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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

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

AI Technical Summary

Problems solved by technology

However, the fluorescent detection reagent product and its application method for detecting the African swine fever virus A137R gene have not been reported yet.

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

Embodiment 1

[0054] Embodiment 1: the design of real-time fluorescence quantitative PCR primer

[0055] A specific primer sequence was designed for the sequence conservation region of the coding gene A137R of the ASFV P11.5 protein, and the sequence design was as follows:

[0056] Forward primer: p11.5-F: 5'-CTTACCAAACTCGACCAGGAGG-3'; (SEQ ID NO.1)

[0057] Reverse primer: p11.5-R: 5'-GTGCATCGTTCCTCAGGGATT-3'; (SEQ ID NO.2)

Embodiment 2

[0058] Example 2: Establishment of Fluorescent Quantitative PCR Detection Method

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

[0060] Genomic DNA of the samples to be tested was extracted using a commercial kit.

[0061] (2) Establishment of fluorescent quantitative PCR reaction system:

[0062] The fluorescence quantification kit used adopts the TB with the item number RR820A from Takara Company Premix Ex Taq TM For reagent II, add 10 μl of TB Green Premix Ex Taq II, 0.8 μl of PCR Forward Primer (10 μM), 0.8 μl of PCR Reverse Primer (10 μM) and 2 μl of genomic DNA extracted in step (1) into a 200 μl fluorescent reaction tube. Using RNase Free dH 2 O supplemented to 20 μl system. placed in Light 96 (Roche Diagnostics) fluorescent quantitative PCR instrument for the reaction, the reaction conditions were 95 ° C pre-denaturation 30 sec, 95 ° C 5 sec, 60 ° C 30 sec, 40 cycles.

[0063] (3) Preparation of standard curve:

[0064] In order to accurately quantify the c...

Embodiment 3

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

[0070] 1. The composition of the kit:

[0071] The kit in this example is a fluorescent quantitative kit for detecting African swine fever virus. The kit contains: the forward primer (10 μM) shown in SEQ ID NO.1 designed in Example 1, the reverse primer (10 μM) shown in SEQ ID NO.2 designed in Example 1, and the fluorescent quantitative PCR reaction reagent , Standards and Controls.

[0072] Standards were prepared as follows:

[0073] The primers shown in SEQ ID NO.3 and SEQ ID NO.4 are used to amplify the genome of African swine fever virus, and the amplified product (SEQ ID NO.9) of 414bp obtained is connected to the pGEM-T vector On the screen, positive clones were screened and plasmid DNA was extracted to prepare a standard.

[0074] Fluorescent quantitative PCR reaction reagents include: SYBR-Green fluorescent...

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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
CPCC12Q1/701C12Q1/6851C12Q2531/113C12Q2563/107C12Q2545/114Y02A50/30
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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