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目的 建立新型发热伴血小板减少综合征布尼亚病毒的TaqMan探针实时荧光RTPCR检测方法并进行评估,为发热伴血小板减少综合征监测中新型布尼亚病毒感染的排查提供实验室检测依据.方法 利用新型布尼亚病毒S片段基因的特异性序列设计引物和探针,探针5'端标记FAM,3'端标记TAMRA,优化反应体系与反应条件,并对不同浓度含有.目的 基因的质粒以及在发热伴血小板减少综合征监测中收集的32份样本和流行性出血热病毒阳性样本进行核酸检测,验证该方法的特异性、敏感性和重复性.结果 该方法所检测的32份可疑病例的临床样本中,3份为阳性,与国家疾病预防控制中心下发的试剂以及商品化试剂盒的检测结果一致;流行性出血热病毒检测结果为阴性.对.目的 基因的检测灵敏度达4×102拷贝/ml;重复性实验中,变异系数为0.125%～0.28%.全部检测过程从样本的核酸提取至检测完成仅需2.5h左右.结论 本文建立的TaqMan探针实时荧光RT-PCR法是一种检测新型发热伴血小板减少综合征布尼亚病毒的特异、快速、敏感的方法.该方法的建立将有益于今后开展发热伴血小板减少综合征监测以及应急临床样本中新型布尼亚病毒感染的排查和快速检测.

Objective To develop and evaluate a real-time RT-PCR method for detecting the novel Bunyavirus, based on TaqMan hybridization probe technology in order to provide a laboratory diagnosis for the novel Bunyavirus infection during the surveillance of Fever with Thrombocytopenia Syndromes. Methods A pair of primers and a probe was designed to identify S segment gene of the novel Bunyavirus. The probe was 5' end labeled with FAM and 3' end labeled with TAMRA. The PCR reaction system and condition were optimized. Different concentration of plasmid DNA containing the target gene, 32 serum samples collected from the surveillance of Fever with Thrombocytopenia Syndromes, and Hantaan virus positive samples were tested using this method to evaluate the specificity, sensitivity and reproducibility of the assay. Results 3 positive samples were detected among 32 clinic samples collected from the suspected cases, conformed to the results obtained from the detection reagents provided by Chinese National Center for Diseases Prevention and Control and the commercial kit, Hantaan virus positive samples tested were negative. The sensitivity of this assay was 4 × 102 Copies/ml. The coefficients of variation (CV) value were 0. 125% ～0. 28% during the reproducibility test. The whole process takes 2. 5h including the extraction of RNA from the sample. Conclusion This real-time RT-PCR method setting up based on TaqMan probe is a specific, rapid and sensitive method for detecting the novel Bunyavirus. The establishment of this method will provide a strong support for quick examination of the novel Bunyavirus infection during the Fever with Thrombocytopenia Syndromes surveillance and the emergent clinical diagnosis.