A kind of kit and method for detecting porcine boca virus

A technology of porcine boca virus and kit, applied in the direction of biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc., can solve the problems of long time and complicated methods, and achieve simple preparation, high sensitivity, The method is simple and fast, and the effect

Inactive Publication Date: 2011-12-21
SHANGHAI ANIMAL EPIDEMIC PREVENTION & CONTROL CENT
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The invention provides a kit and method for detecting porcine Boca virus. The kit and method for detecting porcine Boca virus can quickly and accurately detect porcine Boca virus, and the operati

Method used

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  • A kind of kit and method for detecting porcine boca virus
  • A kind of kit and method for detecting porcine boca virus
  • A kind of kit and method for detecting porcine boca virus

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Experimental program
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Effect test

Embodiment 1

[0066] Embodiment 1 kit composition

[0067] Fluorescent PCR reaction solution 1 tube (25 μL / reaction), the final concentration of dATP, dTTP, dGTP and dCTP in it is 200 μM, Mg 2+ The final concentration is 3.5mM, the final concentration of upstream and downstream primers is 0.2μM, the final concentration of probe is 0.2μM, 1 tube of probe-primer mixture (2μL / reaction), both concentrations are 0.2μM, 1 tube of Taq enzyme (2μL / reaction) , wherein the concentration of Taq enzyme was 0.5U / μL, 1 tube of positive plasmid control (250 μL / tube), and 1 tube of negative plasmid control (250 μL / tube).

[0068] This kit uses a 30 μL reaction system, and the reaction solution is composed of: 25 μL of fluorescent PCR reaction solution, 2 μL of enzyme, 1 μL of probe-primer mixture and 2 μL of template.

Embodiment 2

[0069] The using method of embodiment 2 porcine Boca virus fluorescent PCR detection kit

[0070] 1. Sample processing:

[0071] Please extract the DNA by yourself. The positive control does not need to be extracted, and the sample is added directly.

[0072] 2. Detection of fluorescent PCR

[0073] (1) Take the PCR reaction solution, hot-start Taq enzyme and probe according to the number of samples to be tested n (n=number of samples to be tested + 2), mix them in a centrifuge tube, vibrate on a vortex shaker, and pack in each tube , cover the tube and set aside.

[0074] (2) Now add the negative control solution into an aliquot tube, take the DNA of each sample and add it to the corresponding reaction tube; finally take out the positive control solution and add it to another reaction tube, mark each reaction tube and centrifuge, take it out and put it in a fluorescent PCR instrument .

[0075] (3) Fluorescent PCR reaction conditions: 94°C×3min; 94°C×20sec, 60°C×40sec, 40...

Embodiment 3

[0084] Embodiment 3 kit specificity test

[0085] After processing the following 5 parvovirus-positive tissue samples, 5 PRRS virus-positive tissue samples, and 5 circovirus-positive disease samples that were detected as positive by fluorescent PCR, they were tested by PBov ordinary PCR. Negative. Then, 2 μL was taken as a template for real-time fluorescent PCR detection. Real-time fluorescent PCR detection results such as image 3 .

[0086] 2μL of 15 cases of SPF pig serum were used as templates for real-time fluorescent PCR detection. Real-time fluorescent PCR detection results such as Figure 4 .

[0087] The results of fluorescent PCR showed that only the DNA of porcine Boca virus amplified curves, while the DNA of porcine parvovirus positive tissue samples, porcine blue ear disease virus positive tissue disease samples, and porcine circovirus positive sample samples had no such amplified curves. An amplification curve appears.

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Abstract

The invention belongs to the field of virus diagnosis and detection, solves the technical problems of complicated and long time for detecting porcine boca virus in the prior art, and provides a kit for detecting porcine boca virus, which contains a fluorescent PCR reaction solution, The fluorescent PCR reaction solution contains specific primers, and the sequence of the upstream primer is shown in SEQ ID NO: 1, and the sequence of the downstream primer is shown in SEQ ID NO: 2; it also contains a TaqMan specific probe for porcine boca virus, The sequence of the probe is shown in SEQ ID NO: 3, and it also contains the DNA positive control of the Boca virus plasmid. The present invention also provides a method for detecting porcine Boca virus by using the above-mentioned kit. The kit of the invention can quickly and accurately detect porcine boca virus, has simple operation, short time consumption, high sensitivity and strong specificity, and can qualitatively or quantitatively detect porcine boca virus in various types of specimens.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a virus diagnostic kit, in particular to a kit and method for detecting porcine boca virus. Background technique [0002] Bocaviruses belong to the Parvoviridae family, which is the smallest and simplest class of single-stranded linear DNA viruses, including the Parvovirinae that infects birds and mammals and the Densovirinae that infects arthropods2 a subfamily. The subfamily Parvoviridae includes five genera: Parvovirus, Rhodovirus, Dependovirus, Mink Aleutian and Bocavirus. Parvovirus B19 belongs to the Rhodovirus genus and is the only parvovirus known to infect humans before the discovery of HBoV. In 2005, Sweden's Allander et al. reported that they isolated a new virus from the specimens of infants suffering from respiratory tract infections. After analyzing the gene sequence of the virus, it was found that the new virus was a single-stranded, membrane-less virus. DNA viruses b...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11G01N21/64
Inventor 邓波刘佩红周锦萍华修国崔立王建葛菲菲鞠厚斌刘健张维谊
Owner SHANGHAI ANIMAL EPIDEMIC PREVENTION & CONTROL CENT
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