Bovine rotavirus, coronavirus, and viral diarrhea virus multi-connected RT-PCR detection method

A technology of RT-PCR and bovine viral diarrhea, which is applied in the field of pathogenic virus detection, can solve the problems of large reagent consumables and time consumption, large number of tested samples and difficulties in differential diagnosis, and achieves time-saving and high-efficiency. RT-PCR identification and diagnosis of viral diseases, the effect of accurate results

Active Publication Date: 2019-08-02
INNER MONGOLIA UNIV FOR THE NATITIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The most rapid and effective detection method in the prior art is RT-PCR, and the specific technology used is the FastKing one-step RT-PCR kit, but this method has certain defects. According to the RT-PCR system provided by the kit instruction manual, only Add a pair of specific primers for a specificity check
For the differential diagnosis of these three diseases or when the number of samples to be tested is large, a large amount of reagents, consumables and time will be consumed when there are many detection items, which will also bring corresponding difficulties to the subsequent agarose gel electrophoresis experiment.

Method used

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  • Bovine rotavirus, coronavirus, and viral diarrhea virus multi-connected RT-PCR detection method
  • Bovine rotavirus, coronavirus, and viral diarrhea virus multi-connected RT-PCR detection method
  • Bovine rotavirus, coronavirus, and viral diarrhea virus multi-connected RT-PCR detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] 1. Material preparation:

[0019] (1) Samples to be tested: 8 tubes of bovine anticoagulant blood, and RNA was extracted by conventional methods for later use;

[0020] (2) Specific upstream primers and specific upstream primers: the specific upstream primers are a mixture of three upstream primers, bovine rotavirus upstream primers, bovine coronavirus upstream primers, and bovine viral diarrhea virus upstream primers, and specific downstream primers. The primer is a mixture of 10 μM bovine rotavirus downstream primers, bovine coronavirus downstream primers, and bovine viral diarrhea virus downstream primers; The primer sequence is 5′-TTACACAGGTAGGGGTTCTATTGCC-3′; the upstream primer sequence of bovine rotavirus is 5′-ATGTCTCAGTATTGACGTGACGAGTCT-3′, the downstream primer sequence of bovine rotavirus is 5′-CTACAATCGATCAGTTGCATTGCG-3′; the upstream primer sequence of bovine viral diarrhea It is 5'-ATGGAGTTGATCACAAATGAACTTTTATACA-3', and the downstream primer sequence of ...

Embodiment 2

[0032] 1. Material preparation:

[0033] (1) Samples to be tested: 8 tubes of cow feces with diarrhea, and RNA was extracted by conventional methods for later use;

[0034] (2) Specific upstream primer and specific upstream primer: same as Example 1;

[0035] (3) Reagents used for RT-PCR: 2×FastKing One Step RT-PCR MasterMix, 25×RT-PCEnzyme Mix, RNase-Free ddH2O;

[0036] (4) Electrophoresis gel: 1.5% agarose gel.

[0037] 2. a bovine rotavirus, coronavirus, viral diarrhea virus multiplex RT-PCR detection method, comprising the following steps:

[0038] (1) Use the RNA of the sample to be detected as the RNA template, and use the specific upstream primer and the specific downstream primer as primers to perform RT-PCR amplification to obtain the amplified product; wherein the RT-PCR amplification system and the RT-PCR amplification Condition: with embodiment 1;

[0039] (2) Afterwards, the amplified product is detected by 1.5% agarose gel electrophoresis and the result is r...

Embodiment 3

[0044] 1. Material preparation:

[0045] (1) Samples to be tested: 5 parts of lymph nodes, spleen and intestinal hemorrhage in diarrhea cattle organs, and RNA was extracted by conventional methods for later use;

[0046] (2) Specific upstream primer and specific upstream primer: same as Example 1;

[0047] (3) Reagents used for RT-PCR: 2×FastKing One Step RT-PCR MasterMix, 25×RT-PCEnzyme Mix, RNase-Free ddH2O;

[0048] (4) Electrophoresis gel: 1.5% agarose gel.

[0049] 2. a bovine rotavirus, coronavirus, viral diarrhea virus multiplex RT-PCR detection method, comprising the following steps:

[0050] (1) Use the RNA of the sample to be detected as the RNA template, and use the specific upstream primer and the specific downstream primer as primers to perform RT-PCR amplification to obtain the amplified product; wherein the RT-PCR amplification system and the RT-PCR amplification Condition: with embodiment 1;

[0051] (2) Afterwards, the amplified product is detected by 1.5%...

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Abstract

The invention discloses a bovine rotavirus, coronavirus, and viral diarrhea virus multi-connected RT-PCR detection method, which is characterized in that according to the detection method, RNA of a to-be-detected sample is taken as an RNA template, and a specificity upstream primer and a specificity downstream primer designed according to hereditary characteristics of the bovine rotavirus, coronavirus, and viral diarrhea virus are taken as a primer to perform RT-PCR proliferation to obtain a proliferation product. Then, electrophoresis detection is performed on the proliferation product with 1.5% agarose gel, and a result is recorded; according to the results of the electrophoresis detection, whether the to-be-detected sample contains the bovine rotavirus, coronavirus, and viral diarrhea virus is determined. The bovine rotavirus, coronavirus, and viral diarrhea virus detection method achieves has the beneficial effect of accurately, rapidly and efficiently distinguishing and diagnosingthe three viral disease with RT-PCR.

Description

technical field [0001] The invention belongs to the technical field of pathogenic virus detection, and in particular relates to a multiplex RT-PCR detection method for bovine rotavirus, coronavirus and viral diarrhea virus. Background technique [0002] Bovine rotavirus disease is an acute gastrointestinal infectious disease of various young animals and infants caused by rotavirus, characterized by mental fatigue, anorexia, vomiting, diarrhea, and dehydration. The pathogen of this disease is rotavirus, and the virus particles are round and wheel-shaped. The virus has strong resistance to external factors, and is still infectious after half a year in feces and milk that does not contain antibodies. The feces of patients and latently infected persons contain a large amount of rotavirus, which can be transmitted to susceptible humans and animals through the digestive tract. There is a certain cross-infection effect between humans and various animals. As long as the virus pers...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2600/16C12Q2537/143C12Q2521/107Y02A50/30
Inventor 史量全刘锴马德慧郭双殷杰王永强赵志炎
Owner INNER MONGOLIA UNIV FOR THE NATITIES
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