Polymerase chain reaction detection of small round‐structured viruses from two related hospital outbreaks of gastroenteritis using inosine‐containing primers

Abstract

Two outbreaks of gastroenteritis in the UK which occurred nine days apart at Lymington and Southampton hospitals were investigated. The clinical and epidemiological features of both outbreaks were characteristic of small round-structured virus (SRSV) infection with rapid onset of diarrhoea and/or nausea and vomiting and propagation of the outbreaks by secondary spread. SRSV particles were observed by immune electron microscopy (EM) in 60% of faecal samples from both outbreaks and no other pathogens were detected. The index case for the second outbreak was a patient who was admitted with diarrhoea and vomiting after being discharged from Lymington hospital during the first outbreak. The possibility that the two outbreaks were caused by the same strain of SRSV was investigated by the polymerase chain reaction (PCR). New inosine-containing PCR primers were designed to amplify the RNA polymerase region of SRSV cDNA from genetic groups I and II. The PCR using the group II primers achieved a higher detection rate for SRSVs in faecal samples (68% of samples positive from both outbreaks) than immune EM. SRSVs were not detected using the group I primers or using conventional degenerate PCR primers. The nucleotide sequences of PCR amplicons from both outbreaks were identical providing molecular epidemiological evidence for the involvement of a single SRSV strain. Comparison of the RNA polymerase region of this virus with the equivalent regions of genetic group I (69.4-75.0% amino acid identify) and genetic group II (88.9-100% amino acid and 77.1-88.1% nucleotide identity) SRSVs revealed that the causative SRSV was a distinct member of genetic group II.

Evidence for airborne transmission of Norwalk-like virus (NLV) in a hotel restaurant

“…Diagnostic RT-PCR was performed using RNA polymerase gene primers [14] as described previously [15]. For DNA sequencing the PCR reaction was scaled up to 100 µl using antibody inactivated Taq .…”

Genetic diversity of noroviruses in Brazil

“…Since the 1990s, the development of amplification protocols for different genomic regions, denominated regions A and B (ORF-1), C, D and E (ORF-2), has resulted in a NoV diagnostic breakthrough in several countries (Ando et al 1995, Green et al 1995, Noel et al 1997, Fankhauser et al 2002, Kojima et al 2002, Vennema et al 2002, Vinjé et al 2004. A global electronic network surveillance of NoV, Noronet (noronet.nl), was established following an agreement between three networks involved in molecular surveillance of NoV: the Australian and New Zealand NoV Surveillance Network, the Foodborne Viruses in Europe Network and the CaliciNet in USA.…”

Precise Characterization of Norovirus (Norwalk-Like Virus)-Specific Monoclonal Antibodies with Broad Reactivity

“…However, due to the great diversity of nucleotide sequences throughout the whole genome of NV and the capsid protein, neither ELISA nor RT-PCR detects all types of NV (3,4,5,9). In addition, the very limited numbers of particles shed in patient fecal material makes detection by EM difficult (2,9). In cases of NV infections with homologous strains, genomic RNA detection by RT-PCR and antigen-antibody detection by ELISA can yield excellent results (1,3,4,7,8).…”