 10 min. Take 10 μL of the PCR product and detect it by 2% agarose gel electrophoresis.

[0052] Conventional single-plex PCR sensitivity detection reaction system: Green Taq Mix (Vazyme) 12.5 μL, upstream primer mix 0.5 μL, downstream primer mix 0.5 μL, ddH 2 O 8.5 μL, add 3 μL PEDV, TG...

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Abstract

The invention discloses a multiple RT-PCR quick detection primer group for five porcine enteroviruses. The primer group comprises five pairs of PCR primers which respectively aim at the five porcine enteroviruses PEDV, TGEV, PDCoV, PRoV and PKV and respectively have base sequences shown in a sequence table from SEQ. ID. NO.1 to SEQ. ID. NO.10. According to this, the inventor further develops a corresponding quick detection kit. The primer group has the advantages of high specificity, high sensitivity, short time consumption and low cost when being applied in a same reaction tube to detect single or mixed infection of the five porcine enteroviruses at the same time. A quick, simple and convenient tool is provided for detection of swinery mixed infection, a solid foundation is laid for clinical quick identification and detection and lab epidemiological investigation, timely formulating of treatment plans in porcine industry is facilitated, and swinery death rate and economic loss are reduced.

Description

technical field [0001] The invention belongs to the technical field of porcine enterovirus nucleic acid detection, in particular to a multiple RT-PCR rapid detection kit for five porcine enteroviruses and an application thereof. Background technique [0002] In recent years, with the intensive and large-scale development of pig farming, the mixed infection of multiple pathogens in pig herds has become more and more common. Porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine Deltacoronavirus (PDCoV) and porcine rotavirus (PRoV) are the main pathogens of viral enteric disease in suckling piglets due to their pathological The similarities in clinical symptoms and epidemiology of the changes make it difficult to distinguish these diseases. At the same time, according to the inventor's monitoring of porcine ridge virus (PKV), it is found that its detection rate in PEDV-positive pigs is extremely high, and it is speculated that it i...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701C12Q2537/143C12Q2521/107
Inventor 欧阳康钟莲黄伟坚王若木陆颖吴雨殊刘雪婷
Owner GUANGXI UNIV
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