关键词: 口蹄疫病毒, 荧光RT-PCR, 体外转录cRNA, 检测

Abstract:

【Objective】 Foot-and-mouth disease (FMD) is a serious political and economic disease worldwide and is caused by foot-and-mouth disease virus (FMDV). In order to improve the diagnostic speed and accuracy of FMDV, especially for the early detection of FMDV transmission on the boundary quarantine, the one-step real-time quantitative RT-PCR (RT-qPCR) method was established to detect Euroasiatic serotype (EAT) and South Africa serotype (SAT) FMDV simultaneously. 【Method】 Aiming to amplify the conserved 5' untranslated region (UTR) of FMDV genomic sequence, the specific primers(U6/L6)and probe(P6) for EAT, primers (AY48U/L) and probe (AY48P) for SAT of FMDV were designed to establish a multiplex qPCR detection method for FMDV. The amplification solution and reaction conditions were optimized using plasmid inserted with target DNA fragment as template. To offer control for the established qPCR assay, the respective cRNAs were achieved by in vitro transcription using T7 promoter ligated PCR amplicons as template. The sensitivity and specificity of the established assay were then verified using the subsequent cRNA as template. The field samples were adopted to examine the efficiency of the final established FMDV one-step multiplex RT-qPCR detection method. 【Result】 The amplification efficiency was both up to 90% in FMDV multiplex qPCR assay using EAT and SAT plasmids as targets. The in vitro transcription cRNAs, tested to be without plasmid DNA residual, were used to be as positive control for EAT and SAT FMDV one-step multiplex RT-qPCR. The detection limit was 7.6×10-9ng cRNA for SAT and 6.3×10-8ng cRNA for EAT, respectively. Specificity test indicated that EAT and SAT FMDV could be distinguished efficiently using the developed method. The examining result was in accordance with the actual infection status of the field samples.【Conclusion】The established EAT and SAT FMDV one-step multiplex RT-qPCR assay will supply an effective approach to distinguish EAT from SAT FMDV contamiantion and for thd use of FMDV epidemiology investigation.

Key words: foot-and-mouth disease virus, real-time RT-PCR, in vitro transcription cRNA, detection