RT-PCR技术在流行性出血热病毒分型诊断中的应用

Diagnosis Study on Patients with EHF Virus I、II type by RT-PCR

目的 运用逆转录聚合酶链反应 (RT- PCR)技术对流行性出血热 (EHF)病人标本进行研究 ,以建立一项有效的 EHF病毒分型诊断方法。方法 收集发病 10 d以内的早期 EHF病人血浆、外周血单个核细胞 (PBMC)、尿沉淀细胞。采用异硫氰酸胍 -酚 -氯仿一步抽提各种标本的总RNA。两对特异性引物分别互补于汉滩病毒 ( 型 )及汉城病毒 ( 型 ) M基因片段的 G1区。分别在两对引物的正链引导下反转录合成特异性 c DNA,用相应的引物对进行 PCR扩增。产物经琼脂电泳 ,标准分子量对照鉴定。同时以 Vero- E6细胞及鼠脑传代的标准病毒株进行阳性对照 ,以正常人血浆作阴性对照。结果 19例病人的 30份标本均能被 I型或 型引物扩增出特异性产物 ,其中I型阳性 13例 , 型阳性 4例 ,I、 型同时阳性 2例 ,同一病人的不同标本结果一致。结论 标准病毒株 RT-

Objective A study wase made on patient samples using a advanced reverse transcription polymerase chain reaction (RT PCR) for the target of constructing a gene typing diagnosis method of epidemic hemorrhagic fever(EHF).Methods Patient samples include plasma,peripheral blood mononuclear cells(PBMC) and uric tract cells(UTC),being collected 1～10 days after onset of symptoms.Total RNA was extracted from the samples by guanidinum thiocyanata phenol chloroform method. Two sets of primers were designed from the M gene segment of Hantaan virus(type I) or Seoul virus (type II). The purified RNA was used to reverse transcription under the guide of every sense primer.Then,the specific sequence of cDNA was amplified by PCR.And the products of the PCR were verified by agarose electrophorosis.At the same time,standard virus strains serotype I,serotype II were detected. Results All 30 samples of 19 patients were amplified by type I or type II primer pairs,showing samples of plasma,PBMC and UTC can be used for the RT PCR.In 19 patients,there were 13 infected by type I virus and 4 by type Ⅱ,The samples of other 2 patients could be amplified by the 2 sets of primers.Conclusion RT PCR is a simple,sensitive,specific and reliable method for gene typing diagnosis of EHF. [