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
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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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