Broadly reactive reverse transcriptase polymerase chain reaction for the diagnosis of SRSV-associated gastroenteritis.
A limitation to date of reverse transcriptase polymerase chain reactions (RT-PCRs) for the detection of small, round structured viruses (SRSVs) 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 samples collected from 136 separate incidents of gastroenteritis occurring in the UK between 1992 and 1994 were examined by RT-PCR employing a single primer pair (N1/E3). SRSV RNA was detected in samples from 93 of 101 (91%) incidents shown to be SRSV-associated by electron microscopy (EM) and in 5 of 35 (14%) SRSV-negative incidents. Amplification products were tested by Southern blot hybridisation with a pool of four digoxigenin (DIG)-labelled oligonucleotides derived from genomic sequence data of SRSV SPIEM types UK 1 to 4. Products from approximately 5% of amplified strains did not hybridise. The N1/E3 primer pair were shown to be SRSV-specific by their failure to amplify other faecal viruses including other human caliciviruses with typical calicivirus morphology. Hybridisation of PCR products with the individual oligonucleotides 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, 51/60 (85%) with the UK3 probe, and 27/60 (45%) reacted with the UK4 probe. All PCR products that hybridised with the UK4 probe hybridised with 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 environmental samples.
Green
J
J
Enteric and Respiratory Virus Laboratory, Central Public Health Laboratory, London, United Kingdom.
Gallimore
C I
CI
Norcott
J P
JP
Lewis
D
D
Brown
D W
DW
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D017931:DNA Primers; D004196:Disease Outbreaks; D005243:Feces / Q000821:virology; D005759:Gastroenteritis / Q000175:diagnosis* / Q000453:epidemiology / Q000821:virology; D006801:Humans; D015994:Incidence; D008969:Molecular Sequence Data; D009663:Norwalk virus / Q000235:genetics / Q000302:isolation & purification*; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D012194:RNA-Directed DNA Polymerase; D006113:United Kingdom / Q000453:epidemiology
D017931:DNA Primers; D015345:Oligonucleotide Probes; D012194:RNA-Directed DNA Polymerase