羊口疮病毒环介导等温扩增高效检测方法的建立  

Establishment of a loop-mediated isothermal amplification(LAMP) for rapid detection of ORFV

作　　者：王海峰[1] 张七斤[1] WANG Hai-feng;ZHANG Qi-jin(College of Veterinary Medicine,Inner Mongolia Agricultural University,Hohhot,Inner Mongolia 010018,China)

机构地区：[1]内蒙古农业大学兽医学院

出　　处：《中国兽医学报》2018年第7期1282-1286,1306,共6页Chinese Journal of Veterinary Science

基　　金：国家重点研发计划资助项目(2017YFD0500903)

摘　　要：羊口疮（ORF）的快速诊断对于该病的防控至关重要，而环介导等温扩增（LAMP）技术可以满足对羊口疮病毒（ORFV）高效性的现场检测需求。本试验对ORFV VIR基因扩增，通过优化反应条件，对LAMP检测ORFV进行了显色反应、特异性和灵敏性的检测，并进行了后续的临床检测。结果显示，利用优化的LAMP染色法，将呈现绿色的阳性组与橙色的阴性组区分开。对扩增的质粒和阳性对照样品的检测呈现出了阳性条带，而对山羊痘、绵羊痘、牛传染性鼻气管炎和牛副流感3型病毒的检测并无阳性条带。优化的LAMP检测ORFV的灵敏度比传统PCR高出1000倍。对15份临床样品检测的结果表明，优化的LAMP电泳和染色法对ORFV的检出率均为100％，与传统PCR检测结果相一致。上述结果表明优化的LAMP对ORFV的检测具有特异性、高灵敏性且更加方便等优点，从而更适用于现场的高效检测。Rapid diagnosis of ORF is essential for the prevention and control of this disease,and the loop mediated isothermal amplification（LAMP） technique can meet the needs of on-site detection of the ORF virus（ORFV）. In the study,the VIR gene amplification of ORFV was carried out,and the chromogenic reaction,specificity and sensitivity of LAMP were detected using optimized condi- tions. The clinical detection of ORF was carried out by using optimized LAMP. The results showed that using the optimized LAMP, the positive group in the color green was separated from the negative group in the color orange. Detection of the amplified plasmid and positive control samples showed positive bands. But there were no positive bands in samples of goat pox virus, sheep pox virus,infectious bovine rhinotracheitis virus and bovine parainfluenza type 3 virus. The sensitivity of the optimized LAMP to detect ORFV was 1 000 times higher than that of the traditional PCR. The detection results of 15 clinical samples showed that the detection rate of ORFV was 100% using optimized LAMP, which was consistent with the results of conventional PCR. In conclusion, the re- sults showed that the optimized LAMP has the advantages of specificity,high sensitivity and con- venience for ORFV detection,so it is more suitable for high efficiency on-site detection.

关 键 词：羊口疮病毒 环介导等温扩增技术 临床检测 

分 类 号：S852.65[农业科学—基础兽医学]