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Triple real-time fluorescent quantitative PCR kit for detecting African swine fever wild strains and gene deletion strain

A real-time fluorescence quantitative, gene deletion technology, applied in recombinant DNA technology, microbial determination/inspection, biochemical equipment and methods, etc., to achieve the effects of high agility, fast detection process, and short detection time

Inactive Publication Date: 2020-04-17
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is only a kit for detecting African swine fever wild virus at present, and a kit for detecting African swine fever gene deletion virus has not been reported

Method used

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  • Triple real-time fluorescent quantitative PCR kit for detecting African swine fever wild strains and gene deletion strain
  • Triple real-time fluorescent quantitative PCR kit for detecting African swine fever wild strains and gene deletion strain
  • Triple real-time fluorescent quantitative PCR kit for detecting African swine fever wild strains and gene deletion strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Design of triple real-time fluorescent quantitative PCR detection primers for African swine fever gene deletion strains and wild strains

[0046] According to CD2V, P72, MGF-360 14L gene of African swine fever virus (ASFV) highly conserved and have the sequence of specific region, the present inventor designs the specific primer and Taqman probe that do not interfere with each other in combination with self experience. needle, thus providing a kind of PCR primer for distinguishing ASFV wild-type strain and gene-deleted strain, specifically:

[0047] The primer sequences are as follows:

[0048] ASFV-VP72-F: 5'-CCTCTCCTATGCAACATTCATGATT-3'

[0049] ASFV-VP72-R: 5'-TTCGCTGCGTATCATTTTCATC-3'

[0050] ASFV-CD2V-F: 5'-GAAGAAGAACAATGTCAGCATGATG-3'

[0051] ASFV-CD2V-R: 5'-CGACTGTAAGGCTTAGGAAGTAATGG-3'

[0052] ASFV-MGF-360 14L-F: 5'-GGGTTCGGATACAGGCGTTA-3'

[0053] ASFV-MGF-360 14L-R: 5'-CGTGTTCCTGCCGTGTATCTAA-3'

[0054] The probe sequence is:

[0055] ASFV...

Embodiment 2

[0058] Embodiment 2: Triple real-time fluorescent quantitative PCR detection

[0059] 1.1 Extraction of DNA

[0060] The DNA extraction kit used in the present invention is AxyPrep Body Fluid Virus DNA / RNA Mini Kit of AXYAGEN Company.

[0061] 1.2 Primers and probes

[0062] The primers and probes used in the present invention were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0063] 1.3 Fluorescent quantitative PCR amplification reagents

[0064] The 2x Probe Taq Mix used in the present invention was purchased from Beijing Quanshijin Biological Co., Ltd.

[0065] 1.4 Positive plasmid

[0066] In the present invention, the partial gene sequences of ASFV CD2V, P72, and MGF-360 14L published in the GenBanK database are artificially synthesized, connected with the pMD18T cloning vector, transformed into E. On the LB medium plate, culture at 37°C for 12-16 hours. After the bacteria were picked, screened and identified by sequencing, the plasmids were extracted after...

Embodiment 3

[0071] Example 3: Sensitivity detection of triple fluorescent PCR

[0072] The three kinds of positive plasmids in Example 2 were respectively serially diluted to 10^7copies, 10^6copies, 10^5copies, 10^4copies, 10^3copies, 10^2copies, 10^1copies, 10^0copies, each dilution gradient Do 3 replicates to test the sensitivity and minimum detection limit. Using OriginPro 8.5 software to perform linear regression on the data and draw a standard curve, it can be seen that the linear relationship between the Ct values ​​of the three genes is greater than 0.99, the repeatability between the repetitions is good, and the minimum detection limit of the three genes can reach 10copies / reaction.

[0073] figure 1 , figure 2 and image 3 Figure a in the figure shows the amplification curves of the three genes with different copy numbers, and the copy numbers of templates corresponding to 1-8 are 10^7copies, 10^6copies, 10^5copies, 10^4copies, 10^3copies, 10^ 2copies, 10^1copies, 10^0copi...

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Abstract

The invention provides a triple real-time fluorescent quantitative PCR detection primer for detecting an African swine fever wild strain and a gene deletion strain, and a kit and a detection method thereof. The triple fluorescent quantitative detection kit is developed and researched for three genes CD2V, VP72 and MGF-360 14L of the African swine fever virus by utilizing a multiple fluorescent PCRtest means, and whether a sample is infected with the African swine fever virus and whether gene deletion exists in the infected virus or not can be determined at the same time. The method can detecta large number of samples at the same time, provides an effective tool for scientifically and reasonably preventing and controlling African swine fever, guarantees the healthy development of the pigindustry, and has the advantages of convenience in operation, high sensitivity, strong specificity, short detection time and the like.

Description

technical field [0001] The invention belongs to the technical field of biological kits, and in particular relates to a specific primer, a probe, a triple PCR detection kit and a detection method for detecting African swine fever wild strains and gene deletion strains. Background technique [0002] African swine fever (ASF) is a highly contagious disease caused by African swine fever virus (ASFV). Disorder, accompanied by severe thrombocytopenia and lymphopenia, infected animals usually die within 10d to 14d after infection, and the case fatality rate is as high as 100%. Since it was first diagnosed in August 2018, African swine fever has shown an infection trend in most areas of my country, affecting pigs of all ages, causing hemorrhagic fever, and the mortality rate of the most acute and acute infection is as high as 100%, which constitutes a serious threat to my country's pig industry. huge threat. The ASFV genome is double-stranded DNA with a size of 170-190kb. Viral ge...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q1/701C12Q2600/16C12Q2600/166C12Q2531/113C12Q2537/143C12Q2561/101C12Q2545/113
Inventor 周丹娜田永祥胡贤旺郭锐梁婉段正赢杨克礼袁芳艳刘威高婷刘泽文
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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