Scientia Agricultura Sinica ›› 2011, Vol. 44 ›› Issue (21): 4499-4507.doi: 10.3864/j.issn.0578-1752.2011.21.019

• VETERINARY SCIENCE • Previous Articles     Next Articles

Development of Loop-Mediated Indirect PCR for Simultaneously Detecting Three Species of Viruses in Pigs

 ZHENG  Ming, BIAN  Chuan-Zhou, WANG  Lao-Qi   

  1. 1.郑州牧业工程高等专科学校生物技术实验室，郑州 450011
  • Received:2011-07-29 Online:2011-11-01 Published:2011-09-16

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Abstract: 【Objective】 The aim of this study is to establish a method of loop-mediated indirect PCR assay for simultaneous identification of porcine circovirus type 2 (PCV2), classical swine fever virus (CSFV) and porcine reproductive and respiratory syndrome virus (PRRSV) in pigs. 【Method】 According to the nucleotide sequences of PCV2, CSFV and PRRSV from GenBank, three pairs of specific probes were designed. By PCR, the Lectin gene fragment of soybean was labeled with different probes to be used as reporter genes aiming to the nucleotide sequences of PCV2, CSFV and PRRSV, respectively. After hybridizing with the target gene, gap filling and cyclizing, these probe-labeled reporter genes were amplified by reverse PCR with a pair of primers to detect the three species of viruses indirectly. The loop-mediated indirect PCR assay was developed, and the specific PCR products were 564 bp for PCV2, 434 bp for CSFV and 338 bp for PRRSV, respectively. The assay will be useful for the specific detection of the three species of viruses, not only for single virus but also for multiple viruses. 【Result】 The specificity and sensitivity of the loop-mediated indirect PCR assay were analyzed for single virus and multiple viruses. The results showed that the specificity was high, and no amplification was achieved to the other pathogeneses. The detection limits of the loop-mediated indirect PCR assay for multiple viruses was 0.8 pg?μL-1 for PCV2, 25 pg?μL-1 for CSFV and 6.2 pg?μL-1 for PRRSV, respectively, which was the same with the results of single virus detection. Twenty clinical samples were comparatively detected. The data showed that the results of the loop-mediated indirect PCR assay, not only for single virus but also for multiple viruses, were in accordance with the results of conventional PCR.【Conclusion】 The loop-mediated indirect PCR developed in this study is a simple, fast and specific method for detecting PCV2, CSFV and PRRSV in clinical samples, and can be used to detect all these three species of viruses at the same time.

Key words: porcine circovirus type 2, classical swine fever virus, porcine reproductive and respiratory syndrome virus, loop-mediated indirect PCR

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