8种猪呼吸道和繁殖障碍病病原体GeXP检测方法的建立

doi:10.3864/j.issn.0578-1752.2015.24.014

Abstract

Abstract: 【Objective】A GeXP-multiplex PCR assay was developed to simultaneously detect eight swine reproductive and respiratory viruses, including reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), porcine circovirus 2 (PCV2), swine influenza virus (SIV) (including H1 and H3 subtypes), porcine parvovirus (PPV), pseudorabies virus (PRV) and Japanese encephalitis virus (JEV). 【Method】Nine pairs of specific primers were designed based on the conserved sequences of eight pathogens available in the GenBank database. Each gene-specific primer was fused at the 5’ end of a universal sequence to generate 9 pairs of specific chimeric primers. The GeXP-single PCR assay was performed using a single cDNA/DNA template to determine the feasibility of the primers. The GeXP-mutiplex PCR assay was performed using a single cDNA/DNA template and 9 pairs of the specific chimeric primers to determine the specificity and accuracy of the GeXP-mutiplex PCR assay. Serial dilution from 103 copies/μL to 10 copies/μL of in vitro transcripted RNA of target genes (SIV-H1, SIV-H3, PRRSV, CSFV and JEV) and plasmid of PCV-2, PPV and PRV were used to examine the single template sensitivity of GeXP multiplex PCR assay; Serial dilution from 104 copies/μL to 10 copies/μl of all viral targets were used to examine the sensitivity of eight pathogens of GeXP multiplex PCR assay. The GeXP assay was evaluated using 23 clinical specimens and compared with the single PCR assay. 【Result】The corresponding specific fragments of genes were amplified by the GeXP -single PCR assay and the GeXP-mutiplex PCR assay. The results confirmed the feasibility of the primers and the specificity and accuracy of the GeXP assay. The GeXP assay achieved a sensitivity of 10 copies/µL for a single pathogen. The sensitivity of the GeXP assay to detect the eight pathogens simultaneously was 103 copies/µL. The GeXP assay successfully detected twenty-three clinical samples and the results of the GeXP assay were consistent with the results by the single PCR assay.【Conclusion】The GeXP assay was established successfully for simultaneously detecting eight swine reproductive and respiratory pathogens; These results showed that the GeXP assay is a specific, sensitive and high-throughput test to detect swine reproductive and respiratory pathogens. This assay provides a new test for the simultaneous differentiation of eight swine reproductive and respiratory diseases.

Key words: swine reproductive and respiratory diseases, genomeLab gene expression profiler (GeXP), multiplex PCR, universal primers, specific chimeric primers

ZHANG Min-xiu, XIE Zhi-xun, DENG Xian-wen, XIE Zhi-qin, XIE Li-ji, HUANG Li, HUANG Jiao-ling . Establishment of a GeXP Analyser-Based Multiplex PCR Assay for Detection of Eight Reproductive and Respiratory Swine Pathogens[J].Scientia Agricultura Sinica, 2015, 48(24): 4996-5006.

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