Kit, primer and probe for detecting bovine rotavirus

A bovine rotavirus and kit technology, which is applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of insufficient sensitivity and specificity of PCR amplification technology, and achieve high Sensitive, highly specific effects

Active Publication Date: 2020-09-29
INNER MONGOLIA AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, 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 detecting bovine rotavirus
  • Kit, primer and probe for detecting bovine rotavirus
  • Kit, primer and probe for detecting bovine rotavirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Kit

[0054] A detection kit, its composition is as shown in table 2:

[0055] Table 2

[0056]

[0057]

[0058] The using method of the embodiment kit of the present invention is:

[0059] 1. Sample processing:

[0060] 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.

[0061] 2. Preparation of amplification reagents:

[0062] Take out the corresponding PCR reaction solution from the kit, melt and mix at room temperature, centrifuge at 2000rpm for 10s, prepare the template PCR reaction solution for 1 person as follows: 10μL 2×Reaction Buffer+0.5μL DNA Polymerase+0.5μL RT Enzyme Mix+1.0μL upstream and downstream primers (10μmol / L)+0.5μL probe+5.5μL RNase-Free ddH 2 O, the above prepared PCR master mix was dispensed into each PCR tube in a...

Embodiment 2

[0074] The preparation of embodiment 2 bovine rotavirus TaqMan quantitative RT-PCR standard items

[0075] Entrust Sangon Bioengineering (Shanghai) Co., Ltd. to synthesize the recombinant plasmid pMD19T-BRV. Mainly refer to BRV Sun9 isolate (AB-374146.1), KJ9-1 isolate (HM-988974.1), KJ19-2 isolate (MF-940662), KV0418 isolate (EU873011.1), KV0426 isolate published on GenBank (EU873012.1) and the VP6 full gene sequence of the M-1 isolate (HM235508.1), synthesize a BRV VP6 gene sequence with a full length of 207bp, specifically as shown in SEQ ID NO: 4, and connect the sequence to pMD TM 19-T Vector cloning vector to obtain recombinant plasmid pMD19T-BRV.

[0076] 1. Recombinant plasmid pMD19T-BRV identification of conventional PCR products

[0077] The synthesized plasmid DNA pMD19T-BRV was used as a template, and the upstream and downstream specific primers were used for PCR amplification. Such as figure 1 , a band with a size of about 207bp was obtained, indicating that t...

Embodiment 3

[0083] Embodiment 3 Quantitative RT-PCR reaction system and parameter optimization

[0084] BRV quantitative RT-PCR reaction parameters are shown in Table 3, and BRV quantitative RT-PCR 20 μL reaction system is shown in Table 4:

[0085] Table 4

[0086]

[0087] Use CFX96 Real-time PCR instrument to amplify BRV positive plasmid samples and BRV total RNA, such as figure 2 As shown, there is a good amplification effect.

[0088] 2.2.4.2 Primer concentration optimization

[0089]The template is the standard positive recombinant plasmid DNA of BRV, absorb 0.6 μL (concentration 300nmol / L), 0.7 μL (concentration 350nmol / L), 0.8 μL (concentration 400nmol / L), 0.9 μL (concentration 450nmol / L) of the upstream and downstream primers respectively. ), 1 μL (concentration 500nmol / L) to optimize the concentration of BRV primers. For reaction parameters and systems, refer to Table 3 and Table 4.

[0090] Experimental results such as image 3 shown. From image 3 It can be seen th...

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Abstract

The invention relates to the field of virus detection, and in particular relates to a kit, primer and probe for detecting bovine rotavirus. The invention provides a kit for detecting bovine rotavirus;the kit contains a nucleic acid amplification reagent; the nucleic acid amplification reagent contains a primer and a probe for detecting bovine rotavirus; the primer comprises an upstream primer asshown in SEQ ID NO:1 and a downstream primer as shown in SEQ ID NO:2; and the probe comprises a probe as shown in SEQ ID NO:3. The kit provided by the invention has the technical advantages of high sensitivity and high specificity.

Description

technical field [0001] The invention relates to the field of virus detection, in particular to a kit, primers and probes for detecting bovine rotavirus. Background technique [0002] Rotavirus (RV) is one of the main pathogens causing diarrhea in children and young animals, and has a great impact on various mammals and birds in nature. Bovine rotavirus (BRV), also known as calf diarrhea virus, belongs to the genus 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. The main symptoms include loss of appetite, lethargy, watery diarrhea, and even death. Symptomatic infection, mild self-limiting diarrhea and severe diarrhea with severe dehydration and electrolyte imbalance, the incidence rate of calves infected with the virus can be as high as 90% to 100%. The mortality rate of calves is increased, and it will also seriously affect the growth and development of calves, there...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/701C12Q1/6851C12Q2531/113C12Q2563/107C12Q2545/113
Inventor 周伟光丽平张志丹希尼尼根徐晓静彭雪松
Owner INNER MONGOLIA AGRICULTURAL UNIVERSITY
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