应用反转录-聚合酶链反应检测口蹄疫病毒  

A POLYMERASE CHAIN REACTION FOR THE DETECTION OF FMDV IN TISSUES AND O-P SAMPLES

作　　者：朱彩珠[1] 卢永干[1] 邱孝高[1] 赵启祖[1] 谢庆阁[1] 张维德[1] 员美蓉[1] 杨福荣[1] 张强[1] 

机构地区：[1]中国农业科学院兰州兽医研究所

出　　处：《病毒学报》1998年第3期272-278,共7页Chinese Journal of Virology

摘　　要：建立了一种适用于检测动物（猪、牛、羊）组织（肌肉、淋巴结、脊髓、扁桃体和蹄冠皮）和牛食道－咽部分泌物（Ｏ－Ｐ液）中的口蹄疫（ＦＭＤ）病毒核酸（ＲＮＡ）的反转录－聚合酶链反应（ＲＴ－ＰＣＲ）技术。引物对是人工合成的两条２０ｍｅｒ寡核苷酸片段，它们的序列相应于ＦＭＤ病毒结构蛋白ＶＰ１基因后２／３区段，在４个血清型之间基本一致（保守）。ＰＣＲ产物经琼脂糖凝胶电泳检测。试验结果表明，ＲＴ－ＰＣＲ具有良好的特异性，属该４个血清型的各个毒株都获得了预计长度的ＤＮＡ片段；而相关的小ＲＮＡ病毒科的猪水泡病（ＳＶＤ）和柯赛奇Ｂ５（Ｃｏｘ．Ｂ５）病毒（特异性对照），及未感染的细胞培养物和动物组织（空白对照）都是阴性。与常规检测方法相比，该ＲＴ－ＰＣＲ的检测灵敏度提高６－１２倍，最高可检测２０ＴＣＩＤ５０（细胞毒）或０．１６～０．３２ＬＤ５０（组织毒）的病毒量，二次扩增还可提高检测灵敏度１００倍。因为直接利用组织样品，检测试验快速简便，可在２４小时内获得结果。应用该方法已检测了２３２份组织样品和５８份Ｏ－Ｐ液样品。A reverse transcription polymerase chain reaction (RT-PCR) technique was developed and applied to detect food-and-mouth disease (FMD) virus RNA in animal (swine,sheep and cattle) tissues (tonsil,lymph node,spinal cord,muscle,skin of foot) and in bovine oesophageal-pharyngeal secreta (O-P fluid).The pair of primers used in the RT-PCR were 2 synthetic 20-mer oligonucleotides,spanning to the 3′ side 2/3 of VP1 region of viral genome,and their sequences were almost the same among 4 types (A,O,C,Asia-1) of FMDV.PCR-amplified DNA segments were determined by electrophoresis on 1% agarose gel.The results proved that the RT-PCR has a good specificity,being able to amplify DNA of expected length regardless of the serotype of the virus.But all the negative results were given from the related picornavirus (SVDV and coxsackievirus B5,as specific controls),or the uninfected cell cultures and tissues (background controls).In comparison with the routine methods,the RT-PCR technique was able to detect 20TCID 50 of viral RNA extracted from cell cultures, or 0.16-0.32 LD 50 from tissues,at least 6-12 times more sensitive than infectivity assay.The sensitivity of detection was increased by 100 times if a second-round of PCR was performed.And the technique was rapid and simple because the tissue samples were directly assayed and the results could be obtained within 24 hours. Using this technique, 232 tissues and 58 O-P samples have been examined and the results were reliable and satisfactory.

关 键 词：RT-PCR FMDV RNA萃取 动物组织 O-P液 

分 类 号：S852.65[农业科学—基础兽医学] S854.43[农业科学—兽医学]