Detection of the Hantavirus RNA in Eulaelaps Stabular is Uising a Nested Reverse Transcriptase Polymerase Chain Reaction(nested RTPCR)

用HTn病毒姬鼠型M基因型特异性引物，异硫氰酸胍一步法提取rnA，nESTEdrTPCr检测鼠肺和厩真厉螨体内76118株病毒rnA，扩增产物经琼脂糖凝胶电泳和点印迹杂交证实。结果表明，姬鼠型76118株病毒液和叮刺感染病毒乳鼠后第10天厩真厉螨组织悬液接种乳鼠第9天鼠肺、叮剌后第10天和30天螨，均检出病毒rnA。30只螨样本提取rnA也能扩增出病毒rnA。家鼠型ur株感染样本不能被扩增。该方法具有较高特异性和敏感性，操作简单，结合原位分子杂交，可用于螨媒传播HfrSV的研究。double polymerase chain reaction,using nested primers of M genome segmant of Hantaan virus,was established and applied to detect the viral RNA extracted from the experimental mites and mice by a single step of acid guanidinium thiocyanatephenolchloroform.The results were showed in agarose gel electrophoresis and dotblot hybridization by digoxigenin enzyme linked immunosorbent assay system.The virus was detected in lungs of mice at the 9th day postinoculation of the tissue suspension of the mites infected with Hantaan virus,and in the mites at 10 and 30 days post infection.No virus was detected from mites with Seoul strain infection.This indicated that the nested RTPCR can be used as a simple,sensitive,specific method with in situ hybridization for study of transmission of HFRSV by gamasid mites.国家自然科学基