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Multiplex pcr rapid diagnostic kit for five kinds of porcine diarrhea virus and its application

A technology of rapid diagnosis and kits, applied in the direction of resistance to vector-borne diseases, biochemical equipment and methods, and microbial measurement/inspection, etc., can solve problems such as difficult early detection, low sensitivity, misjudgment and misjudgment, and achieve Reduce pig mortality and economic loss, high specificity, low cost effect

Active Publication Date: 2020-09-29
杭州洪桥中科基因技术有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Conventional etiological and serological testing is difficult to distinguish and identify the above-mentioned diseases at the same time. At the same time, there are defects such as cumbersome operation, time-consuming and labor-intensive, and low sensitivity. Especially when multiple pathogens are infected, it is difficult to effectively detect early and easily produce Misjudgment Misjudgment

Method used

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  • Multiplex pcr rapid diagnostic kit for five kinds of porcine diarrhea virus and its application
  • Multiplex pcr rapid diagnostic kit for five kinds of porcine diarrhea virus and its application
  • Multiplex pcr rapid diagnostic kit for five kinds of porcine diarrhea virus and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1, kit structure

[0049] A multiplex real-time fluorescent quantitative PCR rapid diagnostic kit for five kinds of porcine viruses, consisting of multiplex PCR reaction mixture, virus primers, TGEV virus standard, GAR virus standard, GCR virus standard, PEDV virus, PCV2 virus standard, positive control Products, negative controls and boxes.

[0050] There are corresponding container holes in the box body, which are used to place quantitative PCR reaction solution tubes, five virus primer tubes, TGEV virus standard tubes, GAR virus standard tubes, GCR virus standard tubes, and PEDV virus standard tubes respectively. tube, PCV2 virus standard tube, positive control tube and negative control tube.

[0051] The multiplex PCR reaction mixture contains PCR reaction buffer (containing magnesium chloride and deoxyribonucleotide triphosphate mixture, etc.) and heat-resistant DNA polymerase; the viral primers are five kinds of viral primers, the upstream and downstre...

Embodiment 2

[0052] Embodiment 2, the present invention detects five kinds of virus-specific experiments

[0053] Step 1: Primer Synthesis

[0054] The primer sequences of 5 kinds of viruses designed in the present invention (see Table 1) were synthesized by China Shanghai Sangon Biological Company Technical Service Company, and the synthesis amount was 3 OD per primer.

[0055] Step 2: Specific detection of multiplex PCR system

[0056] According to the multiplex PCR reaction system, take 17 copies of the same PCR reaction master mix (i.e., 19 μL of multiplex PCR reaction mixture and 1 μl of 5 virus primer mixtures), and add 1 μL of positive sample cDNA / DNA template 1: GCR; 2: GAR; 3: TGEV; 4: PEDV; 5: PCV2; 6: GCR, GAR, TGEV, PEDV and PCV2; 7: GCR and TGEV; 8: GAR and PEDV; 9: TGEV and PCV2; 10: GCR and PCV2; 11: GCR, GAR, and TGEV; 12: TGEV, PEDV, and PCV2; 13: GCR, TGEV, and PCV2; 14: GCR, TGEV, PEDV, and PCV2; 15: GAR, TGEV, PEDV, and PCV2; 16: Deionized water negative control ;17:...

Embodiment 3

[0057] Embodiment 3, the present invention detects five kinds of virus sensitivity experiments

[0058] Step 1: Primer Synthesis

[0059] With the first step in embodiment 2.

[0060] Step 2: Sensitivity Testing

[0061] The multiplex PCR sensitivity detection system is as follows: 19 μL PCR reaction solution (multiple PCR reaction mixture), 10 μM TGEV, GAR, GCR, PEDV and PCV2 upstream and downstream primer mixture 1 μL, 1 μL 10-fold serial dilution of TGEV, GAR, GCR, PEDV and PCV2 plasmid standard (5.0×10 8 ~5.0×10 1 copies / μL) template, add ddH 2 O to the total volume of the reaction system was 25 μL; the reaction was carried out in a Bio-Rad S1000 PCR amplification instrument, and the reaction parameters were denaturation at 95°C for 3 minutes; 40 cycles of denaturation at 95°C for 20 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s; the last 72 ℃ 5min. Take 5 μL of PCR product, mix with 1 μL LoadingBuffer, and detect by 2% agarose electrophoresis.

[00...

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Abstract

The invention discloses a multi-PCR (polymerase chain reaction) rapid diagnostic kit for five porcine diarrhea viruses. The multi-PCR rapid diagnostic kit comprises five pairs of PCR primers, PCR reaction liquid, a PCR standard substance, a positive control substance and a negative control substance. The invention further discloses application of the kit in simultaneous specific detection of infection of the five porcine diarrhea viruses. The five porcine diarrhea viruses include TGEV (transmissible gastroenteritis virus), GAR (porcine group A rotaviruses), GCR (porcine group C rotaviruses), PEDV (porcine epidemic diarrhea virus) and PCV2 (porcine circovirus 2).

Description

technical field [0001] The invention belongs to the field of virus nucleic acid detection, in particular to a multiplex PCR rapid diagnostic kit for five kinds of porcine diarrhea virus, which can simultaneously detect porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV) and porcine transmissible gastroenteritis in the same reaction tube Transmissible gastroenteritis virus (TGEV), porcine rotavirus type A (Porcine group A rotaviruses, GAR), porcine rotavirus type C (Porcine group Crotaviruses, GCR), porcine circovirus type 2 (Porcine circovirus 2, PCV2 ) five kinds of porcine viruses, suitable for rapid detection of five kinds of porcine viruses in clinical and scientific research. Background technique [0002] In recent years, with the development of large-scale and intensive pig farming, the occurrence of multi-pathogen mixed infection and secondary infection in pig herds has become more and more common. Porcine epidemic diarrhea virus (PEDV), porcine t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2537/143C12Q2545/113Y02A50/30
Inventor 姜永厚刘高鹏
Owner 杭州洪桥中科基因技术有限公司
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