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Detection method for detecting bovine coronavirus

A coronavirus, a technology to be detected, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, and resistance to vector-borne diseases, etc., can solve the problem of qualitative and quantitative detection of untraceable mutations, complex processes, and low accuracy, etc. problems, to achieve high specificity, high sensitivity, and high precision

Pending Publication Date: 2021-06-15
XINJIANG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

ddPCR overcomes the shortcomings of traditional PCR and fluorescent quantitative PCR systems, such as difficulty in accurately determining gene copy number, inability to qualitatively and quantitatively detect trace mutations, low accuracy, high cost, limited throughput, and complicated process. It has high-throughput detection, accurate Higher degree, more digitization, saving time and so on

Method used

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  • Detection method for detecting bovine coronavirus
  • Detection method for detecting bovine coronavirus
  • Detection method for detecting bovine coronavirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The design and synthesis of embodiment 1, BCoVddPCR detection primer and probe

[0029] Referring to the whole genome sequence of BCoV recorded in GenBank in NCBI, the N gene of BCoV was selected as the target gene, and a pair of specific primers and probes were designed using Primer premier 5.0 software, which were synthesized by Shanghai Bioengineering Co., Ltd. The specific sequence is as follows:

[0030] Upstream primer: 5'-GATCTACTTCACGCGCATCC-3' (SEQ ID NO.2);

[0031] Downstream primer: 5'-GTGGCTTAGTGGCATCCTTG-3' (SEQ ID NO.3);

[0032] Probe: 5'-FAM-TGGCTCTACTGCGCGATCCTGCA-BHQ1-3' (SEQ ID NO.4);

Embodiment 2

[0033] The preparation of embodiment 2, BCoV standard positive plasmid

[0034] Total virus RNA was extracted by Trizol method, and reverse-transcribed into cDNA using HiFiScript cDNA Synthesis Kit reagent (Kangwei Century Biotechnology Co., Ltd.). Use 2× Hieff Canace Gold PCR Master Mix high-fidelity enzyme premix (Shanghai Yisheng Biological Co., Ltd.) to amplify the BCoV N gene and perform 1% agarose gel electrophoresis, use The Gel Extraction Kit agarose gel recovery kit (Shanghai Yisheng Biological Co., Ltd.) was used to recover the target gene. use Easy Vector System I Vector Kit (Promega) ligated the recovered product, and transformed the ligated product into Escherichia coli DH5α competent cells by heat shock. Use 2x Taq PCR Mix reagent (Tiangen Biochemical Technology (Beijing) Co., Ltd.) for bacterial liquid PCR identification, and use positive bacterial liquid Plasmid Mini Kit (Shanghai Yisheng Biological Co., Ltd.) was used to extract the plasmid, and the extr...

Embodiment 3

[0035] Embodiment 3, the establishment of BCoV TaqMan fluorescent quantitative PCR detection method

[0036] Using the primer set and probe described in Example 1, and using the standard positive plasmid prepared in Example 2 as a template, configure a qPCR 20 μL reaction system as follows:

[0037] SuperRealPreMix (Probe) 10μL, upstream primer (10pmol / L) 0.8μL, downstream primer (10pmol / L) 0.8μL, probe (10pmol / L) 0.9μL, 50×ROX Reference Dye 0.2μL, RNase-free waste 5.3μL , template 2 μL; qPCR reaction conditions are: 95 ° C pre-denaturation 15 min; 95 ° C denaturation 10 s, 60 ° C annealing 30 s, a total of 40 cycles; 1.0 × 10 10 ~1.0×10 0 Copies / μL standard positive plasmid was used as a template for qPCR reaction, and a negative control was set up. The obtained amplification curve was as follows: figure 1 As shown, the template concentration from left to right is 1.0×10 10 ~1.0×10 0 copies / μL, the result shows that the minimum detection limit of this method is 10copies / μ...

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Abstract

The invention relates to a detection method for detecting bovine coronavirus based on a microdroplet type digital PCR technology. The detection method comprises a target fragment such as SEQ ID NO.1, a specific primer pair and a probe. A bovine coronavirus microdroplet type digital PCR detection kit has the sensitivity of 1copies / [mu] L, and can only be used for specifically detecting bovine coronavirus. When the kit is used for detecting clinically collected bovine diarrhea samples, the result shows that the positive rate of the kit is higher than that of existing qPCR, detection of trace viruses can be achieved, and nucleic acid can be absolutely quantified without a standard curve. The detection method can realize high-specificity, high-sensitivity and absolute quantitative detection on the bovine coronavirus, and has the advantages of high-throughput detection, higher accuracy, higher digitization, time saving and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method and application of primers and probes for detecting bovine coronavirus based on droplet digital PCR technology and a kit thereof. Background technique [0002] Bovine coronavirus (BCoV) belongs to Mandoviridae, Coronaviridae, Coronaviridae, and Betacoronavirus belongs to subgroup B. BCoV is a single-stranded, positive-strand RNA virus with a genome size of 27,000-32,000 nt, which is the largest genome among known RNA viruses. The genome of BCoV mainly encodes five proteins, nucleocapsid protein (N), spike protein (S), major envelope protein (M), minor envelope protein (E) and hemagglutinin esterase (HE). ), the nucleocapsid protein encoded by the N gene is highly conserved. BCoV mainly infects cattle, 1-week-old calves are most susceptible, and can cause persistent subclinical infection in adult cattle. Respiratory tract infection and fecal-oral in...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/701C12Q1/6851C12Q2563/159C12Q2563/107Y02A50/30
Inventor 姚刚付强马义诚任强林张杨马雪连苏战强陈卓夏瑞阳舒展李鑫李紫仟阿依努尔·木萨
Owner XINJIANG AGRI UNIV
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