Detection of enteric viruses in treated wastewater sludge using cell culture and molecular methods

Abstract

Two continuous cell lines, BGM and CaCo-2 were compared for the detection of viruses in mesophilic treated sludge (MIS) and secondary disinfected wastewater effluent (WWE) samples. Samples were inoculated in both cell lines and examined microscopically for cytopathogenic effect (CPE) for 14 days. Enumeration by the most probable number (MPN) method and statistical analysis revealed significantly greater MPN values for CaCo-2 than in BGM cells for WWE. Statistical analysis of MTS and WWE samples showed that CaCo-2 cells were more sensitive than BGM (p=0.0287). This suggests that CaCo-2 cells are more sensitive for the detection of enteroviruses in environmental samples. M-PCR was developed to detect and differentiate human adenovirus (Ad) from enteric adenovirus (Ead) from seeded environmental samples. Two sets of primers hexAA1885/1913 and K402/403 (308 bp and 152 bp amplicons respectively) were chosen for combination in M-PCR. The optimum MgC12 concentration was 1.25 mM with primer concentration of 100 pmol for hexAA1885/1913 and 50 pmol for K4021403 primers. Optimum primer annealing temperature was 60° C. Sensitivity of M-PCR was 10^2 TC1D50 for Ead and Ad mixed and 10^0 TCID50 for individual viruses per reaction. M-PCR has potential in the rapid and specific identification of these types of viruses in environmental samples.