应用反转录-套式PCR方法检测肾综合征出血热病人血清中的汉坦病毒特异性RNA

HANTAVIRUS RNA DETECTION IN CLINICAL SERA OF HEMORRHAGIC FEVER WITH RENAL SYNDROME USING REVERSE TRANSCRIPTION-NESTED PCR

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.