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RT-CPA detection primer and kit for swine transmissible gastroenteritis virus

A technology of RT-CPA and detection primers, which is applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc. It can solve the problems of high detection cost, long operation time, discomfort and rapid detection, etc., and achieve detection The effect of low cost, high sensitivity, and simple determination of detection results

Active Publication Date: 2022-03-18
GANSU ANIMAL HUSBANDRY & VETERINARY MEDICINE INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the traditional virus isolation and identification method can be used as the final diagnosis basis for the diagnosis of the disease, it takes a long time to operate and the process is complicated, which is not suitable for rapid detection requirements.
Technologies such as IEM, nucleic acid probes, and PCR have the advantages of high sensitivity and specificity, but they require relatively high test conditions, require expensive equipment and professional technicians, and the operation is relatively complicated, and the detection cost is high. Only professional detection Laboratories can only be equipped, and it is difficult for grassroots units to carry out

Method used

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  • RT-CPA detection primer and kit for swine transmissible gastroenteritis virus
  • RT-CPA detection primer and kit for swine transmissible gastroenteritis virus
  • RT-CPA detection primer and kit for swine transmissible gastroenteritis virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] A porcine transmissible gastroenteritis virus RT-CPA detection kit, comprising: ReactionBuffer; BstDNA polymerase (NEB); AMV reverse transcriptase (Promega); dNTPs; MgSO 4 ; Betaine (Sigma); cross primers SD-CPF, SD-CPR; stripping primers SD-F, SD-R; detection primers SD-DF, SD-DR; nucleic acid detection test strips.

[0064] SD-CPF (SEQ ID No.1): 5'-GTAAAAGCTAGGTAGTAACACTAATA GTCACGTTAATAACATT-3';

[0065] SD-CPR (SEQ ID No.2): 5'-TAATAGTCACGTTAATAACATTGTAAA AGCTAGGTAGTAACAC-3';

[0066] SD-F (SEQ ID No.3): 5'-CAAGTTGAAAACACAGCT-3';

[0067] SD-R (SEQ ID No.4): 5'-CATACCAAGACCAATAGTT-3';

[0068] SD-DF (SEQ ID No.5): 5'-(Biotin)AAATGCTCTCAAATTACTG-3';

[0069] SD-DR (SEQ ID No. 6): 5'-(Fitc)AACACTCTTATTGACAAGAC-3'.

Embodiment 2

[0071] Optimization of the concentration and ratio of different primers in a porcine transmissible gastroenteritis virus RT-CPA detection kit

[0072] 1. Processing of samples to be tested:

[0073] Aseptically collect samples, add Hank , The s solution was fully ground with a grinder, added with double antibody, freeze-thawed repeatedly 3 to 5 times, then centrifuged at 2000r / min for 10 to 20min, absorbed the supernatant, and stored at -20°C for later use.

[0074] 2. The reaction system of RT-CPA amplification

[0075] Use 20 μL reaction system, add 1 μL of viral RNA template, ReactionBuffer 2.0 μL, SD-CPF / R, SD-F / R, SD-DF / R, MgSO in the PCR tube 4 1.2mM, dNTPs1.0mM, Betaine1.6M, BstDNA polymerase 8U, AMV reverse transcriptase 5U, nuclease-free water to make up to 20μL. Among them, cross primers (SD-CPF, SD-CPR), stripping primers (SD-F, SD-R), and detection primers (SD-DF, SD-DR) use different concentration ratios:

[0076] 1) SD-CPF / R: 1.0 μM each, SD-F / R: 0.5 μM each,...

Embodiment 3

[0087]Different concentrations of MgSO in a porcine transmissible gastroenteritis virus RT-CPA detection kit 4 Optimization

[0088] 1. Processing of samples to be tested:

[0089] The preparation method is the same as in Example 2.

[0090] 2. The reaction system of RT-CPA amplification:

[0091] Using a 20 μL reaction system, add 1 μL of viral RNA template, 2.0 μL of Reaction Buffer, 1.0 μM of SD-CPF / R, 0.6 μM of SD-F / R, 0.4 μM of SD-DF / R, and MgSO in a PCR tube. 4 , dNTPs1.0mM, Betaine1.6M, BstDNA polymerase 8U, AMV reverse transcriptase 5U, nuclease-free water to make up to 20μL.

[0092] where MgSO 4 The concentrations are 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8mM, respectively.

[0093] 3. Reaction conditions for RT-CPA amplification:

[0094] The reaction tube was incubated at 63°C for 60min and then inactivated at 85°C for 2min.

[0095] 4. Judgment of test results:

[0096] Take 10 μL of the amplification product, electrophoresis with 1.0% agarose gel, and place it ...

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Abstract

The invention provides an RT-CPA detection primer and kit for swine transmissible gastroenteritis virus, and belongs to the technical field of swine virus disease virus detection, the RT-CPA detection primer comprises SD-CPF, SD-CPR, SD-F, SD-R, SD-DF and SD-DR; and the nucleotide sequences are shown as SEQ ID No.1-6 in sequence. The primer provided by the invention is good in specificity and can effectively detect the swine transmissible gastroenteritis virus; the method is rapid and efficient, the detection time is about 90 min, and the method is very suitable for on-site rapid detection of TGEV; the method does not depend on expensive instruments and equipment and professional detection personnel, and the detection cost is low; the detection result is simple, objective and visual to judge; the sensitivity is high, and 10 copies / mu L of TGEV can be detected.

Description

technical field [0001] The invention belongs to the technical field of porcine viral disease detection viroids, and in particular relates to an RT-CPA detection primer and a kit for porcine transmissible gastroenteritis virus. Background technique [0002] Porcine transmissible gastroenteritis was first reported by Doyle and Hutchings in the United States in 1946, and the disease occurred and spread in many countries and regions one after another. The disease has been reported in our country since the late 1950s. Since 2001, porcine transmissible gastroenteritis has occurred and spread in some areas of our country from time to time, causing certain harm to the pig industry. In recent years, diarrhea in nursery piglets caused by infectious gastroenteritis has become more common, seriously affecting the growth and health of piglets, especially a large number of piglets aged 1 to 4 weeks died, causing huge economic losses to farmers. Porcine transmissible gastroenteritis (TGE)...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2521/107C12Q2521/101Y02A50/30
Inventor 陈伯祥赵子惠成伟伟李元新杨明
Owner GANSU ANIMAL HUSBANDRY & VETERINARY MEDICINE INST
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