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Primer pair, probe and kit for specific detection of porcine lymphotropic herpesviruses III

A herpes virus and primer pair technology, which is applied in the field of PCR primer pairs, probes and kits for detecting type III porcine lymphotropic herpes virus, can solve the problems of poor epidemiology and achieve high testing efficiency and application Effects with a wide range and high sensitivity

Inactive Publication Date: 2015-04-15
惠州出入境检验检疫局检验检疫综合技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Little is known about the epidemiology of the virus

Method used

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  • Primer pair, probe and kit for specific detection of porcine lymphotropic herpesviruses III
  • Primer pair, probe and kit for specific detection of porcine lymphotropic herpesviruses III
  • Primer pair, probe and kit for specific detection of porcine lymphotropic herpesviruses III

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Design and synthesis of primers and probes

[0038] Download all homologous gene sequences of type III porcine lymphotropic herpes virus from GenBank, and the primers and probes are synthesized by Invitrogen. The sequence of the type porcine lymphotropic herpes virus standard strain (GeneBankNo.AY170315) and The homologous sequence is compared with the homologous gene sequence of type I and type II (type I GeneBankNo.AY177148 and AF478169, Type II GeneBankNo.AF191043, AY170314 and AY170317 ） , Use MEGA software for homology comparison, use primer Express3.0 software to design primers and TaqMan probes,

[0039] The length of the amplified target fragment is 170bp.

[0040] The nucleotide sequence of the upstream primer is shown in PLHV3 D F1;

[0041] The nucleotide sequence of the downstream primer is shown in PLHV3 D R1;

[0042] The nucleotide sequence of the probe used with the above primers is shown in PLHV3 D P1. The 5'end of the probe is labeled with reporte...

Embodiment 2

[0043] Example 2 The extraction steps of total DNA are as follows:

[0044] Add the same amount of anticoagulant buffer solution or use an anticoagulant blood collection tube to collect 3~5Ml pig blood samples, mix them, and store at 2~8℃ for 3 days. Centrifuge the blood sample at 5000r / min for 5min, remove the supernatant, suck the white blood cells on the upper layer of red blood cells, and transfer them to a centrifuge tube without pollution or biological toxicity. Follow the instructions of Beijing Tiangen Biotechnology Company Viral Genomic DNA / RNA Extraction Kit (DP315) to extract viral DNA (the DNA extraction in this experiment is based on the DP315 kit, and other commercial viral DNA extraction kits are applicable), use directly or Store in the refrigerator at -20℃ for later use.

Embodiment 3

[0045] Example 3 Establishment of real-time fluorescent quantitative PCR amplification method

[0046] 1. Real-time fluorescent quantitative PCR reaction system

[0047] Use total DNA as template to perform real-time fluorescent quantitative PCR reaction, that is, a 20μL reaction system contains: 10× fluorescent quantitative PCR buffer 2μL, 0.5μL dNTPs (25mmol / L), 0.5μL primer PLHV3 D F1 (10umol / L) , 0.5μL primer PLHV3 D R1 (10umol / L), 1.2μL magnesium ion (25mmol / L), 0.3μL fluorescent probe (20umol / L), 1μL DNA template, 0.2μL Taq enzyme (5U), and DEPC treated water Make up to 20μL.

[0048] 2. Real-time fluorescence quantitative PCR reaction conditions

[0049] After putting the sample tube into the 7500 fluorescent PCR machine of ABI company, set the following conditions to proceed: 95℃, 2min; 90℃, 10s; 60℃, 40s for a total of 40 cycles. Collect data after each cycle. After the reaction, the result is judged according to the amplification curve and the standard curve.

[0050] 3. E...

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Abstract

The invention provides primers and a probe for real-time fluorescent quantitive PCR (Polymerase Chain Reaction) detection of porcine lymphotropic herpesviruses III through gene sequence analysis and comparison of genes of porcine lymphotropic herpesviruses III. The nucleotide sequences of the primers and probe are shown as follows: SEQIDNO.1-3. The invention further provides a method of detecting the porcine lymphotropic herpesviruses III and a detection kit. The detection kit and a detection reagent provided by the invention have the advantages of accuracy in detection, high sensitivity, strong specificity and simplicity and rapidness, and have a good detection capacity.

Description

technical field [0001] The invention relates to biological detection technology, in particular to a pair of PCR primers, a probe and a kit for detecting type III porcine lymphotropic herpes virus. Background technique [0002] Porcine Lymphotropic herpesviruses 3 (PLHV3) type Ⅲ belongs to the genus Gammaherpesvirus of the family Herpesviridae. An infectious disease that can infect pigs, and the virus may be transmitted to humans through allografting of cells, tissues and organs of pigs. There are three subtypes of porcine lymphotropic herpes disease, namely PLHV-1, PLHV-2 and PLHV-3. PLHV-1 and PLHV-2 were discovered by EhLers et al. in 1999, and PHLV-3 was discovered by Chmie Lewicz in 2003. Little is known about the epidemiology of the virus. As a member of lymphotropic viruses, PLHV sequence fragments can often be detected in peripheral blood cells of blood, lymph nodes, spleen and tonsils and other tissues. Pigs are the most promising donors in animal replacement sou...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/686C12Q1/70C12Q2561/101C12Q2563/107C12Q2545/114
Inventor 朱事康周宇佟铁铸刘星李春萍于飞吕飞刘中勇林志雄
Owner 惠州出入境检验检疫局检验检疫综合技术中心
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