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Primer pair, probe and kit for detecting African swine fever virus and application of primer pair, probe and kit

An African swine fever virus and primer pair technology, applied in the field of bioengineering, can solve the problems of extremely high primer design requirements, huge economic and time costs, etc., and achieve the effects of short detection time, improved detection sensitivity, and broad application prospects

Inactive Publication Date: 2021-08-06
黄婉秋 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, RPA technology itself has extremely high requirements for primer design, and primer design and screening require huge economic and time costs.
[0009] At present, the African swine fever epidemic is still prevalent in many countries around the world and has not been effectively controlled. In order to improve the ASF detection rate, reduce the missed diagnosis rate, and reduce the false negative rate, we urgently need to develop a high-sensitivity and rapid Accurate African Swine Fever Nucleic Acid Detection Technology

Method used

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  • Primer pair, probe and kit for detecting African swine fever virus and application of primer pair, probe and kit
  • Primer pair, probe and kit for detecting African swine fever virus and application of primer pair, probe and kit
  • Primer pair, probe and kit for detecting African swine fever virus and application of primer pair, probe and kit

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Primer and probe design and screening of embodiment 1 ASFV virus p72 gene

[0046] By searching the PubMed nucleic acid database, it was found that the gene sequence of ASFV p72 (B646L) was well conserved, but there were still individual base mutations ( figure 1 ). Taking GenBank: AY578706.1 as the reference sequence, the distribution of mutation bases was analyzed ( figure 2 ), and designed primers and probes in fragments with low frequency of base mutation (Table 1).

[0047] Table 1 Primers and probes of ASFV p72 gene

[0048]

[0049] Appropriate primer sequences are an important part of RPA high-efficiency amplification. First, probes are designed in sequences with three consecutive thymines (T), and the probes are modified with FAM fluorescein. Eleven pairs of primers were designed at different positions at both ends of the probe to screen out the most efficient combination of primers and probes (Table 1). During primer screening, pair the first forward p...

Embodiment 2

[0053] Example 2 The nestRPA detection of preferred primers and probe combinations for p72 gene to ASFV plasmid DNA

[0054] The plasmid DNA containing the ASFV p72 gene sequence was further diluted to 50 copies / uL, 20 copies / uL, 5 copies / uL, 2 copies / uL, and 1 copy / uL for testing the ability of nestRPA to detect the ASFV p72 gene . It was found that using the preferred primer pair and probe combination of the p72 gene to carry out nestRPA detection, the positive plasmid DNA containing the ASFVp72 gene fragment ( Figure 5 , Figure 5 The value unit in the legend is copy / uL), indicating that the nestRPA new technology can significantly improve the detection sensitivity of ASFV virus nucleic acid p72 gene.

Embodiment 3

[0055] Example 3 The nestRPA detection of the preferred primers and probe combinations of the p72 gene to the ASFV standard product DNA

[0056] With ASFV standard product DNA (pseudovirus culture fluid, containing p72 gene), its DNA content is 5.9 * 10 3 copies / uL, to verify the sensitivity of the p72 gene preferred primers. Take 200uL of the pseudovirus culture solution rewarmed and mixed at room temperature, use the column extraction method to extract DNA, and dissolve it in 20uL of enzyme-free water. According to the initial DNA content of the pseudovirus culture solution, the concentration of the standard DNA extraction solution is calculated to be about 5.9 × 10 4 copy / uL, that is, the p72 gene DNA concentration is 5.9×10 4 copy / uL.

[0057] The standard DNA extract was diluted 10 times, 100 times, 1000 times, 10000 times, 12500 times, 20000 times, 25000 times, 40000 times, 50000 times, 80000 times and 100000 times sequentially by doubling dilution method. Utilize th...

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Abstract

The invention discloses a primer pair, a probe and a kit for detecting African swine fever virus. The kit comprises the primer pair and the probe which are designed for an African swine fever virus p72 gene sequence shown in SEQ ID NO: 1 or a part of the sequence. The kit for detecting the African swine fever virus is based on a nested RPA detection technology (nestRPA), can detect 1 copy / microliter ASFV virus nucleic acid fragments at least, has ultrahigh sensitivity, does not need expensive detection equipment, and is short in detection time which only needs 12min. The technology can early discover African swine fever, provides a new technical support for early isolation and early treatment, and has a very good application prospect.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a primer pair, a probe, a kit and an application thereof for detecting the P72 gene of African swine fever virus based on nested RPA (nestRPA) technology. Background technique [0002] African swine fever (Infection with African swine fever virus, ASF) is a kind of acute, hemorrhagic, acute severe infectious disease. ASF is a highly contagious and highly pathogenic animal disease. It is mainly transmitted in pigs, and its intermediate host is the soft tick of the genus Ornidoderma under the family Molluscidae. After pigs are infected by ASFV, the skin, abdominal viscera and even heart hemorrhage will appear rapidly, resulting in acute hemorrhagic death. The bodily fluids, blood, and meat products of sick pigs are contagious. In 1921, the African swine fever epidemic was first reported in Kenya. Since then, the epidemic has always existed in sub-Saharan Afri...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6848C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6848C12Q2521/507C12Q2549/119C12Q2563/107
Inventor 黄婉秋黄健宋小冬
Owner 黄婉秋
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