African swine fever virus nucleic acid amplification primer, detection method and kit

An African swine fever virus and nucleic acid technology, which is applied in biochemical equipment and methods, microbial determination/test, DNA/RNA fragments, etc. problems, to achieve the effect of saving the cost of detection kits, high sensitivity and low price

Inactive Publication Date: 2010-12-22
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] ①Hemadsorption (HAD) test: It is one of the most classic quarantine methods for ASF. This method is highly sensitive, but its disadvantages are time-consuming, cumbersome operation and hi

Method used

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  • African swine fever virus nucleic acid amplification primer, detection method and kit
  • African swine fever virus nucleic acid amplification primer, detection method and kit
  • African swine fever virus nucleic acid amplification primer, detection method and kit

Examples

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Effect test

Embodiment 1

[0083] Example 1: Detecting with the recombinant plasmid pcDNA-p72 carrying the African swine fever virus p72 gene as the detection target

[0084] 1. Design of primers and probes: 22 strains of ASFV (o1, BA71, BEN, cam, cro1.2, cro3.5, DR2, E70, E75, F6, ht, K1, ker, M1, Malawi, mk, Pr4, Pr5, ten, vic, wb, Za) p72 gene (Accession NO. AY578701.1, FJ174348.1, EF121428.1, AF511452.1, AY578690.1, AY578691.1, L76727. 1. AY578692.1, AY578693.1, AY578694.1, AY578695.1, AY578696.1, AY578697.1, AY578699.1, AY261361.1, AY578700.1, AY578702.1, AY578703.1, AY5 AY578705.1, AY578707.1, AY578708.1) were compared and analyzed, and a highly conserved region with a length of 651bp (905-1555bp) was found as the sequence to be tested, and PCR primers and labeled probes were designed and synthesized:

[0085] Forward primer 5'-CGTATCTGGACATAAGACGT-3' (SEQ ID NO: 1);

[0086] Reverse primer 5'-ATGCGTCTGGAAGAGCTGT-3' (SEQ ID NO: 2);

[0087] Biotin Capture Probe 5'-Biotin-TTTTTTTTTTGCGCAAGAGGGGG...

Embodiment 2

[0093] Example 2: 10 African swine fever virus genomes were detected as detection targets

[0094] 1. Design of primers and probes: DNAstar analysis software compared 100 published ASFV p72 genes of different genotypes, and determined that the 1516-1836 nucleotide sequence was highly conserved, and designed and synthesized 2 pairs of PCR primers, biological Two pairs of prime-labeled capture and alkylthiol-labeled detection probes:

[0095] F1: 5'-ATAAGCTTTCAGGATAGAG-3' (SEQ ID NO: 5);

[0096] R1: 5'-CATAATCCGTGTCCCAACT-3' (SEQ ID NO: 6);

[0097] F2: 5'-CACGCAGAAATAAGCTTTC-3' (SEQ ID NO: 9)

[0098] R2: 5'-CGTGTCCCAACTAATATAA-3' (SEQ ID NO: 10)

[0099] Capture probe C1: 5'-biotin-TTTTTTTTTTAGGGTATGTAAGAGCTGCAG-3' (the 5' end of SEQ ID NO: 7 is modified with biotin);

[0100] Capture probe C2: 5'-biotin-TTTTTTTTTTGGTATTCCTCCCGTGGCTTC-3' (the 5' end of SEQ ID NO: 11 is modified with biotin);

[0101] Detection probe D1: 5'-GATACCATGAGCAGTTACGGTTTTTTTTTT-O-(CH 2 ) 3 -SH...

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Abstract

The invention relates to an African swine fever virus nucleic acid amplification primer, a detection method and a kit. The primer pair and the specific nucleic acid probe are selected from SEQ ID NO: 1-12. The detection method comprises the following steps: (a) amplifying nucleic acid in the sample to be detected by using a PCR primer; (b) labeling the detection probe labeled by alkyl sulfydryl group on nano gold particles; (c) hybridizing the capture probe labeled by biotin and the detection probe labeled by nano gold in step (b) with a metamorphic PCR product; (d) adding the hybrid system in step (c) to a Streptavidin-coated ELISA plate to capture the hybrid compound; (e) carrying out silver enhancement to capture nano gold; (f) and stoping the silver enhancement reaction, and visually inspecting the grey scale judgment result. The invention has the characteristics of high detection sensitivity, strong detection specificity and low detection cost, can effectively eliminate false positive or false negative result when the PCR method is used for detecting African swine fever virus, and quickly detect the African swine fever virus nucleic acid.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an African swine fever virus nucleic acid amplification primer, detection method and kit. Background technique [0002] African swine fever is an acute, febrile infectious disease caused by African swine fever virus. It is characterized by a short course of disease and a mortality rate of up to 100%. It is classified as a type 1 disease. Since there is no African swine fever in my country, it cannot be directly detected in the diseased pigs. [0003] 1. Existing African swine fever (ASF) detection technology at home and abroad [0004] ①Hemadsorption (HAD) test: It is one of the most classic quarantine methods for ASF. This method is highly sensitive, but its disadvantages are time-consuming, cumbersome operation and high technical requirements. Diagnosis of the strain. [0005] ② Serological detection technology: Since the specific antibody of African swine fever virus (ASFV) in t...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 孙怀昌张鑫宇田瑞鹏夏晓莉
Owner YANGZHOU UNIV
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