Double FQ-PCR detection kit for identifying PCV (porcine circoviruses) type 2 and type 3

A porcine circovirus and kit technology, applied in the field of molecular biology, can solve problems such as low homology and loss of pig industry

Inactive Publication Date: 2018-08-10
HENAN CENT FOR ANIMAL DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In June 2015, a new type of porcine circovirus type 3 (PCV3) was first reported in the United States. This virus can also cause porcine circovirus-associated disease (PCVAD). In 2016, PCV3 virus infection was also detected in many provinces in my country. severe losses to the pig industry
The virus genome is 2.0kb in full length and encodes two main proteins: Rep and Cap proteins, among which PCV3 has low homology with the Rep protein and Cap protein genes of PCV2

Method used

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  • Double FQ-PCR detection kit for identifying PCV (porcine circoviruses) type 2 and type 3
  • Double FQ-PCR detection kit for identifying PCV (porcine circoviruses) type 2 and type 3
  • Double FQ-PCR detection kit for identifying PCV (porcine circoviruses) type 2 and type 3

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 The design of primers and probes for PCV2, PCV3 TaqMan MGB probe dual real-time fluorescence quantitative (FQ-PCR) detection and the establishment of detection methods

[0045] 1.1 Materials

[0046] 1.1.1 Positive standards and strains

[0047] PCV2 and PCV3 positive standard samples were amplified by the Henan Animal Disease Prevention and Control Center by PCR to amplify the PCV2 and PCV3 target genes respectively, and the target genes were cloned into the pGEM-T Easy vector to obtain PCV2 and PCV3 positive standard products; other control viruses, bacteria or Positive recombinant plasmids were provided by Henan Animal Disease Prevention and Control Center.

[0048] 1.1.2 Instruments and reagents

[0049] Fluorescent PCR instrument, product of American ABI Company, model ABI7500; PCR amplification instrument, product of German Biometra Company; gel imaging analysis system, product of American Alpha Innotech Company; constant temperature water bath oscilla...

Embodiment 2

[0097] Example 2 PCV2, PCV3 double real-time fluorescent quantitative PCR (FQ-PCR) detection kit

[0098] The dual FQ-PCR detection kit of the present invention includes PCV2 detection primer pair and probe (SEQ ID NO: 1-3), PCV3 detection primer pair and probe (SEQ ID NO: 4-6).

[0099] The detection kit also includes one or more of dNTPs, reverse transcriptase, Taq DNA polymerase, PCR reaction buffer, positive standard, negative control and the like.

[0100] The specific application of the detection kit is as follows:

[0101] (1) Sample requirements

[0102] 1. Apply proper technique to collect samples.

[0103] 2. The sediment and suspended matter in the sample may affect the test results and should be removed by centrifugation.

[0104] 3. Sample processing and collection should not be left at room temperature for more than 6 hours; if not tested within 6 hours, the sample should be placed in a refrigerator at 2-8 °C; if it needs to be stored or transported for more t...

Embodiment 3

[0126] Example 3 Application of PCV2, PCV3 double real-time fluorescent quantitative PCR (FQ-PCR) detection technology

[0127] PCV2, PCV3 dual FQ-PCR detection method established according to the present invention and PCV2 / PCV3 general PCR detection method established before (PCV2-P7: 5'-GGT GCC CGC TGC CAC ATC-3', PCV2-P8: 5' -GGGAAAGGG TGACGA ACTG-3'; PCV3-P9:5'-ATGAGACACAGAGCTATATTCA-3', PCV3-P10:5'-TTAGAGAACGGACTTGT AACG-3'; PCV2 reaction system is calculated as 25μl: the final concentration of primers P7 and P8 is 0.4 μmol / L, 2.5mmol / L dNTPs 2μL, 10×Ex Taq enzyme buffer 2.5μL, 5U / μL Ex Taq DNA polymerase 0.2μL, DNA template 2μL, deionized water to make up to a total volume of 25μL; the amplification reaction conditions are : 94°C for 5 minutes; 94°C for 30 seconds, 56°C for 30 seconds, 72°C for 10 minutes, a total of 40 cycles; PCV3 reaction system is the same as PCV2, the amplification reaction conditions are: 94°C for 5 minutes; 94°C for 30 seconds, 52°C 30 seconds, 1...

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Abstract

The invention provides a double FQ-PCR detection kit for identifying PCV (porcine circoviruses) type 2 and type 3. By aiming at the characteristic of low Rep protein and Cap protein gene homology of PCV2 and PCV3, a pair of specific primers and a TaqMan MGB probe (SEQ ID NO:1-6) are designed by using Rep protein gene of PCV2 and Cap protein gene of PCV3 as amplification target regions; the real-time fluorescence quantitative PCR technology is used; the identification detection of the PCV2 and the PCV3 is realized. The detection kit provided by the invention is applicable to viral nucleic aciddetection in samples such as nose swab, oral cavity secreta, faeces, serum, lungs, lymph gland and kidneys of a suspected PCV infectious pig; the sensitivity can reach 1.0*10<1> copy/mu L; the crossedreaction with other pathogene capable of generating mixed infection or with similar of infection symptoms do not exist.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a double FQ-PCR detection kit for distinguishing porcine circovirus type 2 and type 3. Background technique [0002] Porcine circovirus (Porcine circovirus, PCV) is a covalently closed, single-stranded circular DNA virus, which is the smallest animal virus discovered so far. Before 2015, only two genotypes of PCV1 and PCV2 were found worldwide. PCV1 generally does not cause pig disease. Porcine circovirus-associated disease (PCVAD) caused by PCV2 is currently a major disease that endangers the pig industry. The clinical manifestations are weaned piglet multisystem wasting syndrome (PMWS), respiratory and intestinal diseases, Reproductive disorders and porcine dermatitis and nephrotic syndrome (PDNS). In June 2015, a new type of porcine circovirus type 3 (PCV3) was first reported in the United States. This virus can also cause porcine circovirus-associated disease (PCVAD). In 20...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2600/16C12Q2531/113C12Q2537/143C12Q2561/101
Inventor 闫若潜班付国赵雪丽王东方王华俊马震原王淑娟谢彩华曹伟伟房大学李桂莲仲伟平闫志玲张代宝
Owner HENAN CENT FOR ANIMAL DISEASE CONTROL & PREVENTION
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