Real-time fluorescent PCR primer and detection kit for A137R gene of African swine fever virus and application of real-time fluorescent PCR primer and detection kit

An African swine fever virus and kit technology, which is applied in the field of detection kits and fluorescent PCR primers of African swine fever virus A137R gene to achieve the effects of improving detection efficiency, reducing loss and efficient detection

Pending Publication Date: 2022-01-28
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the fluorescent detection reagent product and its application method fo

Method used

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  • Real-time fluorescent PCR primer and detection kit for A137R gene of African swine fever virus and application of real-time fluorescent PCR primer and detection kit
  • Real-time fluorescent PCR primer and detection kit for A137R gene of African swine fever virus and application of real-time fluorescent PCR primer and detection kit
  • Real-time fluorescent PCR primer and detection kit for A137R gene of African swine fever virus and application of real-time fluorescent PCR primer and detection kit

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0054] Example 1: Design of real-time fluorescent quantitative PCR primers

[0055] The sequence of primer sequences for the sequence conserved region of the coding gene A137R of the ASFV P11.5 protein, the sequence design is as follows:

[0056] Positive primers: P11.5-f: 5'-cttaccaaactcgaccaggagg-3 '; (SEQ ID NO.1)

[0057] Reverse primers: P11.5-R: 5'-gtgcatcgttcctcaggggett-3 '; (SEQ ID No.2)

Example Embodiment

[0058] Example 2: Establishment of fluorescence quantitative PCR detection method

[0059] (1) Extraction of viral genome DNA:

[0060] The sample genomic DNA to be tested is extracted with a commercial kit.

[0061] (2) Fluorescence Quantitative PCR Reaction System Establishment:

[0062] The fluorescent quantitative kit used is TB from TAKARA's number of items RR820A. Premixex TAQ TM The II reagent was sequentially added to the 200 μl of the fluorescent reaction tube, PCRFORWARD Primer (10 μm) 0.8 μL, PCR Reverse Primer (10 μm) 0.8 μL, and the sample genomic DNA extracted in step (1), Use RNase Free DH 2 O Supplements to 20 μL of system. Light 96 (RocheDiagnostics) Fluorescence Quantitative PCR instrument was reacted, and the reaction conditions were 95 ° C for pre-degeneration of 30 SEC, 95 ° C for 5 sec, 60 ° C 30SEC, 40 cycles.

[0063] (3) Preparation of standard curves:

[0064] In order to accurately quantify the copy number of the virus in the sample, a plasmid containi...

Example Embodiment

[0069] Example 3: The composition of the kit, the optimization and specificity, sensitivity and repetition of experimental parameters

[0070] 1. Composition of the kit:

[0071] The kit of the present embodiment is a fluorescent quantitative kit for detecting African swine fever viruses. The kit contains: the forward primer (10 μm) shown in SEQ ID NO.1 design, and the reverse primer (10 μm), a fluorescent quantitative PCR reaction reagent is shown in Example 1. , Standards and contrasts.

[0072] The standard is prepared by the following method:

[0073] The genome of African swine fever virus was amplified by primers shown in SEQ ID NO.3 and SEQ ID NO.4, and the amplification product was connected to the PGEM-T vector. The positive cloning was screened and the plasmid DNA was extracted, that is, the standard product was prepared.

[0074] Fluorescence quantitative PCR reactants include: Sybr-Green fluorescent dyes and RNase Free DH 2 O.

[0075] The controls were divided into po...

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Abstract

The invention discloses a fluorescent quantitative PCR kit for detecting African swine fever virus and application. The fluorescent quantitative PCR kit contains primers as shown in SEQ ID NO.1 and SEQ ID NO.2, a fluorescent quantitative PCR reaction reagent, a standard substance and a reference substance. The fluorescent quantitative PCR kit disclosed by the invention can be used for detecting the African swine fever virus, is high in specificity, is only specifically combined with the African swine fever virus, and has no cross reaction with other swine viruses; the kit has the advantage of high detection sensitivity, has an excellent linear relationship in a copy range of 1 * 10 < 1 > to 1 * 10 < 8 > of a standard substance, can efficiently detect cotton swabs of African swine fever virus pigs and virus-carrying pigs without clinical symptoms, and improves the detection efficiency.

Description

technical field [0001] The invention belongs to the technical field of virus detection, in particular to a fluorescent PCR primer, a detection kit and an application of African swine fever virus A137R gene. Background technique [0002] African swine fever (ASF) is an acute, virulent, and highly contagious animal disease caused by African swine fever virus (ASFV). The World Organization for Animal Health (OIE) lists ASF as a statutory reportable animal disease. my country will also It is listed as a class of animal epidemics that need to be guarded against. The disease has a short course of disease, a wide range of infection, and the morbidity and mortality can be as high as 100%. It is extremely harmful to the pig industry and has seriously affected the development of my country's pig industry and the international people's livelihood. At present, there is no effective way to control African swine fever. Detection and removal of infected animals is the only way to control an...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6851C12Q2531/113C12Q2563/107C12Q2545/114
Inventor 秦爱建尹丹吕卉鲍春晖钱琨邵红霞叶建强缪发明扈荣良
Owner YANGZHOU UNIV
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