HFRS患者血清中HV-RNA的PCR检测及扩增产物的测序分析

HV-RNA detection by reverse transcription-polymerase chain reaction and sequencing of amplified products

目的 探讨 RT- PCR用于 HFRS临床标本中病毒基因检测的若干影响因素及西安地区近年主要流行汉坦病毒(HV)毒株的分子流行病学特点 .方法 设计合成了两套分别互补于 HV M基因片段和 S基因片段的引物 ,建立了检测HFRS患者临床血清标本中 HV基因的 RT- nested PCR方法 ,并对扩增的部分 HV靶基因进行了测序分析和比较 .结果 119份 HFRS患者血清 (经临床和 IFA确诊 )经用 S基因片段引物的 RT- nested PCR检测 ,阳性率分别为 :6 0 .5 %(<1wk) ,34 .1% (1～ 2 wk) ,4.0 % (2～ 3wk) ,0 % (>3wk ) ,31份 HFRS急性期患者血清用 M基因片段引物的RT- nested PCR检测 ,仅 12份阳性 ,阳性率 40 .0 % .将 PCR扩增的 6个靶基因序列与 76～ 118株 S基因片段相应的核苷酸序列进行分析比较 ,同源性为 87.2 %～ 96 .0 % ;其推导的氨基酸序列与 76～ 118株核衣壳蛋白相应的氨基酸序列比较 ,同源性为 89.0 %～ 96 .0 % .结论 S基因片段引物的PCR扩增效果优于 M基因片段引物 .6份 S基因片段引物扩增产物的测序结果显示 ,西安地区引起

AIM To explore and verify the possibility of ap-plication of RT-PCR for diagnosis of hemorrhagic fever with renal syndrome (HFRS), and to study the variations of hantaviruses (HV). METHODS We synthesized two sets of PCR primers complemented to M gene and S gene of HV respectively and conducted RT-nested PCR for detecting clinical serum samples. RESULTS The serum samples from 119 HFRS patients (IFA positive) were tested by S gene primers and RT-PCR. The positive rates were 60.5% (<1 wk), 34.1% (1~2 wk), 4.0% (2~3 wk), and 0% (>3 wk) respectively. The serum samples from 31 acute HFRS patients were detected by M gene primers and RT-PCR; only 12 samples were positive (40.0%). Compared with coding region of S gene of HV strain 76~118, the identity of the nucleotides between them was from 87.2% to 96.0%, and that of the deduced amino acid between them was from 89.0% to 96.0 %. CONCLUSION It is suggested that the primers complemented to S gene are more efficient than the ones complemented to M gene. Based on the results of DNA sequencing, our study shows that Hantaan virus is a predominant serotype harbored and transmitted in Xi'an district, Shaanxi province.