实时荧光RT-PCR检测新型布尼亚病毒核酸方法的研究与应用

Research and application of real-time fluorescent RT-PCR in detection of novel Bunyavirus nucleic acid

目的研究实时荧光定量反转录-聚合酶链(Real-timeRT-PCR)检测新型布尼亚病毒的方法,用于新布尼亚病毒的核酸检测,为发热伴血小板减少综合征(SFTS)的早期诊断提供科学依据。方法以新布尼亚病毒L基因为靶基因设计引物以及TaqMan探针,建立Real-timeRT-PCR方法,并对岱山县72份临床疑似病人血清、血浆、30份正常人群血清进行了检测鉴定,并验证了方法的特异性、敏感性和重复性。结果建立的Real-time RT-PCR具有良好的特异性和敏感性,CV<1.0%。在102份检测标本中,72份临床疑似病人血清、血浆,阳性48份,30份正常人群血清阴性。结论本研究建立的Real-time RT-PCR方法具有较高的特异性、敏感性和重复性,可用于新布尼亚病毒核酸的快速检测、在发热伴血小板减少综合征的早期诊断上具有重要意义。

Objective To develop a real - time fluorescent RT - PCR method for detecting novel Bunyavirus nucleic acid, so as to provide scientific basis for early diagnosis of severe fever with thrombocytopenia syndrome. Methods Primers and TaqMan probe were designed based on the L gene of novel Bunyavirus. Real time RT - PCR was established to identify novel Bunyavirus in serum samples of 72 clinical suspected patients and 30 healthy people, and the specificity, sensitivity and reproducibility of the method were verified. Results The developed real - time RT - PCR method had good specificity and sensitivity. The repro- ducibility experiments implied the intraassay coefficient of variation was lower than 1.0%. In 102 samples, 48 samples were positive from 72 clinical patients, while all the 30 serum samples from healthy people were negative. Conclusion The devel- oped real time RT - PCR method exhibited higher specificity, sensitivity and reproducibility, so it could be used for rapid nucleic acid detection of novel Bunyavirus in human. It was also of significance for the early diagnosis of fever with thrombocytopenia syndrome.