应用聚合酶链反应对我国不同来源肾综合征出血热病霉的型别分析

TYPING OF HFRS VIRUSES IN CHINA BY POLYMERASE CHAIN REACTION

应用聚合酶链反应（ＰＣＲ），对分离自不同地区、不同宿主来源的２６株肾综合征出血热病毒进行了分型，其中包括４个型别的国际标准株，用异硫氰酸胍－酚－氯仿方法从感染的Ｖｅｒｏ－Ｅ６细胞中提取总ＲＮＡ，设计了５对寡核苷酸引物，一对为汉坦病毒特异性引物；４对为不同型特异性引物。ＰＣＲ分型表明，２６株中除１株Ｒｒ可同时被汉坦（ＨＹＮ）和Ｓｅｏｕｌ（ＳＥＯ）两型特异性引物扩增外，其余２５株分别只被４个型别引物中的一个所扩增，依次为ＨＴＮ１６株，ＳＥＯ７株，Ｐｕｕｍａｌａ（ＰＵＵ）１株，ＰｒｏｓｐｅｃｔＨｉｌｌ（ＰＨ）１株。ＰＣＲ分型的结果与空斑减少中和试验完全一致，表明ＰＣＲ可以对肾综合征出血热病毒准成分型。应用限制性内切酶分析了扩增产物，结果与理论基本一致，证实了扩增产物的特异性。

his paper reports the use of polymerase chain reaction (PCR)for typing 26isolates of Hantavirus from different geographic regions and different hosts.Total cellular RNA was ex-tracted from virus infected Vero-E6 cell monolayers by the acid guanidium thiocyanate-phenol-choroform method .One set of primer was designed from a conserved re-gion of S genomic segment and used as genus -specific primers.The other 4 sets of primerswere designed from the unique sequences the M segment of each serotype ,such that each set was specific dither to Hantaan ,Seoul,Puumala or Prospect Hill serotype.The results showed that the genus -specific primers could amplify sequences from all 26 Hantaviruses but not from uninfected Vero-E6 cells .Each of the 26 isolates reacted with only one of the 4 sets of Prim -ers, among them 16,7, 1 and isolates belong to Hantaan ,Seoul ,Puumala and Prospect Hilltypes respectively.One isolate named ″Rr″ strain reacted with both Hantaan and Seoul specific primers.Thw PCR products were analyzed by restriction endonucleace digestion for futherconfirmation .The results of PCR were consistent with that of seroloigical typing by plaque re-duction neutralization test (PRNT),As a new sensitive and specific molecular technique, PCR as-say offers a method for the rapid diagnosis and for the accurate typing of new isolates of Hantaviruses.