Norovirus Detection in Shellfish Using a Rapid, Sensitive Virus Recovery and Real-Time RT-PCR Detection Protocol

Greening, Gail E.; Hewitt, Joanne

doi:10.1007/s12161-008-9018-3

Cited by 39 publications

(17 citation statements)

References 32 publications

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“…In this study, the concentrations of HAdV and human NoV in municipal sewage were shown to be highly variable and ranged from 10 4 to 10 7 copies for HAdV-F and from "not detected" to 10 6 copies per liter for NoV GII, respectively, whereas the concentrations of bovine and ovine NoV GIII strains in the two abattoir waste samples were relatively constant at 10 5 copies per liter. Inhibition can be a major problem with RT and PCRs for environmental samples and was described previously in several studies (4,11,15). In this study, inhibition may have generated biased quantitative data or false-negative results, especially for complex samples such as concentrated sewage and shellfish.…”

Section: Discussionmentioning

confidence: 75%

“…Viruses from the water samples were concentrated by hollow-fiber ultrafiltration using Hemoflow HF80S dialysis filters (Fresenius Medical Care, Bad Homberg, Germany) as previously described (19) but with minor modifications to elute the viruses from the solid fraction using a 3% (wt/vol) beef extract-0.05 M glycine solution (pH 9.0). Viruses were recovered from shellfish by using a protease digestion method described elsewhere previously (15). Viruses from sewage and biosolids were concentrated by polyethylene glycol (PEG) 6000 precipitation following virus elution as described previously for water samples (modified from procedures described in references 14, 29, and 48).…”

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confidence: 99%

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Viral Multiplex Quantitative PCR Assays for Tracking Sources of Fecal Contamination

Wolf

Hewitt

Greening

2010

Appl Environ Microbiol

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158

112

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Human and animal fecal pollution of the environment presents a risk to human health because of the presence of pathogenic viruses and bacteria. To distinguish between human and animal sources of pollution, we designed specific real-time reverse transcription (RT)-PCR assays for human and animal enteric viruses, including norovirus genogroups I, II, and III; porcine adenovirus types 3 and 5; ovine adenovirus; atadenovirus; and human adenovirus species C and F, which are excreted by infected humans, pigs, cattle, sheep, deer, and goats, and for the detection of F؉ RNA bacteriophage genogroups I to IV, which are associated with human and animal wastes. The sensitivity of this viral toolbox (VTB) was tested against 10-fold dilution series of DNA plasmids that carry the target sequences of the respective viruses and was shown to detect at least 10 plasmid copies for each assay. A panel of human and animal enteric and respiratory viruses showed these assays to be highly sensitive and specific to their respective targets. The VTB was used to detect viruses in fecal and environmental samples, including raw sewage and biosolids from municipal sewage treatment plants, abattoir sewage, and fecally contaminated shellfish and river water, which were likely to contain animal or human viruses.It is important that sources of fecal pollution are rapidly and accurately identified so that environmental managers can develop strategies to eliminate the source of the pollution in an efficient and cost-effective manner. The identification of fecal sources also allows the potential risk to human health following the consumption of or exposure to contaminated food or water to be estimated. For this purpose, numerous fecal source tracking methods have been developed. Among these are library-dependent methods such as antibiotic resistance testing or molecular fingerprinting (e.g., terminal restriction length fragment polymorphism [T-RFLP] or ribotyping) and library-independent approaches, including the detection of human-specific bacteriophages by plaque assay (e.g., Bacteroides fragilis bacteriophages), the detection of certain chemicals associated with human and nonhuman wastes (e.g., caffeine, laundry brighteners, fecal sterols, and stanols), and the detection of specific genetic markers by PCR. The molecular detection of human and animal viruses and bacteriophages falls into the latter category. Each method has advantages and disadvantages depending on the circumstances and the questions at hand. To date, none has been proposed as a standard method (9,34,40,45).The detection and genotyping of Fϩ RNA bacteriophages have been widely used as a tool for microbial source tracking (MST) (5,8,17,18,31,44). Fϩ RNA bacteriophage genogroup I (GI) and GIV have been associated with animal contamination, and genogroups II and III indicate predominantly human fecal contamination or domestic sewage-associated in-

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Section: Discussionmentioning

confidence: 75%

mentioning

confidence: 99%

Viral Multiplex Quantitative PCR Assays for Tracking Sources of Fecal Contamination

