Primer, probe and kit for specifically detecting type-3 ungulata bocaviruses parvovirus

A technology of parvoviruses and ungulates, applied in the field of biological detection, can solve problems such as the inability to accurately diagnose porcine Boca virus, and achieve the effect of promoting the improvement of testing efficiency, high sensitivity, and high testing efficiency

Inactive Publication Date: 2016-02-03
中华人民共和国惠州出入境检验检疫局
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the prior art is substantially unable to accurately diagnose each genotype of porcine bocavirus

Method used

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  • Primer, probe and kit for specifically detecting type-3 ungulata bocaviruses parvovirus
  • Primer, probe and kit for specifically detecting type-3 ungulata bocaviruses parvovirus
  • Primer, probe and kit for specifically detecting type-3 ungulata bocaviruses parvovirus

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Experimental program
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Effect test

Embodiment 1

[0031] Design and synthesis of embodiment 1 primers and probes

[0032] Download all the complete gene sequences belonging to the type 3 ungulate boca parvovirus population from GenBank, combine the nearly complete gene sequence (KJ755665) obtained by our laboratory with DNAStar software for homology comparison, and use primerExpress5.0 software to design Primers and TaqMan probes were synthesized by Shanghai Sangong Company. The length of the amplified target fragment is 134bp.

[0033] The nucleotide sequence of the upstream primer is shown as PboVF1;

[0034] The nucleotide sequence of the downstream primer is shown as PBoVR1;

[0035] The nucleotide sequence of the probe used in conjunction with the above primers is shown as PboVP1. The 5' end of the probe is labeled with a reporter FAM fluorescent dye, and the other 3' end is labeled with a BHQ1 quencher group.

Embodiment 2

[0036] The extraction steps of embodiment 2 total DNA are as follows:

[0037] Add 2-4ML of PBS solution or normal saline to the feces sample for homogenization, centrifuge at 12000r / min for 5min, and take the supernatant; add 2ML / g of PBS solution after the tissue sample is pulverized, homogenate, freeze-thaw 3-5 times, and centrifuge at 12000r / min After 5 minutes, the supernatant was taken for the following experiments. Extract viral DNA according to the instructions of Beijing Tiangen Biotechnology Company Viral Genomic DNA / RNA Extraction Kit (DP315) (the DNA extraction in this test is only based on the DP315 kit, and other commercial viral DNA extraction kits are applicable), directly use or Store in a -20°C refrigerator for later use, and store in a -70°C refrigerator for long-term storage.

Embodiment 3

[0038] Embodiment 3 Establishment of real-time fluorescent quantitative PCR amplification method

[0039] 1. Real-time fluorescent quantitative PCR reaction system

[0040] Use the total DNA as a template to carry out real-time fluorescent quantitative PCR reaction, that is, in a 20 μL reaction system containing: 10×PCR buffer 2.0 μL, 2.0 μL dNTPs (2.5 mmol / L), 0.5 μL primer PboVF1 (10 μmol / L), 0.5 μL Primer PBoVR1 (10umol / L), 1.0μL magnesium ion (50mmol / L), 0.5μL fluorescent probe (10umol / L), 1.0μL (10ng~50ng / μL) DNA template, 0.2μL Taq enzyme (5U).

[0041] 2. Real-time fluorescent quantitative PCR reaction conditions

[0042] After putting the sample tube into ABI 7500 fluorescent PCR instrument, set the following conditions: 95°C, 3min; 95°C, 5s; 55°C, 15s, 72°C, 40s, a total of 40 cycles. Collect data after each cycle. After the reaction, judge the result according to the amplification curve and the standard curve.

[0043] 3. Establishment of real-time fluorescent qu...

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Abstract

The invention discloses a primer, a probe and a kit for specifically detecting type-3 ungulata bocaviruses parvovirus. The detection kit and the detection agent disclosed by the invention have the advantages of detection accuracy, high sensitivity, high specificity, simplicity, convenience and rapidness and is good in detection property.

Description

technical field [0001] The invention relates to biological detection technology, in particular to a primer, a probe and a kit for specifically detecting type 3 ungulate boca parvovirus. Background technique [0002] Bocavirus belongs to the Bocavirus genus of the Parvoviridae subfamily of the Parvoviridae family. Parvoviruses include Thickoviridae and Parvoviridae, the former mainly infecting insects and the latter mainly infecting vertebrates. The Parvovirus subfamily is further divided into 6 genera including Parvovirus, Bocavirus, Rhodovirus, Relivirus, and Mink Aleutian virus. Porcine Bocavirus (Porcine Bocavirus, PBoV, also known as porcine Boca parvovirus) belongs to the Bocavirus genus, which is a single-strand linear non-enveloped DNA virus with a genome of about 5.2kb, including 3 open reading frames (ORFs), NS1, NP1 and VP1 / VP2, respectively. It mainly attacks the respiratory system and digestive system of pigs. In China, the positive rate of PBoV in pigs with m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2531/113C12Q2527/137C12Q2561/113
Inventor 周宇唐连飞徐鹏宋斯伟朱事康于飞吕飞佟铁铸李春萍刘星刘中勇
Owner 中华人民共和国惠州出入境检验检疫局
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