Extraction and RT-qPCR detection of enteroviruses from solid environmental matrixes: Method decision tree for different sample types and viral concentrations

Abstract

Quantitative RT-PCR methods (RT-qPCR) are becoming increasingly desirable for the detection of enteric viruses in solid environmental matrixes such as sediments, soils and sewage sludge. However, effective methodologies that allow the extraction of high quality RNA ready for molecular quantification continue to be evaluated. In the present study, four different methods for enterovirus extraction from solid environmental matrixes were compared in terms of viral recovery and inhibitor removal. Three indirect methods based on glycine elution and concentration by ultracentrifugation were tested. The main differences between indirect methods were the sample to glycine buffer ratio, and the ultracentrifugation protocol applied. One commercial direct method was also tested. The indirect methods produced better results than the direct method. The ultracentrifugation led to viral losses in samples with high titers; however, as the virus concentration reduced, the ultracentrifugation became increasingly important for viral recovery. Two commercial RNA extraction kits were also evaluated and it was selected the most effective in removing RT-qPCR inhibitors. The results obtained allowed the development of a method decision tree with three versions that are suitable for different samples and viral concentrations.