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Nucleic acid composition for detecting African horse pestivirus and kit thereof

A nucleic acid composition and pestivirus technology, applied in the field of in vitro nucleic acid detection of viruses, can solve the problem of low sensitivity of kits, and achieve the effects of reducing the probability of false negatives, short detection process time, and simple detection steps.

Pending Publication Date: 2021-07-02
百沃特(天津)生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the problem of low sensitivity of detection kits for African horse sickness virus in the related art, the application provides a nucleic acid composition and kit for detecting African horse sickness virus

Method used

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  • Nucleic acid composition for detecting African horse pestivirus and kit thereof
  • Nucleic acid composition for detecting African horse pestivirus and kit thereof
  • Nucleic acid composition for detecting African horse pestivirus and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] The highly conserved region of the African horse sickness virus structural protein 7 gene region published on NCBI was used as the target region, and specific primers and probes were designed using primer design software, as well as internal standard primers and probes. The sequences of primers and probes are shown in Table 1.

[0051] Table 1 Primer and probe sequence information

[0052]

[0053]

[0054] The 5' end of the probe of this application can be labeled with any one of FAM fluorescent group, ROX fluorescent group, HEX fluorescent group, CY3 group and CY5 group; preferably, the 5' end of the detection probe is selected The 5' end of the internal standard probe is labeled with the FAM fluorophore, and the ROX fluorophore is used for labeling. The 3' end of the detection probe is labeled with the MGB1 group, and the 3' end of the internal standard probe is labeled with the MGB2 group.

Embodiment 2

[0056] A test kit for detecting African horse sickness virus, comprising primers and probes, PCRBuffer, DNA polymerase, c-MMLV reverse transcriptase, dNTPs and DEPC H recorded in Example 1 2 O.

[0057] (1) Determination of primer concentration

[0058] In the PCR system, this embodiment sets 5 concentrations of primers, respectively 200nmol / L, 400nmol / L, 600nmol / L, 800nmol / L, 1000nmol / L, with the same concentration of upstream and downstream primers. The specific operation procedure is as follows:

[0059] Prepare PCR reaction solution containing different concentrations of primers, PCR reaction solution also includes 5×Neoscript RT PremixBuffer, 200nmol / L detection probe, 80nmol / L internal standard upstream and downstream primers, 40nmol / L internal standard probe, 10×Neoscript RTase Mix, African horse sickness virus positive inactivated sample and negative sample (enzyme-free and nucleic acid-free water) 15.0 μl, and add sterilized water to make up to 30 μl.

[0060] Afte...

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Abstract

The invention discloses a nucleic acid composition for detecting African horse pestivirus and a kit thereof, and belongs to the technical field of virus in-vitro nucleic acid detection. The nucleic acid composition comprises an upstream primer, a downstream primer and a detection probe, the sequence of the upstream primer is as shown in SEQ ID NO.1, the sequence of the downstream primer is as shown in SEQ ID NO.2, and the sequence of the detection probe is as shown in SEQ ID NO.3. The kit containing the nucleic acid composition can specifically detect the African horse pestivirus in a sample to be detected, and has no cross reaction with eight pathogens such as equine encephalitis virus; the kit has high sensitivity (up to 10 < 3 > copies / mL), good repeatability and controllable quality, and the whole RT-PCR detection process is easy to operate, short in detection time and high in detection efficiency.

Description

technical field [0001] The application relates to the technical field of virus in vitro nucleic acid detection, in particular to a nucleic acid composition and a kit for detecting African horse sickness virus. Background technique [0002] African house sickness (AHS) is a non-contact acute or subacute infectious disease of equine animals caused by double-stranded RNA circoviruses of the Reoviridae family. It is transmitted through the bites of blood-sucking insects such as Culicoides. The main symptoms are fever, subcutaneous edema and viremia, accompanied by tissue and organ hemorrhage in severe cases, horses are the most susceptible, and the case fatality rate can reach 95%. [0003] The mortality rate of AHS is related to the species of infected animals and the serotype or strain of the virus. Among the equine animals, horses are the most sensitive, with a mortality rate of 50% to 95%, followed by mules, with a mortality rate of about 50%. In African regions where AHS ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2600/166
Inventor 马增彬杨若松龚雅云田小杨天意梁健平
Owner 百沃特(天津)生物技术有限公司
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