小反刍兽疫病毒RT-LAMP快速检测和鉴别方法的建立

Establishment of RT-LAMP for the detection and differentiation of peste des petits ruminants virus

采用人工合成方法获得小反刍兽疫病毒N蛋白基因的核酸质粒,并利用该质粒进行小反刍兽疫病毒通用型引物和鉴别型引物的筛选。再经过对中国动物卫生与流行病学中心临床样品进行敏感性试验和对最佳引物进行验证,从而建立鉴别小反刍兽疫病毒新疆株和西藏株的RT-LAMP方法。结果显示,在65℃50min内即可完成对小反刍兽疫病毒的检测和鉴别。特异性、灵敏性试验结果表明,建立的RT-LAMP方法中通用型引物的灵敏度与Real-time RT-PCR相当,鉴别型引物的灵敏度是RT-PCR的10倍,临床样品的检测结果表明,本研究建立的RT-LAMP的准确性高于北京世纪元亨公司研制的小反刍兽疫病毒RTPCR检测试剂盒。

The nucleotide plasmid with peste des petits ruminants virus（PPRV）N protein gene was artificially synthesized instead of extraction from the clinical samples.This plasmid was used to screen the universal primers and the differential primers.RT-LAMP method for the detection and differentiation of peste des petits ruminants virus was established and assessed through the sensitivity and specificity experiments using the clinical samples preserved in China Animal Health and Epidemiology Center.Under the optimized condition,PPRV can be detected and differentiated at 65℃in 50 minutes.The specificity of this assay was examined to detect different PPRV isolates,showing no cross-reaction with other related viruses.The detection limit of the universal primers is similar to that in Real-time RT-PCR and the sensitivity of the differential primers is 10-fold higher than that in the RT-PCR.Also,the accuracy of this method on clinical diagnosis is higher than that using the RT-PCR detection kit of peste des petits ruminants virus produced by Beijing Anheal Laboratories Co.,Ltd.