三种核酸扩增方法检测临床乙脑标本的比较

目的探索三种核酸扩增方法应用于临床乙脑标本检测的敏感性,建立能够适合于临床标本的检测方法,同时比较病毒核酸检测与血清学检测的相关性。方法分别采用普通逆转录PCR、逆转录套式PCR和SyBr Green I实时荧光PCR方法对48份疑似乙脑病例的血清或脑脊液标本进行检测,并比较了三种方法的检出限。结果血清学检测表明,48份标本中有18份标本IgM抗体阳性;3种病毒核酸检测,普通逆转录PCR和SyBr Green I实时荧光PCR方法未能检出阳性标本,而逆转录套式PCR检出15份阳性标本。结论三种核酸检测方法中,逆转录套式PCR最为敏感;单独依赖血清学检测会产生相当一部分假阴性,证明了临床实验室加强乙脑病毒核酸检测对临床诊断具有重要意义。

Comparison of three nucleic acid amplification assays for clinical Japanese encephalitis specimens

Amplification of viral RNA from clinical specimens is another approach for diagnosis of suspected Japanese encephalitis （JE） cases. In this study, the sensitivities of three nucleic acid amplification assays were evaluated, one detection system suitable for clinical JE diagnoses was established. From the 48 specimens that included 18 positive for JEV-IgM, no viral RNA was detected by routine RT-PCR or SyBr Green I RT PCR, however, viral RNA was detected from 15 samples by RT-nested PCR. The fact that only 3 specimens were identified positive both for JEV-IgM and viral RNA revealed that considerable amount of clinical samples were negative for JEV IgM, but positive for viral RNA. Meanwhile, the detection limit of each assay was confirmed as 2.5 10^3pfu/mL for routine RT-PCR, 2.5 10^2pfu/mL for SyBr Green I RT-PCR,2.5 pfu/mL for RT-nested PCR, respectively. These results suggested RT-nested PCR was the most sensitive assay suitable for clinical JE specimens, its application to clinical samples would benefit the diagnosis of JE.