Evaluation of two viral extraction methods for the detection of human noroviruses in shellfish with conventional and real-time reverse transcriptase PCR

Baert, Leen; Uyttendaele, Mieke; Debevere, Johan

doi:10.1111/j.1472-765x.2006.02047.x

Cited by 30 publications

(26 citation statements)

References 28 publications

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“…Of these, two groups (Loisy et al 2005b;Jothikumar et al 2005) used a one-step assay and two used a two-step assay (Nishida et al 2003;Baert et al 2007). We found that the use of specific primers for the RT in two-step RT-PCR assays increased the sensitivity tenfold for both NoV GI and GII compared with the one-step assay.…”

Section: Discussionmentioning

confidence: 95%

“…There are now many real-time RT-PCR methods described for NoV detection in clinical samples (Kageyama et al 2003;Wolf et al 2007;Trujillo et al 2006), but there are still few reports of real-time RT-PCR for NoV detection in shellfish (Nishida et al 2003;Loisy et al 2005b;Jothikumar et al 2005;Baert et al 2007). Of these, two groups (Loisy et al 2005b;Jothikumar et al 2005) used a one-step assay and two used a two-step assay (Nishida et al 2003;Baert et al 2007).…”

Section: Discussionmentioning

confidence: 96%

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

Greening

Hewitt

2008

Food Anal. Methods

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The development and in-house validation of a method for the detection of noroviruses in shellfish are described. The method comprises a simple virus recovery step using proteinase K digestion and extraction of viral ribonucleic acid (RNA) followed by norovirus detection in separate twostep genogroup I (GI) and II (GII) real-time reverse transcription polymerase chain reaction assays. The assay can be completed within 8 h. An internal armored RNA control, which detects the presence of reverse transcription PCR inhibitors, is an integral part of the assay. The method was found to be reliable, fast, robust, reproducible, and sensitive. Sixty New Zealand and imported shellfish samples were tested using the new method, of which 29 (48.3%) samples were associated with recent New Zealand gastroenteritis outbreaks. The nonoutbreak-related samples were submitted for environmental surveillance related to possible sewage contamination events. Norovirus was detected in 30 of 60 (50%) samples, of which 20 were implicated in gastroenteritis outbreaks, and ten were noncommercial shellfish samples from environmental sites. Of the 30 positive samples, 18 (30%) were positive for both GI and GII norovirus, and a further 12 samples (20%) were positive for GII norovirus only. No samples were positive for GI norovirus only. The method is now validated and accredited under ISO 17025. It can be used to monitor norovirus contamination of shellfish in different situations, including outbreak investigations, relaying, environmental monitoring, and following environmental contamination events.

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Section: Discussionmentioning

confidence: 95%

Section: Discussionmentioning

confidence: 96%

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

Greening

Hewitt

2008

Food Anal. Methods

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“…The aqueous phase was taken after centrifugation (8000g, 20min, and 4 0 C) and stored by freezing. A volume of 100µL was purified by the use of an RNeasy Mini kit (Qiagen) according to the manufacturer's instructions (Baert et al, 2007).…”

Section: Methodsmentioning

confidence: 99%

Prevalence of hepatitis A virus in sea food in Iran

Momtaz

Rahimi

Hajian

et al. 2012

Arq. Bras. Med. Vet. Zootec.

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The objective of this study was to determine the prevalence of Hepatitis A Virus (HAV) in sea food samples in the Isfahan and Shahrekord townships in Iran. From September 2010 to April 2011, a total of 300 samples of fresh fish, shrimp, crab and lobster were obtained from randomly selected retail stores in the Isfahan and Shahrekord townships in Iran. The samples were tested for the presence of HAV using a reverse transcriptase-polymerase chain reaction method. Out of the total number of samples examined, 8 (2.7%) were found to be positive for HAV. This virus was detected in 5% and 1.7% of fresh fish and shrimp, respectively. This study shows the importance of sea food as potential sources of HAV infection in people in Iran.

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“…In fact, diverse methods have been developed, but they have been optimized for a limited number of food commodities, including fruits and vegetables (9,20,31,39,50), shellfish (6,46,59), deli ham (16), turkey and roast beef (80), hamburger (77), cheese (38), and water (40). The majority of these methods also include sample elution with a pH 9.5 glycine-buffered solution and subsequent polyethylene glycol (PEG) precipitation.…”

Section: Discussionmentioning

confidence: 99%

Effects of Technological Processes on the Tenacity and Inactivation of Norovirus Genogroup II in Experimentally Contaminated Foods

Mormann¹,

Dabisch²,

Becker³

2010

Appl Environ Microbiol

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Contaminated food is a significant vehicle for human norovirus transmission. The present study determined the effect of physicochemical treatments on the tenacity of infective human norovirus genogroup II in selected foods. Artificially contaminated produce was subjected to a number of processes used by the food industry for preservation and by the consumer for storage and preparation. Virus recovery was carried out by using ultrafiltration and was monitored by using bacteriophage MS2 as an internal process control. Norovirus was quantified by using monoplex one-step TaqMan real-time reverse transcription (RT)-PCR and an external standard curve based on recombinant RNA standards. An RNase pretreatment step was used to avoid false-positive PCR results caused by accessible RNA, which allowed detection of intact virus particles. Significant reductions in titers were obtained with heat treatments usually applied by consumers for food preparation (baking, cooking, roasting). Generally, processes used for preservation and storage, such as cooling, freezing, acidification (>pH 4.5), and moderate heat treatments (pasteurization), appear to be insufficient to inactivate norovirus within a food matrix or on the surface of food. Besides data for persistence in processed food, comparable data for individual matrix-specific protective effects, recovery rates, and inhibitory effects on the PCRs were obtained in this study. The established procedure might be used for other noncultivable enteric RNA viruses that are connected to food-borne diseases. The data obtained in this study may also help optimize the process for inactivation of norovirus in food by adjusting food processing technologies and may promote the development of risk assessment systems in order to improve consumer protection.Norovirus (NV) (formerly Norwalk-like virus) is a member of the family Caliciviridae and is a nonenveloped virus with a single-stranded RNA (ssRNA) genome. Genetically, the human noroviruses are subdivided into three distinct genogroups (genogroup I [GGI], GGII, and GGIV) and into at least 31 genetic clusters or genotypes (53). Noroviruses have emerged as the most common cause of food-borne outbreaks and sporadic cases of acute nonbacterial gastroenteritis worldwide (62,87).In addition to direct person-to-person infection, transmission via environmental contamination (70) and transmission via foods and drinking water (primary and secondary contamination) are known. Food can be contaminated by contact with sewage or sewage water before harvest; e.g., shellfish (3,52,58,60,81) and raspberries (33, 72) have been reported to be vehicles of NV infection. Food is often directly contaminated during production, storage, distribution, and preparation by infected persons (22); e.g., ill or asymptomatic food handlers have been identified as sources of virus contamination of fresh produce and ready-to-eat foods (25, 69). The percentage of cases that can be attributed to food-or waterborne transmission is estimated to be between 16% (56) and 57% (34). Hu...

show abstract

Evaluation of two viral extraction methods for the detection of human noroviruses in shellfish with conventional and real-time reverse transcriptase PCR

Cited by 30 publications

References 28 publications

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

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

Prevalence of hepatitis A virus in sea food in Iran

Effects of Technological Processes on the Tenacity and Inactivation of Norovirus Genogroup II in Experimentally Contaminated Foods

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