Broadly reactive reverse transcriptase polymerase chain reaction for the diagnosis of SRSV‐associated gastroenteritis

Abstract

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.

An epidemiological study of enteric viruses in sewage with molecular characterization by RT-PCR and sequence analysis

“…Because of the known genetic variability of the noroviruses, all the wastewater samples have been analysed with three different sets of previously published norovirus primers (Ando et al 1995;Green et al 1995;Jiang et al 1999).…”

“…For human noroviruses, three set of primers located in the conserved RNA polymerase region were used: the primers designed by Ando et al (1995) yield a 123 bp product, those designed by Green et al (1995) yield a 113 bp product, and those designed by Jiang et al…”

“…were as follows: 40 amplification cycles with denaturation at 948C for 30 s, annealing at 408C for 60 s for one set of norovirus primers (Green et al 1995), annealing at 508C for 30 s for HastV, 60 s for enterovirus, and 80 s for two sets of norovirus primer (Ando et al 1995;Jiang et al 1999), For norovirus, a suspension was made by mixing 100 ml of the stool obtained from a child with diarrhoea due to norovirus (genogroup II) in 900 ml sterile distilled water.…”

Detection of calicivirus from fecal samples from children with acute gastroenteritis in the West Central region of Brazil

“…The cDNA was obtained using the random primer pd(N) 6 (20 µM) (Amersham Biosciences), RT MMLV (200 U/µl) (USB) and ribonuclease inhibitor (20 U/µl) (Invitrogen). The amplification reaction was carried out using Taq DNA polymerase (1,5 U/µl) (Invitrogen) and the primer pairs Ni/E3 (Green et al 1995), 289/290 (Jiang et al 1999). When results with the two primer pairs were inconclusive, the pair primer JV12/13 (Vinjé et al 1997) was used.…”

“…For virus detection, RT-PCR has been extensively used, employing primers designed for different regions of the genome. The most frequently used primers have been designed for the region of the RNA polymerase, which is conserved between the genetic groups (Green et al 1995, Gray et al 1997, Jiang et al 1999, Guo et al 2001.…”