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

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.

Key words:human astrovirus;human sapovirus;duplex fluorescence quantitative RT-PCR method