THE APPLICATION OF A REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION-OLIGONUCLEOTIDE PROBE ASSAY FOR THE DETECTION OF HUMAN ASTROVIRUSES INENVIRONMENTAL WATER

A human astrovirus (HAstV)-specific oligonucleotide probe was utilised for the identification of HAstV-specific sequences in reverse transcr iptase-polymerase chain reaction (RT-PCR) cDNA amplicons using either of two published primers pairs, both of which reportedly detect all kn own HAstV serotypes. The two sets of primers, designated Mon and J7, a mplify 89 bp and 77 bp regions in the conserved 3'-end of the HAstV ge nome, respectively. An overlapping region of 59 bp is amplified by bot h 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 t he RT-PCR-oligonucleotide probe assay was assessed for enhanced detect ion of wild type HAstVs in environmental waters. The PLC/PRF/5 cells w ere 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 th e PLC/PRF/5 cell line, provides a sensitive and specific assay for the detection of wild type HAstVs in environmental waters. (C) 1998 Elsev ier Science Ltd. All rights reserved.