BROADLY REACTIVE REVERSE-TRANSCRIPTASE POLYMERASE CHAIN-REACTION FOR THE DIAGNOSIS OF SRSV-ASSOCIATED GASTROENTERITIS

A limitation to date of reverse transcriptase polymerase chain reactio ns (RT-PCRs) for the detection of small, round structured viruses (SRS Vs) has been that they have detected only a narrow range of SRSVs due to the marked genomic diversity among strains. A total of 331 faecal s amples collected from 136 separate incidents of gastroenteritis occurr ing in the UK between 1992 and 1994 were examined by RT-PCR employing a single primer pair (NI/E3). SRSV RNA was detected in samples from 93 of 101 (91%) incidents shown to be SRSV-associated by electron micros copy (EM) and in 5 of 35 (14%) SRSV-negative incidents. Amplification products were tested by Southern blot hybridisation with a pool of fou r digoxigenin (DIG)-labelled oligonucleotides derived from genomic seq uence data of SRSV SPIEM types UK 1 to 4. Products from similar to 5% of amplified strains did not hybridise. The NI/E3 primer pair were sho wn to be SRSV-specific by their failure to amplify other faecal viruse s including other human caliciviruses with typical calicivirus morphol ogy. Hybridisation of PCR products with the individual oligonucleotide s relating to SRSV SPIEM types UK 1-4 was investigated: 1 of 60 (1.7%) reacted with the UK1 probe, 2/60 (3.4%) reacted with the UK2 probe, 5 1/60 (85%) with the UK3 probe, and 27/60 (45%) reacted with the UK4 pr obe. All PCR products that hybridised with the UK4 probe hybridised wi th the UK3 probe; 6 (10%) failed to hybridise. Identification of this primer pair facilitates routine diagnosis of SRSV infection by RT-PCR and offers the potential for direct detection in food and environmenta l samples. (C) 1995 Wiley-Liss, Inc.