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One-step RT-PCR diagnostic kit for identifying porcine epidemic diarrhea virus vaccine attenuated strain and/or prevalent strain

A porcine epidemic diarrhea and diagnostic kit technology, applied in the field of diagnosis, can solve the problems of easy degradation of template RNA, cumbersome operation, and long amplification reaction time, and achieve the effects of improving sensitivity, reducing reaction time, and good specificity

Pending Publication Date: 2017-06-13
SHANDONG BINZHOU ANIMAL SCI & VETERINARY MEDICINE ACADEMY
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  • Abstract
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  • Application Information

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Problems solved by technology

Li Changlong, Zhu Haixia and others have established the RT-PCR detection method for distinguishing porcine epidemic diarrhea virus vaccine strains from wild strains. Both are two-step RT-PCR detection methods, and there is no detection sensitivity for research. The operation is cumbersome and the amplification Long reaction time, easy degradation of template RNA

Method used

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  • One-step RT-PCR diagnostic kit for identifying porcine epidemic diarrhea virus vaccine attenuated strain and/or prevalent strain
  • One-step RT-PCR diagnostic kit for identifying porcine epidemic diarrhea virus vaccine attenuated strain and/or prevalent strain
  • One-step RT-PCR diagnostic kit for identifying porcine epidemic diarrhea virus vaccine attenuated strain and/or prevalent strain

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Effect test

Embodiment 1

[0027] Example 1 A one-step RT-PCR diagnostic kit for distinguishing porcine epidemic diarrhea virus vaccine attenuated strains and / or epidemic strains, which includes 20 μL RT-PCR one-step enzyme, 250 μL enzyme buffer, 300 μL RNase Freed H 2 O. 40 μL mixed primers, 20 μL positive control, 20 μL negative control, 80 μL DL2000 25 μL 6×Loading Buff, mixed primers include upstream primer P1: 5'-AGTCTGCCAACTTGTCTT-3', downstream primer P2: 5'-AGTAAAAGCAGACTAAAACAAAGCCT-3'. The negative control is sterilized DEPC water, and the positive control is the attenuated strain and / or circulating strain RNA of porcine epidemic diarrhea virus vaccine. The enzyme buffer is composed of dNTP Mixture and One Step EnhancerSolution, the concentration of the dNTP Mixture is 400 μmol / L, and the RT-PCR one-step enzyme is composed of PrimeScript Rtase and DNA Polymerase with a volume ratio of 1:1:1 Composed of RNase Inhibitor. The one-step RT-PCR reaction was carried out in a 25 μL system, 1 μL each ...

Embodiment 2

[0029]The collected feces samples and intestinal contents were made into 10% suspension with PBS (0.01 mol / L, pH 7.2), vortexed, centrifuged at 12 000 r / min for 10 minutes at 4°C, and taken clear for the extraction of viral nucleic acids. The extraction of viral RNA was carried out according to the instructions of AxyPrep Body Fluid Viral DNA / RNA Small Extraction Kit (Corning Life Sciences (Wujiang) Co., Ltd.), and the extracted RNA was stored at -70°C.

[0030] One-step RT-PCR amplification and condition optimization for a single virus.

[0031] The RT-PCR reaction was carried out in a 25 μL system, with 1 μL of P1 and P2 primers (10 μmol / L), 1 μL of PrimeScript 1step Enzyme Mix, 12.5 μL of 2×1 step Buffer, and 1 μL of template (porcine epidemic diarrhea virus vaccine attenuated strain or epidemic strain RNA), plus RNase Free dH 2 From 0 to 25 μL, the reaction program was: 50°C for 20 min; 95°C for 3 min; 94°C for 20 s; annealing temperature 50°C for 20 s; 72°C for 20 s; 40...

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Abstract

The invention provides a one-step RT-PCR diagnostic kit for identifying a porcine epidemic diarrhea virus vaccine attenuated strain and / or prevalent strain. The one-step RT-PCR diagnostic kit comprises 20muL of RT-PCR one-step enzyme, 250muL of a enzyme buffer solution, 300muL of RNase Free dH2O, 40muL of a mixed primer, 20muL of positive control, 20muL of negative control, 80muL of DL2000 and 25muL of 6*Loading Buffer. Compared with the prior art, the kit has the advantages that the operation is simple, inverse transcription and PCR amplification are completed in one step, the reaction time is shortened, degradation of a template RNA is avoided and the detection sensitivity is improved. The amplification results of the primer on porcine transmissible gastroenteritis virus, group A swine rotavirus, classical swine fever virus, porcine reproductive and respiratory syndrome virus and porcine teschovirus are all negative. According to the method, 1pg porcine epidemic diarrhea virus vaccine attenuated strain and 1pg prevalent strain can be detected to the minimum extent; the kit can be widely applied to basic-level detection mechanisms and is of great significance in early differential diagnosis, control and purification of epidemic diarrhea virus diseases on a pig farm.

Description

technical field [0001] The patent of the invention relates to the diagnostic technology in the technical field of veterinary biology, specifically a one-step RT-PCR diagnostic kit for identifying attenuated strains and / or endemic strains of porcine epidemic diarrhea virus vaccines. Background technique [0002] Diarrhea is an important disease that currently threatens the pig industry. There are many reasons for pig diarrhea, including nutritional, bacterial, viral and parasitic factors. Among them, the viral factor is a very important factor. In recent years, the most important virus causing porcine diarrhea is porcine epidemic diarrhea virus (PEDV), which can cause porcine epidemic diarrhea (PED) in pigs. PED is a disease of pigs. It is a highly contagious intestinal infectious disease characterized by watery diarrhea, vomiting, dehydration and loss of appetite. In other seasons, pigs of all ages can be infected, and the incidence of infection in suckling piglets, weaned ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/686C12Q1/701C12Q2521/107C12Q2565/125
Inventor 于新友李峰李天芝沈志强吴家强孙文博
Owner SHANDONG BINZHOU ANIMAL SCI & VETERINARY MEDICINE ACADEMY
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