Direct typing of human enteroviruses from wastewater samples

Abstract

A RT-PCR approach for the direct detection and typing of human enteroviruses in the environment is described in this study. A semi-nested RT-PCR using COnsensus-DEgenerated Hybrid Oligonucleotide Primers (CODEHOP) designed from the VP2 genome region has been developed for the direct typing of enteroviruses in clinical samples (Ibrahim et al., 2013). This CODEHOP/VP2 PCR strategy as well as the CODEHOP/VP1 technique described by Nix et al. (2006), were tested for the detection and typing of enteroviruses in wastewater samples. Virus particles were first extracted and concentrated from wastewater samples by using respectively beef extract and polyethylene glycol 6000, and the presence of enteroviruses was screened by a RT-PCR method using primers from the 5′-end non-coding region (5′NCR). Fifty-two of 172 samples (30.2%) were revealed positive by the 5′NCR method. From these 52 samples, only 19 samples (36.5%) were found positive by at least one of the two CODEHOP techniques, with the following distribution: VP1(+)/VP2(+)=4 (7.7%), VP1(−)/VP2(+)=13 (25%) and VP1(+)/VP2(−)=2 (3.8%). These results illustrate that the direct typing of enteroviruses in environmental samples is insensitive, possibly due to the presence of large amounts of amplification inhibitors; however, the VP2 method was found able to allow the direct detection and typing of c. one-third of the positive environmental samples.