发热伴血小板减少综合征布尼亚病毒实时荧光RT-PCR检测方法的建立

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...

Establishment of a Real-time RT-PCR Method for Detecting the Novel Bunyavirus during the Surveillance of Fever with Thrombocytopenia Syndromes

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.