荧光显色在环介导等温扩增(LAMP)检测猪繁殖与呼吸综合征病毒的应用  

Fluorescent Reagent in Detection of Porcine Reproductive and Respiratory Syndrome virus by Loop-mediated Isothermal Amplification (LAMP)

作　　者：张跃伟[1] 李旭妮[1] 郭盼盼[1] 付萍[1] 李佳禾[1] 黄书林[1] 蒋菲[1] 吴文学[1] 

机构地区：[1]中国农业大学动物医学院,北京100193

出　　处：《农业生物技术学报》2010年第3期508-513,共6页Journal of Agricultural Biotechnology

基　　金：农业部重点项目(No.2008-G57)资助

摘　　要：介绍了一种应用荧光显色的猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)反转录环介导等温扩增(RT-LAMP)的检测方法。该方法使用了针对靶序列M+N基因的一组引物,并且能在63℃等温条件下30min内完成反应。在敏感性检测中RT-LAMP与RT-PCR都能检测103个包含靶基因片段的重组质粒。几种其它病原的核酸也被用来比较这两种方法的特异性,两种方法只能检测PRRSV。在荧光显色剂应用试验中,证实0.05mmol/L钙黄绿素以及0.6mmol/L锰离子可以用于LAMP扩增产物的判断,并且其判断结果与浊度判断结果一致。在对103个临床血液样本的检测中,RT-LAMP与RT-PCR检出的阳性样本数量分别是56个和48个。结果说明,应用荧光显色剂的RT-LAMP检测方法可以快速检测PRRSV。A novel assay method of Porcine reproductive and respiratory syndrome virus （PRRSV） termed reverse transcriptase loop-mediated isothermal amplification （RT-LAMP）, was reported using fluorescent reagent as the LAMP product judgment. With one set of special primers targeting sequence containing the M and N genes together, the LAMP-based assay could be completed within 30 min at 63℃. The detect limit of the assay was 103 copies per reaction determined by using a recombined plasmid containing the target sequence, which was the same as RT-PCR assay. The specificities of two methods were confirmed using genomes of various viruses, which only determined PRRSV. In the fluorescent reagent test, 0.05 mmol/L calcein and 0.6 mmol/L manganese were confirmed to be appropriate in LAMP product judgment, and the result was the same as turbidity test. In detection of Porcine reproductive and respiratory syndrome virus （PRRSV） in blood samples from 103 field piglets, 56 and 48 piglets, respectively, were positive by the RT-LAMP and RT-PCR assays. These results suggest that the RT-LAMP assay with fluorescent reagent provides a useful tool for the diagnosis of PRRSV infections in porcine rapidly.

关 键 词：反转录环介导等温扩增(RT-LAMP) 猪繁殖与呼吸综合征病毒(PRRSV) RT-PCR 荧光显色剂 

分 类 号：S852.65[农业科学—基础兽医学]