Seminested RT-PCR systems for small round structured viruses and detection of enteric viruses in seafood

Häfliger, Daniel; Gilgen, M.; Lüthy, Jürg; Hübner, Philipp

doi:10.1016/s0168-1605(97)00041-x

Cited by 104 publications

(70 citation statements)

References 34 publications

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“…PCR is rapid, highly sensitive, and specific if a well-designed assay is developed. PCR viral detection is less laborious and time-consuming and also more specific and sensitive than cell culture (Table 2) (27,66,79). Results from PCR assays can be obtained within 24 h of sampling, compared to days or weeks of incubation for cell culture assay (62,120).…”

Section: Virus Detection Methodsmentioning

confidence: 99%

Enteric Viruses of Humans and Animals in Aquatic Environments: Health Risks, Detection, and Potential Water Quality Assessment Tools

Fong

Lipp

2005

Microbiol Mol Biol Rev

612

450

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Waterborne enteric viruses threaten both human and animal health. These pathogens are host specific and cause a wide range of diseases and symptoms in humans or other animals. While considerable research has documented the risk of enteric viruses to human health from contact with contaminated water, the current bacterial indicator-based methods for evaluation of water quality are often ineffectual proxies for pathogenic viruses. Additionally, relatively little work has specifically investigated the risk of waterborne viruses to animal health, and this risk currently is not addressed by routine water quality assessments. Nonetheless, because of their host specificity, enteric viruses can fulfill a unique role both for assessing health risks and as measures of contamination source in a watershed, yet the use of animal, as well as human, host-specific viruses in determining sources of fecal pollution has received little attention. With improved molecular detection assays, viruses from key host groups can be targeted directly using PCR amplification or hybridization with a high level of sensitivity and specificity. A multispecies viral analysis would provide needed information for controlling pollution by source, determining human health risks based on assessments of human virus loading and exposure, and determining potential risks to production animal health and could indicate the potential for the presence of other zoonotic pathogens. While there is a need to better understand the prevalence and environmental distribution of nonhuman enteric viruses, the development of improved methods for specific and sensitive detection will facilitate the use of these microbes for library-independent source tracking and water quality assessment tools

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Section: Virus Detection Methodsmentioning

confidence: 99%

Enteric Viruses of Humans and Animals in Aquatic Environments: Health Risks, Detection, and Potential Water Quality Assessment Tools

Fong

Lipp

2005

Microbiol Mol Biol Rev

612

450

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“…Both RT and PCR were carried out with the reaction mixture preparations recommended by the manufacturer. Primer set SRI1 and SRI2 (14) and primer set Mon381 and Mon383 (32) were used to amplify the sequences of the capsid gene of noroviruses genogroup I and II, respectively, giving amplicons of 316 and 322 bp, respectively. The cycling conditions were as follows: one cycle of reverse transcription at 42°C for 15 min and, for PCR, denaturation for 2 min at 94°C; 40 amplification cycles with denaturation for 30 s at 94°C, annealing for 1 min at 50°C, and extension for 1 min at 72°C; and a final cycle of incubation at 72°C for 15 min.…”

Section: Methodsmentioning

confidence: 99%

Characterization of New Recombinant Noroviruses

et al. 2005

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Noroviruses are important etiologic agents of acute gastroenteritis and show great genetic diversity. To characterize more fully previously detected strains that could not be assigned unequivocally to one particular genotype based on the RNA polymerase, we have sequenced a region in the capsid gene and, in some cases, in the junction between open reading frame 1 (ORF1) and ORF2. The results allowed us to identify several recombinant noroviruses: GGIIb viruses were detected for the first time in France in August 2000 and then spread through France and to Europe during the following winter. Here we present the characterization of three other probable GII recombinants which showed different phylogenetic positions depending on their ORF1 and ORF2 sequences. Analysis of the region located between ORF1 and ORF2 by a nucleotide identity window search showed a sudden shift in similarities. Moreover, recombination breakpoints were identified upstream and downstream of the beginning of ORF2 by using a statistical test, thus confirming the involvement of this region in recombination. Unlike GGIIb, the three recombinants described here do not seem to have diffused widely in the community: one was found in a waterborne outbreak, and the other two were found in sporadic cases. Recombination is important for the evolution of RNA viruses and has already been described for noroviruses. Our results suggest that recombination is not a rare phenomenon among noroviruses, but not all these presumed recombinants that formed during RNA replication are able to spread widely.

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“…The great sequence diversity of the NLV genome has made this a challenging undertaking. Primers for previous assays have been taken from open reading frame 1 (ORF1) of the RNA-dependent RNA polymerase (RdRp) gene (1,2,9,13,15,26,27,31,37) or from ORF2 of the capsid protein gene (6,10,11,13,28,38,39).…”

mentioning

confidence: 99%

Broadly Reactive and Highly Sensitive Assay for Norwalk-Like Viruses Based on Real-Time Quantitative Reverse Transcription-PCR

Kageyama

Kojima

Shinohara³

et al. 2003

J Clin Microbiol

1,271

889

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We have developed an assay for the detection of Norwalk-like viruses (NLVs) based on reverse transcription-PCR (RT-PCR) that is highly sensitive to a broad range of NLVs. We isolated virus from 71 NLV-positive stool specimens from 37 outbreaks of nonbacterial acute gastroenteritis and sequenced the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the NLV genome. The data were subjected to multiple-sequence alignment analysis and similarity plot analysis. We used the most conserved sequences that react with diverse NLVs to design primers and TaqMan probes for the respective genogroups of NLV, GI and GII, for use in a real-time quantitative RT-PCR assay. Our method detected NLV in 99% (80 of 81) of the stool specimens that were positive by electron microscopy, a better detection rate than with the two available RT-PCR methods. Furthermore, our new method also detected NLV in 20 of 28 stool specimens from the same NLV-related outbreaks that were negative for virus by electron microscopy. Our new assay is free from carryover DNA contamination and detects low copy numbers of NLV RNA. It can be used as a routine assay for diagnosis as well as for elucidation of the epidemiology of NLV infections.

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Seminested RT-PCR systems for small round structured viruses and detection of enteric viruses in seafood

Cited by 104 publications

References 34 publications

Enteric Viruses of Humans and Animals in Aquatic Environments: Health Risks, Detection, and Potential Water Quality Assessment Tools

Enteric Viruses of Humans and Animals in Aquatic Environments: Health Risks, Detection, and Potential Water Quality Assessment Tools

Characterization of New Recombinant Noroviruses

Broadly Reactive and Highly Sensitive Assay for Norwalk-Like Viruses Based on Real-Time Quantitative Reverse Transcription-PCR

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