人星状病毒和人札幌病毒双重荧光定量RT-PCR检测方法的建立与应用

Development and application of a duplex fluorescence quantitative RT-PCR method for detecting human astrovirus and human sapovirus

摘要:

【背景】 人星状病毒(human astrovirus, HAstV)和人札幌病毒(human sapovirus, HuSaV)是引起人类急性胃肠炎的重要病原体，尤其对婴幼儿、老年人和免疫力低下等人群造成严重的威胁。目前尚无疫苗和特异性手段防控病毒的感染与传播，因此加强检测对防控病毒的传播具有重要的意义。【目的】 建立能同时检测HAstV和HuSaV的双重荧光定量RT-PCR方法，为HAstV和HuSaV的快速检测和流行病学调查提供技术支持。【方法】 针对HAstV和HuSaV保守区域基因序列，设计特异性检测引物和探针并优化反应体系，建立HAstV和HuSaV双重荧光定量RT-PCR检测方法，并分析其特异性、敏感性和重复性。【结果】 HAstV和HuSaV双重荧光定量RT-PCR检测方法针对HAstV和HuSaV最低检测线分别为15 copies/μL和2.1 copies/μL；与其他常见的食源性病毒、致病菌和乳酸菌无交叉反应；批内和批间重复性试验变异系数均小于3.5%。采用人工模拟不同浓度的病毒污染牡蛎样品，结果显示HAstV和HuSaV双重荧光定量RT-PCR检测方法能检测出所有受污染的牡蛎样品，与阳性对照组检测结果差异不显著(P>0.05)；利用所建立的HAstV和HuSaV双重荧光定量RT-PCR检测方法检测不同食品样品的HAstV和HuSaV，其阳性率分别为10.83%和0%。【结论】 本研究建立的HAstV和HuSaV双重荧光定量RT-PCR检测方法具有较好的敏感性、特异性和重复性，适用于食品样品中HAstV和HuSaV的快速检测及大规模流行病学调查。

Abstract:

[Background] Human astrovirus (HAstV) and human sapovirus (HuSaV) are currently the main pathogen of human acute gastroenteritis worldwide, posing a serious threat to human health, especially to infants, the elderly, and immunocompromised people. There is no vaccine or specific means that can prevent the infections and transmission of HAstV and HuSaV. Therefore, developing a rapid and accurate detection method is of great significance for the prevention of HAstV and HuSaV. [Objective] To establish a duplex fluorescence quantitative RT-PCR method for simultaneous detection of HAstV and HuSaV and thus facilitate rapid detection and epidemiological investigations. [Methods] Specific primers and probes were designed according to the conserved sequences of HAstV and HuSaV. The reaction system was optimized, and detection system was established under the optimal reaction conditions. The sensitivity, specificity, and repeatability of the duplex fluorescence quantitative RT-PCR method were evaluated. [Results] The established method had high sensitivity, with the limits of detection being 15 copies/μL and 2.1 copies/μL for HAstV and HuSaV, respectively. The method was able to specifically detect the targets from HAstV and HuSaV and had no cross-reaction with other foodborne viruses, pathogens, or lactic acid bacteria. The coefficients of variation of inner and intra-assay were both less than 3.5%, indicating high repeatability and stability. Furthermore, the established method was employed to detect oysters artificially contaminated with different concentrations of HAstV and HuSaV. The results showed that the method detected all the contaminated samples, with the detection result showcasing no significant difference compared with that in the positive control group (P>0.05). The positive rates of HAstV and HuSaV in the food samples detected by the established method were 10.83% and 0%, respectively. [Conclusion] The duplex fluorescence quantitative RT-PCR method was highly sensitive, specific, and repetitive for the rapid detection of HAstV and HuSaV in food samples and large-scale epidemiological investigations.