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APP, M.hyo, PCV-2 and PRRSV multiplex PCR detection primer, kit and detection method

A PCV-2 and multiplex technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of time-consuming, laborious, low sensitivity, complicated operation, etc., and achieve high sensitivity and high specificity , low cost effect

Active Publication Date: 2015-01-28
重庆海关技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the existing methods for detecting APP, M.hyo, PCV-2 and PRRSV mainly rely on traditional methods such as pathological anatomy, pathogenic bacteria isolation, and serological methods, which are complicated, time-consuming, laborious, and low-sensitivity, making it difficult to directly distinguish APP, M.hyo, PCV-2 and PRRSV

Method used

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  • APP, M.hyo, PCV-2 and PRRSV multiplex PCR detection primer, kit and detection method
  • APP, M.hyo, PCV-2 and PRRSV multiplex PCR detection primer, kit and detection method
  • APP, M.hyo, PCV-2 and PRRSV multiplex PCR detection primer, kit and detection method

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Experimental program
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Effect test

Embodiment 1

[0042] Embodiment 1, the component of kit

[0043] 1. Design and screening of primers

[0044]According to the conserved gene nucleotide sequences of Actinobacillus pleuropneumoniae APP, Mycoplasma hyopneumoniae M.hyo, porcine circovirus type 2 PCV-2 and porcine reproductive and respiratory syndrome virus PRRSV published by GenBank, the software Primer Primer 5.0 software was used to design primers and perform BLAST sequence alignment to verify primer specificity. Primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd.

[0045] The sequence represented by APP SEQ NO.1 is: 5'-TAGTGCTTACCGCATGTAGTGG-3'

[0046] The sequence represented by APP SEQ NO.2 is: 5'-TGGCTGCTCCGCTTTTGG-3'

[0047] The sequence represented by M.hyo SEQ NO.3 is: 5'-GCCGAACAAGCAATCACCAA-3'

[0048] The sequence represented by M.hyo SEQ NO.4 is: 5'-ACCCGAAGAACTTCAACAGCA-3'

[0049] The sequence represented by PCV-2 SEQ NO.5 is: 5'-CGTTACCGCTGGAGAAGG-3'

[0050] The sequence represented by ...

Embodiment 2

[0060] Embodiment 2, single multiplex PCR reaction condition

[0061] The single-plex PCR reaction system of Actinobacillus pleuropneumoniae APP, Mycoplasma hyopneumoniae M.hyo, porcine circovirus type 2 PCV-2 and porcine reproductive and respiratory syndrome virus PRRSV are: each of the upstream and downstream primers is 0.25 μL (20 μmol / L), Premix Taq 12.5 μL, viral genome template or cDNA template after reverse transcription 1 μL, add ddH 2 0 to 25 μL system. The PCR reaction conditions were: 94.0°C pre-denaturation for 5 min; 94.0°C denaturation for 30 s, 58.0°C annealing for 30 s, 72.0°C extension for 30 s, 35 cycles; 72.0°C extension for 7 min; 4.0°C storage.

[0062] refer to figure 1 The results showed that the genomic DNA or cDNA of Actinobacillus pleuropneumoniae APP, Mycoplasma hyopneumoniae M.hyo, porcine circovirus type 2 PCV-2 and porcine reproductive and respiratory syndrome virus PRRSV were in the same PCR reaction system Specific amplification was obtained,...

Embodiment 3

[0063] Embodiment 3, multiple PCR reaction conditions

[0064] The quadruple PCR reaction system of Actinobacillus pleuropneumoniae APP, Mycoplasma hyopneumoniae M.hyo, porcine circovirus type 2 PCV-2 and porcine reproductive and respiratory syndrome virus PRRSV is as follows: mixed upstream and downstream primers 0.25 μL (20 μmol / L), Premix Taq 12.5 μL, viral genome template or reverse-transcribed cDNA four kinds of mixed templates 1 μL, add ddH 2 0 to 25 μL system. The PCR reaction conditions were: 94.0°C pre-denaturation for 5 min; 94.0°C denaturation for 30 s, 56.0-61.0°C annealing for 30 s, 72.0°C extension for 30 s, 35 cycles; 72.0°C extension for 7 min; 4.0°C storage.

[0065] refer to figure 2 The results showed that the genomic DNA or cDNA mixed system of Actinobacillus pleuropneumoniae APP, Mycoplasma hyopneumoniae M.hyo, porcine circovirus type 2 PCV-2 and porcine reproductive and respiratory syndrome virus PRRSV were amplified by gradient PCR. Increase, lane M ...

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Abstract

The invention discloses an APP, M.hyo, PCV-2 and PRRSV multiplex PCR detection primer, a kit and a detection method. In the method, 4 pairs of specific PCR primers are designed according to the gene conservative sequences of 4 pathogens in GenBank, an optimized multiplex PCR system is used for simultaneously detecting one or multiple of APP, M.hyo, PCV-2 and PRRSV swine pathogens, performs non-specific amplification on the genomes of other common pathogens, and the lowest concentration of detected pathogen genomes reaches pg grade. The kit comprises an amplification reaction liquid tube, a positive control tube, a negative control tube and sterilized deionized water. By adopting the invention, single or mixed infection of the APP, M.hyo, PCV-2 and PRRSV pathogens in a sample to be detected within 3 hours, the operation is simple, convenient and quick, the result is accurate and the sensitivity is high.

Description

technical field [0001] The invention relates to the field of animal epidemic molecular biology testing methods and testing reagents, in particular to Actinobacillus pleuropneumoniae APP, Mycoplasma hyopneumoniae M.hyo, circovirus type 2 PCV-2 and porcine reproductive and respiratory syndrome virus A detection primer, a kit and a detection method for PRRSV multiplex PCR detection. Background technique [0002] Porcine respiratory disease syndrome (PRDC) is a general term for porcine respiratory diseases caused by various factors such as viruses, bacteria, mycoplasma, parasites, environmental factors, weaning stress and low immunity of pigs. The clinical manifestations are fever, cough, wheezing, dyspnea, abdominal respiration, increased eye and nose secretions, anorexia, rapid weight loss, rough hair, and sudden death may occur. The disease incidence rate can reach 60%, and the case fatality rate is 20%-90%. After the piglets are weaned, especially the 18-20 week-old piglet...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/686C12Q1/701C12Q2600/112C12Q2537/143
Inventor 李应国聂福平杨俊王昱陈玉燕王国民
Owner 重庆海关技术中心
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