The development of a rapid loop-mediated indirect PCR method for detection and differentiation of highly and lowly pathogenic porcine reproductive and respiratory syndrome virus / 病毒学报

ABSTRACT

The aim of this study is to establish the method of loop-mediated indirect PCR assay for detection of Reproductive and Respiratory Syndrome Virus (PRRSV) infection and differentiation of highly pathogenic PRRSV (HP-PRRSV) and lowly pathogenic PRRSV (LP-PRRSV). Based on the alignments of ORF2 gene sequences and ORFla gene sequences of PRRSV Chinese isolates deposited in GenBank, two pairs of specific probes were designed and labeled to both ends of the soybean Lectin gene fragment by PCR, respectively. The probe-labeled soybean Lectin genes were used to be reporter genes for detection and differentiation of PRRSV. After one round strand displacement reaction, the reporter genes were amplified by reverse PCR. The specific PCR products were 193bp, 355bp for HP-PRRSV and 193bp, 442bp for LP-PRRSV, respectively. The method could detect 5. 6 TCID50/mL LP-PRRSV RNA and 18 TCIDs0/ mL HP-PRRSV RNA, and co-infection did not affect detection sensitivity. No amplification was observed with other porcine originated pathogens including CSFV, PPV, PRV, PCV2, ETEC and Haemophilus parasui. Twenty clinical samples were used for comparative testing with conventional PCR. Fourteen samples were found positive for PRRSV by the loop-mediated indirect PCR, of which 4 were LP-PRRSV, 9 HP-PRRSV and 1 LP/HP-PRRSV co-infection, consistent with the conventional PCR test results. In conclusion, the loop-mediated indirect PCR is a simple, rapid, sensitive and specific etiologic diagnosis tool, and suitable for the differential diagnosis of HP/LP-PRRSV, especially for identification of mixed infection of HP/LP-PRRSV.