The application of a reverse transcriptase-polymerase chain reaction-oligonucleotide probe assay for the detection of human astroviruses in environmental water

A human astrovirus (HAstV)-specific oligonucleotide probe was utilised for the identification of HAstV-specific sequences in reverse transcriptase-polymerase chain reaction (RT-PCR) cDNA amplicons using either of two published primers pairs, both of which reportedly detect all known HAstV serotypes. The two sets of primers, designated Mon and J7, amplify 89 bp and 77 bp regions in the conserved 3′-end of the HAstV genome, respectively. An overlapping region of 59 bp is amplified by both primer pairs and the 30 bp oligonucleotide probe was designed to be homologous to a sequence within this overlapping region. Cell culture amplification in the human primary liver carcinoma cell line, PLC/PRF/5, and the human colonic carcinoma cell line, CaCo-2, prior to using the RT-PCR-oligonucleotide probe assay was assessed for enhanced detection of wild type HAstVs in environmental waters. The PLC/PRF/5 cells were shown to be more effective than the CaCo-2 cells for enhancing the detection of HAstVs. This RT-PCR-oligonucleotide probe assay with the Mon primer pair, in conjunction with cell culture amplification in the PLC/PRF/5 cell line, provides a sensitive and specific assay for the detection of wild type HAstVs in environmental waters.