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PCR primer for detecting African swine fever virus, kit and application thereof

An African swine fever virus and kit technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. Primer and target sequence mismatch, low cost effect

Active Publication Date: 2016-06-22
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, two recent reports showed that the sensitivity and specificity of conventional PCR recommended by OIE were reduced, presumably due to nucleotide mismatch between primers and viral target genes (Gallardo, C., Nieto, R. , Soler, A., Pelayo, V., Fernández-Pinero, J., Markowska-Daniel, I., Pridotkas, G., Nurmoja, I., Granta, R., Simón, A., Pérez, C., Martín,E.,Fernández-Pacheco,P.,Arias,M.,2015.AssessmentofAfricanswinefeverdiagnostictechniquesasaresponsetotheepidemicoutbreaksinEasternEuropeanUnioncountries:howtoimprovesurveillanceandcontrolprograms.J.Clin.Microbiol.53,2555–2565.;Petrovan,V.,Buburuzan,L.,Zaulet,M. , 2015. False positive results using PCR detection method for Africans wine ever virus in wild boards from northern Romanian hunting zones. Turk. J. Vet. Anim. Sci. 39, 287–294.)

Method used

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  • PCR primer for detecting African swine fever virus, kit and application thereof
  • PCR primer for detecting African swine fever virus, kit and application thereof
  • PCR primer for detecting African swine fever virus, kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0042] The screening of experimental example 1 PCR primer

[0043] 1. Experimental method

[0044] 1.1PCR primer design

[0045] According to the 35 complete sequences of the ASFVp72 gene in GenBank ( figure 2 ) and 158 partial sequences (data not shown), design four pairs of specific primers P72-F / P72-R (composed of the nucleotide sequences shown in SEQIDNo.1 and SEQIDNo.2), P72 -2F / P72-2R (made up of the nucleotide sequence shown in SEQIDNo.3 and SEQIDNo.4), P72-3F / P72-3R (made up of the nucleotide sequence shown in SEQIDNo.5 and SEQIDNo.6) and P72-4F / P72-4R (consisting of the nucleotide sequences shown in SEQ ID No. 7 and SEQ ID No. 8) (Table 2).

[0046] Table 2PCR primer sequence

[0047]

[0048] 1.2 Establishment of PCR reaction conditions

[0049] 1.2.1 DNA / RNA extraction and cDNA synthesis

[0050] Genomic DNA was extracted from cell culture or blood samples using DNeasyblood&tissuekit (Qiagen, Germany). For the RNA virus used for specific detection, RNA wa...

experiment example 3

[0081] Experimental Example 3 Clinical Sample Detection

[0082] ASF diagnosis was performed on 62 whole blood samples collected from Uganda between 2010 and 2015. The samples were from clinically infected or clinically healthy domestic pigs and were sent to the Uganda National Animal Disease Diagnostic and Epidemiology Center (NADDEC) for testing. Nucleic acid was extracted from 50 μl of blood sample using DNeasyblood&tissuekit (Qiagen, Germany), and the nucleic acid sample was sent to SVA and detected by fluorescent quantitative PCR as described above. A small amount of DNA samples were sent to Harbin Veterinary Research Institute (HVRI) in China, and all samples were tested using the PCR method of the present invention and the two PCR methods validated by OIE.

[0083] Among 62 clinical samples, 8 samples were detected as positive for ASFV by the PCR method of the present invention, which was consistent with the detection results of fluorescent quantitative PCR, and the Ct...

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Abstract

The invention discloses a PCR primer for detecting an African swine fever virus, a kit and application thereof, and belongs to the detection field of the African swine fever virus. According to a conserved region of an ASFV p72 gene on the GenBank, four pairs of primers are designed, the primer with strong specificity and high sensitivity is screened out from the four pairs of primers, and the primer is composed of nucleotide sequences as shown in SEQ ID No.1 and SEQ ID No.2. The invention further discloses a kit prepared from the primer and used for detecting the African swine fever virus, and a corresponding PCR detection method is established. The detection method established by the invention is more specific and sensitive in comparison with two African swine fever virus PCR detection methods recommended by OIE; and the clinical sample detection result shows that the PCR detection method disclosed by the invention is simple in operation, low in cost, good in specificity and high in sensitivity, and can be effectively applied to the screening and fast diagnosis of the African swine fever.

Description

technical field [0001] The present invention relates to PCR primers for detecting African swine fever virus (Africans swine fever virus, ASFV), also relates to the kit prepared by said PCR primers and its application in detection of African swine fever virus, belonging to the detection field of African swine fever virus . Background technique [0002] African swine fever (African swine fever, ASF) is a severe infectious disease of domestic pigs and wild boars caused by African swine fever virus (ASFV). The country has caused enormous economic damage (Costard, S., Mur, L., Lubroth, J., Sanchez-Vizcaino, J.M., Pfeiffer, D.U., 2013. Epidemiology of Africanswinefevervirus. VirusRes. 173, 191–197.). ASFV is a large enveloped double-stranded DNA virus belonging to the African swine fever virus family (Asfarviridae) (Dixon, L.K., Chapman, D.A., Netherton, C.L., Upton, C., 2013. Africanswines ever virus replication and genomics. Virus Res. 173, 3–14.). [0003] ASFV can cause hem...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/701C12Q2531/113
Inventor 仇华吉罗玉子孙元孟星宇李素李永锋
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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