西尼罗病毒的逆转录重组酶介导扩增检测方法

Development of a reverse transcription recombinase aided amplification assay for detection of West Nile virus

摘要:

【背景】随着西尼罗热在全球范围内广泛流行，西尼罗热传入我国的风险加大。研究西尼罗病毒的快速检测方法，为西尼罗病毒检测建立方法储备。【目的】建立西尼罗病毒逆转录重组酶介导扩增 (reverse transcription recombinase aided amplification，RT-RAA)检测方法。【方法】根据西尼罗病毒基因保守区序列设计引物和探针，建立西尼罗病毒RT-RAA检测方法，并评价该方法的重复性、特异性和灵敏度。【结果】 RT-RAA方法的整个扩增反应过程温度恒定，反应温度为39 °C，检测时间较短(20 min内)，并且检测限可达10 copies，与基孔肯雅病毒、登革病毒、乙型脑炎病毒、黄热病毒等蚊媒病毒无交叉反应，具有良好的特异性，样本检测符合预期。【结论】建立的西尼罗病毒RT-RAA方法具有快速、特异以及灵敏的特点，可用于西尼罗病毒的口岸快速检测和流行病学监测。

Abstract:

[Background] West Nile fever is widely prevalent in the world, the risk of West Nile fever spreading to China has increased. The rapid detection method of West Nile virus (WNV) was studied to establish the method reserve for West Nile virus detection. [Objective] To establish a reverse transcription recombinase aided amplification (RT-RAA) assay for detection of WNV. [Methods] The primers and probes were designed based on the conserved genome sequence of WNV. An RT-RAA assay of WNV was constructed, and the reproducibility, specificity and sensitivity of the assay were evaluated. [Results] The RT-RAA assay had a constant temperature throughout the amplification reaction, the reaction temperature was 39 °C. The detection time was less than 20 minutes and the detection sensitivity could reach 10 copies. The method had excellent specificity as it didn’t have cross amplification with other arbovirus such as chikungunya virus, dengue virus, Japanese encephalitis virus and Yellow fever virus. The results of sample detection also meet the expectation. [Conclusion] The RT-RAA assay of WNV established in our study was rapid, specific and sensitive. It can be used for rapid detection and epidemiological surveillance of WNV at the port.