肾综合征出血热患者血清中汉坦病毒的检测及其序列分析

HV RNA DETECTION BY REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION AND SEQUENCING OF AMPLIFIED PRODUCTS

目的 为了进一步探讨RT -PCR用于HFRS临床标本中病毒基因检测的可行性及陕西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 本研究设计并合成了两套分别互补于HVM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 -nestedPCR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病人血清 (经临床和IFA确诊 )经用S基因片段引物的RT -nestedPCR检测 ,阳性率分别为 :6 0 .5 % (<1w) ,34.1% (1～ 2w) ,4% (2～ 3w) ,0 % (>3w) ,31份HFRS急性期病人血清用M基因片段引物的RT -nestedPCR检测 ,仅 12份阳性 ,阳性率 40 %。结论 表明S基因片段引物的扩增效果优于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 ,陕西西安地区HFRS感染的主要毒株为汉滩型 ,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 %。

Aim In order to explore and verify the possibility of application of RT-PCR for diagnosis of hemorrhagic fever with renal syndrome(HFRS), and to study the variations of hantaviruses(HV),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. The serum samples from 119 HFRS patients (IFA positive) were tested by S gene primers and RT-PCR. The positive rates were 60 5%(<1w), 34.1%(1～2w),4%(2～3w),0%(>3w) respectively. The serum samples from 31 acute HFRS patients were detected by M gene primers and RT-PCR, only 12 samples were positive (40%).It suggests that the primers complemented to S gene are more efficient than the ones complemented to M gene. Basing on the results of DNA sequencing, it shows that Hantaan virus is a predominant serotype harbored and transmitted in Xi'an district, Shaanxi province. Compared with coding region of S gene of HV strain 76-118, the identity of the nucleotides between them is from 87.2% to 96.0%, identity of the deduced amino acid between them is from 89.0% to 96.0%.