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Primer set for detecting bovine norovirus and bovine coronavirus and double RT-PCR method

A coronavirus and primer set technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., to achieve good repeatability, strong sensitivity, and accurate detection results

Active Publication Date: 2018-11-30
INST OF ANIMAL HUSBANDRY & VETERINARY MEDICINE HENAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows researchers to identify different genes that control or regulate certain biologically important processes such as cell growth and tissue development during embryogenesis. These techniques help scientists study how these organisms work together more effectively than other methods like traditional experiments.

Problems solved by technology

This patented technical problem addressed in this patents relates to improving upon traditional tests such as enzyme immunoassay or Western Blot analysis for accurate identification between different types of viruses like cowpoxyneckvirus and porcinervis sinis due to their similarity across generational origins.

Method used

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  • Primer set for detecting bovine norovirus and bovine coronavirus and double RT-PCR method
  • Primer set for detecting bovine norovirus and bovine coronavirus and double RT-PCR method
  • Primer set for detecting bovine norovirus and bovine coronavirus and double RT-PCR method

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Embodiment 1

[0034] Embodiment 1: the construction of the double RT-PCR method that detects bovine norovirus and bovine coronavirus

[0035] 1. Design and synthesis of primer sets for detecting bovine norovirus and bovine coronavirus

[0036]The sequences of bovine norovirus and bovine coronavirus announced in GenBank were carried out homologous comparison analysis, and the ORF1 gene of bovine norovirus (accession number in Genbank was NC_029645) and bovine coronavirus (accession number in Genbank were NC_029645) were selected respectively. A segment of the ORF1ab gene of U00735) is the target sequence amplified by each virus, and Premier 5.0 is used to design primers for the above target sequence. The designed amplification primers of the two viruses were analyzed by DNAstar to avoid the formation of stable primer dimers; at the same time, the homology or complementarity of the amplified sequences of each pair of primers was analyzed. Analysis, to avoid higher homology or complementarity...

Embodiment 2

[0052] Embodiment 2: specificity experiment

[0053] Utilize the double RT-PCR method of detecting bovine norovirus and bovine coronavirus that the embodiment of the present invention 1 constructs to bovine norovirus, bovine coronavirus, BRV (bovine rotavirus), BAstV (bovine astrovirus), BVDV ( Bovine viral diarrhea virus), BKV (bovine crest virus), BToV (bovine toroidal virus) were detected, and a negative control (using ddH 2 (0), verify the specificity of the double RT-PCR method that the present invention establishes, and specific method is as follows:

[0054] Extract respectively BRV (bovine rotavirus), BAstV (bovine astrovirus), BVDV (bovine viral diarrhea virus), BKV (bovine crest virus), BToV (bovine ring strain) according to the method described in step 2 in Example 1. Virus) RNA, reverse transcription respectively to obtain each viral cDNA;

[0055] Using the mixed cDNA of bovine norovirus and bovine coronavirus, the cDNA of bovine norovirus, the cDNA of bovine co...

Embodiment 3

[0056] Embodiment 3: Sensitivity experiment

[0057] It is 23.8ng / μL to measure the DNA concentration of bovine norovirus by the ultra-micro nucleic acid protein analyzer, and the DNA concentration of bovine coronavirus is 54.45ng / μL, according to the formula (6.02×10 23 copy number / mole)×(nucleic acid concentration ng / μL×10 -9 ) / (nucleic acid length×660)=standard plasmid copy number copies / μL, the calculated DNA copy number of bovine norovirus is 6.71×10 9 copies / μL, the copy number of bovine coronavirus DNA is 1.38×10 10 copies / μL; 0.5 μL of bovine coronavirus DNA and 0.5 μL of bovine norovirus DNA were added to 4 μL of sterile double-distilled water at the same time, and mixed evenly to obtain a mixture of bovine coronavirus DNA and bovine norovirus DNA (bovine norovirus DNA) In the mixture of coronavirus DNA and bovine norovirus DNA, the copy number of bovine norovirus DNA is 6.71×10 8 copies / μL, the copy number of bovine coronavirus DNA is 1.38×10 9 copies / μL); then t...

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Abstract

The present invention discloses a primer set for detecting bovine nornovirus and bovine coronavirus, the primer set comprises a primer for detecting the bovine nornovirus and a primer for detecting the bovine coronavirus, and the nucleotide sequences of the primer set are as shown in SEQ ID NO. 1-SEQ ID NO. 4. The invention also discloses a double RT-PCR method for detecting the bovine norovirus and the bovine coronavirus, and the steps are as follows: (1) synthesizing the primer set for detecting the bovine norovirus and the bovine coronavirus; (2) extracting RNA in a to-be-tested sample, andperforming inverse transcription on the extracted RNA as a template to obtain cDNA; (3) performing PCR amplification reaction on the cDNA obtained in the step (2) as a template by use of the primer set synthesized in the step (1); and (4) analyzing a PCR amplification product. The double RT-PCR method is simple, rapid, economical and efficient, and can greatly save detection time and improve detection efficiency.

Description

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Claims

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Application Information

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Owner INST OF ANIMAL HUSBANDRY & VETERINARY MEDICINE HENAN ACAD OF AGRI SCI
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