A primer set and dual RT-PCR method for the detection of bovine norovirus and bovine coronavirus

A coronavirus and primer set technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., to achieve the effect of improving detection efficiency, accurate detection results, and good repeatability

Active Publication Date: 2021-12-10
INST OF ANIMAL HUSBANDRY & VETERINARY MEDICINE HENAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In 2017, bovine norovirus was detected for the first time in China, and the epidemiological investigation of bovine norovirus in my country and the co-infection with other pathogens have not been reported in China

Method used

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  • A primer set and dual RT-PCR method for the detection of bovine norovirus and bovine coronavirus
  • A primer set and dual RT-PCR method for the detection of bovine norovirus and bovine coronavirus
  • A primer set and dual RT-PCR method for the detection of bovine norovirus and bovine coronavirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Construction of a double RT-PCR method for the detection of bovine norovirus and bovine coronavirus

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

[0036]The sequences of bovine norovirus and bovine coronavirus published in GenBank were compared and analyzed, and the ORF1 gene of bovine norovirus (the accession number in Genbank was NC_029645) and the bovine coronavirus (the 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 Primer 5.0 is used to design primers for the above target sequence. The amplification primers of the two designed 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 high homology or complementarity betwee...

Embodiment 2

[0052] Example 2: Specificity Experiment

[0053] Utilize the double RT-PCR method for detecting bovine norovirus and bovine coronavirus constructed in Example 1 of the present invention to detect bovine norovirus, bovine coronavirus, BRV (bovine rotavirus), BAstV (bovine astrovirus), BVDV ( Bovine viral diarrhea virus), BKV (bovine crista virus), BToV (bovine ring torovirus) were detected, and a negative control (with ddH 2 (2), verify the specificity of the double RT-PCR method established by the present invention, and the concrete method is as follows:

[0054] BRV (Bovine Rotavirus), BAstV (Bovine Astrovirus), BVDV (Bovine Viral Diarrhea Virus), BKV (Bovine Crest Virus), BToV (Bovine Cyclovirus) were extracted according to the method described in Step 2 in Example 1. virus) RNA, respectively reverse transcription to obtain each virus cDNA;

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

Embodiment 3

[0056] Example 3: Sensitivity experiment

[0057] The DNA concentration of bovine norovirus was 23.8 ng / μL, and the DNA concentration of bovine coronavirus was 54.45 ng / μL, according to the formula (6.02×10 23 copy number / mol)×(nucleic acid concentration ng / μL×10 -9 ) / (nucleic acid length × 660) = standard plasmid copy number copies / μL, the number of copies of bovine norovirus DNA is calculated to be 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 to obtain a mixture of bovine coronavirus DNA and bovine norovirus DNA (bovine coronavirus DNA and 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 the mixture of bovine noro...

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Abstract

The invention discloses a primer set for detecting bovine norovirus and bovine coronavirus. The primer set is composed of primers for detecting bovine norovirus and primers for detecting bovine coronavirus. The core of the primer set is The nucleotide sequence is shown in SEQ ID NO.1-SEQ ID NO.4. The invention also discloses a dual RT-PCR method for detecting bovine norovirus and bovine coronavirus, the steps are as follows: (1) synthesizing the primer set for detecting bovine norovirus and bovine coronavirus; (2) extracting the primers to be tested The RNA in the sample is reverse-transcribed using the extracted RNA as a template to obtain its cDNA; (3) using the cDNA prepared in step (2) as a template, the primer set synthesized in step (1) is used for PCR amplification reaction; (4) ) to analyze PCR amplification products. The double RT-PCR detection method is simple, fast, economical and efficient, can greatly save detection time and improve detection efficiency.

Description

technical field [0001] The invention belongs to the field of molecular biology detection, and in particular relates to a primer set and a double RT-PCR method for detecting bovine norovirus and bovine coronavirus. Background technique [0002] After calves are infected with Bovine Norovirus (BNoV), the clinical symptoms are mainly diarrhea, anorexia, dehydration, etc. Moreover, Bovine Norovirus is often mixed with Bovine Coronavirus (BCoV), and feeding Cattle industry brings huge economic losses. Both bovine norovirus and bovine coronavirus infection can cause disease in calves within 3 weeks of age, and the clinical symptoms and necropsy lesions caused by the virus are similar. misdiagnosed. Therefore, it is particularly necessary to establish a diagnostic method that can detect these two pathogens simultaneously. [0003] Traditional methods for diagnosing bovine norovirus and bovine coronavirus include electron microscopy, virus isolation, antigen-antibody ELISA detect...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701C12Q2537/143C12Q2521/107Y02A50/30
Inventor 师志海兰亚莉王文佳王璟施巧婷徐照学
Owner INST OF ANIMAL HUSBANDRY & VETERINARY MEDICINE HENAN ACAD OF AGRI SCI
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