摘要
采用5′端生物素标记汉滩病毒特异性寡核苷酸探针,结合磁性分离技术和异硫氰酸胍-酚一步法(胍酚法)提取病毒RNA,进行反转录-套式PCR,检测肾综合征出血热(HFRS)病人血清样本,胍酚法能检测血清中至少几个PFU病毒的RNA,比磁珠法敏感10倍,整个检测过程在5小时完成。应用该方法对137份汉滩型和汉城型HFRS病人血清进行检测分型,总阳性率达60.50%。其中,病程在7日以内(急性期)的病人血清仍能检测到22.73%阳性率。扩增产物经打点杂交检测证实为特异性扩增,并能准确分型。与空斑减少中和试验(PRNT)分型的符合率为100%。对20份正常人及24份非HFRS患者血清进行PCR扩增,结果均为阴性。将全部试剂做成试剂盒,为基层单位早期诊断肾综合征出血热病人提供了操作简便、特异、敏感、快速、直接的诊断方法。
The magenetic separation technique combined with a biotinylated primer which specifically hybridizes to the 5′end of M genome segment of hantavirus and a modified guanidine thiocyanate-phenol method were both tried to extract hantavirus RNA from mimic HFRS clinical sera,then the extracted RNA were detected with reverse-transcription and nested PCR (RT-nested PCR)method。Several plaque-forming units of viral RNA were detectable using RT-nested PCR by the guanidine-phenol RNA extraction method,which is ten times more sensitive than the magnetic RNA separation technique.The whole detection procedure could be completed within 5 hours.137 HFRS clinical sera were assayed by Hantaan virus specific and Seoul virus specific RT-nested PCR.The total positive rate was 60.5%.According to the days after onset of disease the hantavirus RNA detection rate was 100%,57.14% and 22.73% on the days of less than 7(acute phase),8-14 and more than 15 respectively.The result of typing of sera by RT-nested PCR was consistant with that by plague reduction neutralization test(PRNT).The specificity of PCR products were confirmed by dot hybridization.All the 20 normal human sera and 24 non-HFRS patients′ sera were negative.For convenient use and anti-contamination the reagents were organized into diagnostic kits,that provide a specific,sensitive,rapid and direct method for the early diagnosis of HFRS patients.
出处
《病毒学报》
CAS
CSCD
北大核心
1997年第2期119-125,共7页
Chinese Journal of Virology
基金
国家863高技术计划
欧盟科学研究发展部资助
关键词
肾综合征出血热
汉坦病毒
RNA
聚合酶链反应
Hemorrhagic fever with renal syndrome
Magnetic separation method for RNA
RT-nested PCR