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Porcine bocavirus detection and parting enrichment multiple PCR rapid diagnostic kit

A porcine boca virus, rapid diagnosis technology, applied in recombinant DNA technology, biochemical equipment and methods, microbial determination/inspection, etc., to achieve the effect of short detection time, high specificity and high specificity

Inactive Publication Date: 2016-07-27
ZHEJIANG SCI-TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because there is a large nucleotide variation in porcine bocavirus, there are many false negative results in designing the detection primers for porcine bocavirus for different reference strains.

Method used

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  • Porcine bocavirus detection and parting enrichment multiple PCR rapid diagnostic kit
  • Porcine bocavirus detection and parting enrichment multiple PCR rapid diagnostic kit
  • Porcine bocavirus detection and parting enrichment multiple PCR rapid diagnostic kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1 kit structure

[0044] see figure 1 , an enrichment multiplex PCR rapid diagnostic kit for simultaneous and specific detection of three porcine Boca virus infections, including: PCR reaction solution, PBoVG1 virus standard, PBoVG2 virus standard, PBoVG3 virus standard, positive control, negative control , a box body, the box body is provided with container holes, which are respectively used to place PCR reaction solution tubes, PBoVG1 virus standard tubes, PBoVG2 virus standard tubes, PBoVG3 virus standard tubes, positive control tubes and negative control tubes, etc., wherein There are 3 tubes of PCR reaction solution (including PCR reaction buffer, TaqDNA polymerase, deoxyribonucleotide triphosphate mixture) (marked as ), PBoVG1 virus standard product is 1 tube (marked as ▲), PBoVG2 virus standard product is 1 tube (marked as ●), PBoVG3 virus standard product is 1 tube (marked as ■), positive control substance is 3 tubes (marked as ), 3 tubes of nega...

Embodiment 2

[0045] Embodiment 2 preparation of plasmid standard

[0046] Step 1: Primer Synthesis

[0047] The three groups of porcine bocavirus genogroup-specific combined primers and universal primer sequences (see Table 1) designed by the present invention were synthesized by Shanghai Sangon Biological Company Technology Service Company, and the synthesis amount was 2OD per primer.

[0048] Step 2: Total viral DNA / RNA extraction

[0049] The pig feces samples containing PBoVG1, PBoVG2 and PBoVG3 virus sources were placed in a 1.5mL centrifuge tube, and the virus nucleic acid was extracted from the pig feces samples with the AxyPrep Body Fluid Virus DNA / RNA Small Kit (AXYGEN) according to the product instruction manual to obtain Viral DNA (153 ng / μL).

[0050] The third step: PCR amplification

[0051] PCR reaction system (total reaction volume 25μL): 10×PCRbuffer 2.5μL, 25mM MgCl 2 2.5 μL, 2.5 mM dNTP 2 μL, TaqDNAPolymerase 1.25 U, with each group of porcine boca virus DNA extracte...

Embodiment 3

[0054] Embodiment 3 The present invention detects three kinds of porcine boca virus gene group specific experiments

[0055] Step 1: Primer Synthesis

[0056] With the first step in embodiment 2.

[0057] The second step: specific detection

[0058] Prepare 8 identical reaction solutions according to the enrichment multiplex PCR reaction system, that is, 10×PCR buffer 2.0μL, 25mM MgCl 2 2.0 μL, 2.5 mM dNTP 1.6 μL, TaqDNA Polymerase 2.5U, 3 pairs of combination primers and 1 pair of universal primers described in Table 1, wherein the final concentration of the upstream and downstream combination primers of PBoVG1, PBoVG2 and PBoVG3 is 0.01 μM, and the universal primers UF and UR The final concentration is 0.2μM each, and 1.0×10 5 Copies / μL PBoVG1, PBoVG2, PBoVG3, PBoVG1+PBoVG2+PBoVG3, PBoVG1+PBoVG2, PBoVG1+PBoVG3, PBoVG2+PBoVG3 plasmid standard 1.0μL, add 1.0μL deionized water as a negative control to the eighth copy, add ddH 2 O to a total volume of 20 μL of the reaction s...

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Abstract

The invention discloses a porcine bocavirus detection and parting enrichment multiple PCR rapid diagnostic kit, namely an enrichment multiple PCR rapid diagnostic kit for simultaneous detection of all known porcine bocavirus genotypes. The kit comprises a PCR solution, a standard substance and a reference substance. A kit body is provided with container holes for containing a PCR solution tube, a PBoV G1 virus standard substance tube, a PBoV G2 virus standard substance tube, a PBoV G3 virus standard substance tube, a positive reference substance tube, a negative reference substance tube and a primer tube respectively. Through one PCR, whether a sample to be detected is a porcine bocavirus or not and which genotype of PBoV G1, PBoV G2 and PBoV G3 the sample to be detected belongs to can be quickly identified. The kit is high in specificity and sensitivity, easy, convenient and fast to use, and suitable for rapid detection and classified studies on the porcine bocavirus in clinics and scientific research, and has high application value.

Description

technical field [0001] The invention belongs to the field of viral nucleic acid detection, in particular to a multiplex PCR rapid diagnostic kit for simultaneously detecting all known porcine boca virus genotypes, capable of simultaneously detecting all known porcine boca virus genotypes in the same reaction tube, applicable Rapid detection of porcine bocavirus in clinical and scientific research. Background technique [0002] Since porcine bocavirus (PBoV) was first identified in Swedish pigs with weaned multisystemic wasting syndrome (PMWS) in 2009, a growing number of studies have reported the emergence of a range of porcine bocaviruses in pig herds , and is widely prevalent in the world, but its pathogenicity, genotyping, and differential diagnosis methods are still relatively weak. [0003] Porcine Bocavirus is a single-stranded linear non-enveloped DNA virus with a genome size of 4786-5905 bp. There is no unified classification standard for its genotyping. The reporte...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2537/143
Inventor 姜永厚郑晓文刘高鹏
Owner ZHEJIANG SCI-TECH UNIV
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