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Kit, primer and probe for simultaneously detecting bovine viral diarrhea virus, bovine rotavirus and bovine coronavirus

A technology for bovine viral diarrhea and bovine rotavirus, applied in the direction of using vectors to introduce foreign genetic material, methods based on microorganisms, microorganisms, etc., can solve the problems of insufficient sensitivity and specificity of PCR amplification technology, and achieve high sensitivity , easy operation, good repeatability

Active Publication Date: 2020-12-18
INNER MONGOLIA AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing PCR amplification technology still has the defects of insufficient sensitivity and specificity

Method used

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  • Kit, primer and probe for simultaneously detecting bovine viral diarrhea virus, bovine rotavirus and bovine coronavirus
  • Kit, primer and probe for simultaneously detecting bovine viral diarrhea virus, bovine rotavirus and bovine coronavirus
  • Kit, primer and probe for simultaneously detecting bovine viral diarrhea virus, bovine rotavirus and bovine coronavirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Kit

[0061] A detection kit, its composition is as shown in table 2:

[0062] Table 2

[0063]

[0064] Wherein, the mixed recombinant plasmids are recombinant plasmids respectively containing the nucleotide sequences shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.

[0065] The using method of the embodiment kit of the present invention is:

[0066] 1. Sample processing:

[0067] Carry out nucleic acid extraction (commercial kits are recommended for RNA extraction), and the negative control and samples are processed at the same time. It is recommended to test the extracted nucleic acid immediately, otherwise please store it below -20°C.

[0068] 2. Preparation of amplification reagents:

[0069] The triple fluorescent quantitative RT-PCR reaction solution system was prepared as follows for one person: 12.5 μL 2×One Step RT-PCR BufferⅢ+0.5 μL TaKaRa Ex Taq HS+0.5 μL PrimeScript RT Enzyme MixⅡ+0.24 μL BVDV upstream and downstream primers (50 μmol...

Embodiment 2

[0079] 1. Preparation of BVDV, BRV and BCoV triple fluorescent quantitative RT-PCR standards

[0080] Entrust BGI (Beijing) Co., Ltd. to synthesize recombinant plasmids pMD19T-BVDV, pMD19T-BRV and pMD19T-BCoV.

[0081] (1) Refer to the BVDV1 C24V isolate (AF091605.1), 1-SD1 isolate (M96751.1), LN-1 isolate (KT896495.1), SD-15 isolate (KR866116.1) published on GenBank , NADL isolate (AJ133738.1), SuwaCp isolate (KC853441.1) and BVDV2 11F011 isolate (KC963968.1), JZ05-1 isolate (GQ888686.2), XJ-04 isolate (FJ527854.1) , New York'93 isolate (AF502399.1), and the 5'UTR gene sequence of C413 isolate (AF002227.1), synthesize a BVDV gene sequence (SEQ ID NO: 10) with a full length of 148bp, and connect the sequence to On the pMD19-T Vector cloning vector, the recombinant plasmid pMD19T-BVDV was obtained.

[0082] (2) Refer to the BRV LS00005_OSU isolate (KR052766.1), WI79-9 isolate (GU565062.1), TOPORF11#1 isolate (KF729678.1), B10925 isolate (EF554125.1), PTRV published on GenBank...

Embodiment 3 3

[0088] Embodiment 3 Triple fluorescent quantitative RT-PCR reaction system

[0089] The reaction parameters of the triple fluorescent quantitative RT-PCR are shown in Table 3; the 25 μL reaction system of the triple fluorescent quantitative RT-PCR is shown in Table 4:

[0090] Table 4

[0091] components volume 2×One Step RT-PCR BufferⅢ 12.5μL TaKaRa Ex Taq HS 0.50 μL PrimeScript RT Enzyme MixⅡ 0.50 μL BVDV / BCoV upstream and downstream primers (50μmol / L) 0.24 μL BRV upstream and downstream primers (50μmol / L) 0.20 μL BVDV probe (10μmol / L) 1.2μL BRV probe (10μmol / L) 1.0 μL BCoV probe (10μmol / L) 0.7μL RNase-Free ddH 2 o

5.24μL template 2μL

[0092] Establishment of fluorescent quantitative RT-PCR standard curve:

[0093] Dilute the above concentration to 1 x 10 10 The copy number BVDV standard, BRV standard and BCoV standard were mixed in equal volumes, and the standard mixture was diluted...

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PUM

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Abstract

The invention relates to the field of virus detection, and particularly relates to a kit, a primer and a probe for simultaneously detecting bovine rotavirus, bovine rotavirus and bovine coronavirus. The nucleotide sequences of the primer and the probe in the kit are shown as SED ID NO: 1-SEQ ID NO: 9. The kit has the technical advantages of being easy and convenient to operate, high in specificity, high in sensitivity, good in repeatability and capable of simultaneously achieving qualitative detection and accurate quantification of BVDV, BRV and BCoV.

Description

technical field [0001] The invention relates to the field of virus detection, in particular to a kit, primers and probes for simultaneous detection of bovine rotavirus, bovine viral diarrhea virus and bovine coronavirus. Background technique [0002] Bovine viral diarrhea virus (BVDV) is the causative agent of bovine viral diarrhea (BVD), which mainly causes viral diarrhea in cattle. The main clinical manifestations are fever, mucosal erosion, ulcers, and leukopenia. , persistent infection, coughing, miscarriage or malformed fetuses in pregnant cows, etc. In addition to mainly infecting cattle, the virus can also cause infection in sheep, pigs, deer and a variety of wild animals. Huge economic loss. [0003] Bovine rotavirus (BRV), also known as calf diarrhea virus (Calf diarrheavirus), belongs to the genus Rotavirus (Rotavirus) of the family Reoviridea. Bovine rotavirus mainly infects calves less than 1 month old, and calves less than 1 week old are the most susceptible. ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/63C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6851C12N15/63C12Q2600/16C12Q2600/166C12Q2531/113C12Q2537/143C12Q2563/107C12Q2545/114
Inventor 徐晓静孙亚杰周伟光关平原希尼尼根温永俊张七斤张志丹
Owner INNER MONGOLIA AGRICULTURAL UNIVERSITY
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