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Multiple-connection probe amplification detection kit, primer and probe for simultaneously detecting five cow disease viruses

A technology for multiple ligation of probes and bluetongue virus, applied in the field of inspection and quarantine, can solve the problems of limitation, long time, and heavy detection workload.

Active Publication Date: 2015-10-28
PEOPLES REPUBLIC OF CHINA BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the currently established methods are single-target detection for one pathogen. In the actual detection, it needs to be repeated many times to complete the purpose of detecting different pathogens. The detection workload is large and the time is long, which cannot meet the rapid clearance of large-scale animal quarantine. the actual needs of
And it is difficult to realize the differential diagnosis of mixed infections and diseases with similar symptoms in actual samples
While establishing single-target pathogen detection technology, veterinary institutions in various countries have also successively developed multiplex PCR and multiplex fluorescent PCR that can simultaneously detect common cattle viruses. Fluorescence PCR is due to the mutual interference of the fluorescence emitted by the fluorescent groups used by different probes, and the limitation of fluorescence PCR instrument to the fluorescence resolution of different wavelengths also limits the development of fluorescence PCR multiple detection technology

Method used

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  • Multiple-connection probe amplification detection kit, primer and probe for simultaneously detecting five cow disease viruses
  • Multiple-connection probe amplification detection kit, primer and probe for simultaneously detecting five cow disease viruses
  • Multiple-connection probe amplification detection kit, primer and probe for simultaneously detecting five cow disease viruses

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Embodiment 1, preparation and use of kit

[0071] 1. The composition of the kit:

[0072] (1) MLPA buffer, purchased from The MRC-Holland Company, which includes KCl, Tris-HCl, EDTA and PEG-6000, pH 8.5.

[0073] (2) Probes, which include long and short probes for bluetongue virus, bovine viral diarrhea virus, bovine enzootic leukemia virus, bovine infectious rhinotracheitis virus and foot-and-mouth disease virus. The sequences of each probe are shown in Table 1, wherein, the 5' end of said SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10 is phosphorylated; each probe The concentration is 1uM, which can be divided or mixed together;

[0074] (3) Ligating reaction solution, which includes: Ligase-65 buffer A and Ligase-65 buffer B, both purchased from MRC-Holland Company; the formula of the connecting reaction solution is shown in Table 3;

[0075] (4) PCR reaction solution, which includes universal primers P1 and P2 as shown in the sequence ta...

Embodiment 2

[0109] Embodiment 2, the sensitivity test of kit

[0110] 1. Materials

[0111] The inactivated bluetongue virus, bovine viral diarrhea inactivated virus, and bovine infectious rhinotracheitis inactivated virus are all preserved in the laboratory. The inactivated bovine endemic leukemia virus is provided by the China Veterinary Drug Administration. The O-type foot-and-mouth disease inactivated Viruses were provided by Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0112] 2. Method

[0113] 1) Preparation of recombinant plasmid DNA in vitro

[0114] Preparation of bluetongue virus NS3 gene-positive recombinant plasmid: clone bluetongue virus NS3 gene, reclaim the PCR amplification product, the length is 492bp, connect with pGEM-T vector (purchased from PromeGA company), transform JM109 competent cells, The plasmid DNA was extracted by alkaline lysis method, and the positive recombinant plasmid of bluetongue virus NS3 was obtained after ide...

Embodiment 3

[0130] Embodiment 3, the specificity test of kit

[0131] 1. Materials

[0132] Table 5 Viruses and nucleic acids used in the research process of specificity test

[0133] Virus

source

bluetongue virus

The lab saves

bovine infectious rhinotracheitis virus

The lab saves

bovine viral diarrhea virus

The lab saves

[0134] bovine enzootic leukemia virus

Provided by China Veterinary Drug Administration

Type O foot and mouth disease inactivated virus

Lanzhou Veterinary Research Institute

[0135] 2. Method

[0136] Use any set of probes from bluetongue virus, bovine viral diarrhea virus, bovine enzootic leukemia virus, bovine infectious rhinotracheitis virus and foot-and-mouth disease virus to detect the nucleic acids of the other four viruses by MLPA respectively, to verify the specificity of the method.

[0137] 3. Results

[0138] Using any set of probes in the five viruses for MLPA detectio...

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Abstract

The invention discloses a multiple-connection probe amplification detection kit, primer and probe for simultaneously detecting the bluetongue viruses, the infectious bovine rhinotracheitis viruses, the bovine viral diarrhea viruses, the enzootic bovine leucosis viruses and the foot and mouth disease viruses. The multiple-connection probe is shown in sequence tables from SEQ ID NO:1 to SEQ ID NO:10. The primer is shown in sequence tables from SEQ ID NO:11 to SEQ ID NO:12. The primer, the probe and / or the multiple-connection probe amplification detection kit including the primer and the probe can detect the five vital cow disease pathogenies including the bluetongue viruses, the infectious bovine rhinotracheitis viruses, the bovine viral diarrhea viruses, the enzootic bovine leucosis viruses and the foot and mouth disease viruses at the same time, the detection time and cost are saved, and epidemic diseases can be diagnosed in time.

Description

technical field [0001] The invention provides a multiple connection probe amplification detection kit and primers and probes for detecting bluetongue, bovine viral diarrhea, bovine endemic leukemia, bovine infectious rhinotracheitis, and foot-and-mouth disease viruses. The purpose of realizing one sampling, one analysis and simultaneous detection of five kinds of cattle diseases belongs to the field of inspection and quarantine. Background technique [0002] Bluetongue virus (BTV), bovine viral diarrhea virus (BVDV), bovine enzootic leukemia virus (EBLV), infectious bovine rhinotracheitis virus (IBRV), foot-and-mouth disease virus (FMDV) The main common pathogens of disorders and digestive tract diseases are the most serious pathogens that harm the cattle industry. They are defined as cross-border pathogens by the World Organization for Animal Health (OIE) and are also important quarantine objects in the international trade of animals and animal products in my country. . [...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/701C12Q2600/16
Inventor 史喜菊马贵平柏亚铎郝俊虎乔彩霞刘全国李炎鑫李冰玲
Owner PEOPLES REPUBLIC OF CHINA BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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