Wolf

Hewitt

Greening

2010

Appl Environ Microbiol

Self Cite

158

112

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show abstract

“…Foodborne pathogenic viruses can be present in different food matrices (Koopmans et al 2002;Croci et al 2008;Greening and Hewitt 2008), and pork meat products could constitute a significant route of indirect zoonotic transmission of enteric viruses. Hepatitis E virus (HEV) could be transmitted to humans by close contact with infected swine or from the consumption of contaminated raw or undercooked pork, wild boar liver or deer meat (Cook et al 2006;Tei et al 2003Tei et al , 2004Mishiro 2004).…”

Section: Introductionmentioning

confidence: 99%

Evaluation of Extraction Methods for Efficient Detection of Enteric Viruses in Pork Meat Products

et al. 2010

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We report an in-house protocol for extraction and purification of nucleic acids of enteric viruses, which gives more consistent results than representative commercial methods. The protocol uses 4 M guanidine thiocyanate, 0.5% N-lauroylsarcosine sodium salt, and 25 mM sodium citrate pH 7.0 supplemented with 0.14 M β-mercaptoethanol for lysis of virus particles. The addition of TRIzol followed by chloroform-based separation of the aqueous phase is used to purify nucleic acids from the lysate. RNA precipitation is performed using lithium chloride. This protocol was compared with QIAGEN's RNeasy Kit and bioMérieux's NucliSens method, by evaluating the ability of each to detect enteric viruses in a complex food matrix. Three different pork products, i.e., cooked ham, liver, and Spanish fermented sausage ("chorizo") were artificially contaminated with decreasing numbers of murine norovirus 1 and human adenovirus 2. The extracted and purified viral nucleic acids were detected by real-time polymerase chain reaction (PCR) assays. Whereas the two commercial extraction methods did not facilitate robust results (quantification was only possible with some viruses and/or some matrices), when coupled with the in-house protocol the linearity and the efficiency of the quantitative reverse transcription-PCR (qRT-PCR) assays were close to 1 in all the food matrices, independent of the virus. Scalability of the in-house method was evaluated by analysis of 1 and 2.5 g of spiked pig liver samples, and quantification was possible on 1 g samples contaminated with any of the two model viruses. Therefore, the in-house protocol facilitates robust qRT-PCR-based quantitative detection of viruses in pork products, and is moreover relatively cheap and simple to perform.

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“…Thus, detection of the presence of enteric virusesparticularly norovirus, hepatitis A and E and adenovirusin foods is an important issue in food safety, and a rapid and robust diagnostic methodology is needed (Croci et al 2008;Greening and Hewitt 2008;Cook and Rzezutka 2006). Because at present most of these viruses cannot, or with difficulty, be cultured and integrated cell culture real-time PCR methods are useful but too time-consuming for the quick results required by the food industry, a detection approach based on nucleic acid amplification is necessary (Rodríguez-Lázaro et al 2007;Bosch et al 2011).…”

Section: Introductionmentioning

confidence: 99%

Construction and Analytical Application of Internal Amplification Controls (IAC) for Detection of Food Supply Chain-Relevant Viruses by Real-Time PCR-Based Assays

et al. 2011

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Internal amplification controls (IACs) were constructed for incorporation into real-time nucleic acid amplification assays for bovine polyomavirus, hepatitis A virus, hepatitis E virus, human adenovirus, human norovirus genogroup I, human norovirus genogroup II, murine norovirus and porcine adenovirus. The addition of optimised amounts of IAC into the assays did not affect the limits of detection for each specific target virus. A poorly performed extraction of viral nucleic acids was simulated, and the effectiveness of IACs in identifying failed assays was demonstrated. The IACs constructed in this study can be reliably used in their specific assays to provide a robust control that can be routinely applied in the analysis of foods for viruses.

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Norovirus Detection in Shellfish Using a Rapid, Sensitive Virus Recovery and Real-Time RT-PCR Detection Protocol

Cited by 39 publications

References 32 publications

Viral Multiplex Quantitative PCR Assays for Tracking Sources of Fecal Contamination

Viral Multiplex Quantitative PCR Assays for Tracking Sources of Fecal Contamination

Evaluation of Extraction Methods for Efficient Detection of Enteric Viruses in Pork Meat Products

Construction and Analytical Application of Internal Amplification Controls (IAC) for Detection of Food Supply Chain-Relevant Viruses by Real-Time PCR-Based Assays

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