Development of an extraction and concentration procedure and comparison of RT-PCR primer systems for the detection of hepatitis A virus and norovirus GII in green onions

158

Several hepatitis A virus (HAV) and norovirus (NV) outbreaks due to consumption of berries and vegetables have been reported during recent years. To facilitate the detection of enteric viruses that may be present on different fresh and frozen products, we developed a rapid and sensitive detection method for HAV, NV, and rotavirus (RV). Initial experiments focused on optimizing the composition of the elution buffer, improving the viral concentration method, and evaluating the performance of various extraction kits. Viruses were extracted from the food surface by a direct elution method in a glycine-Tris (pH 9.5) buffer containing 1% beef extract and concentrated by ultrafiltration. Occasionally, PCR inhibitors were present in the processed berry samples, which gave relatively poor detection limits. However, this problem was overcome by adding a pectinase treatment in the protocol, which markedly improved the sensitivity of the method. After optimization, this concentration method was applied in combination with real-time reverse transcription-PCR (RT-PCR) using specific primers in various types of berries and vegetables. The average detection limits were 1 50% tissue culture infective dose (TCID 50 ), 54 RT-PCR units, and 0.02 TCID 50 per 15 g of food for HAV, NV, and RV, respectively. Based on our results, it is concluded that this procedure is suitable to detect and quantify enteric viruses within 6 h and can be applied for surveillance of enteric viruses in fresh and frozen products.

Section: Discussionsupporting

confidence: 88%

Procedure for Rapid Concentration and Detection of Enteric Viruses from Berries and Vegetables

Butot

Putallaz

Sánchez

2007

158

“…Molecular detection methods such as reverse transcriptase PCR (RT-PCR) are commonly used for detecting NoV in various foods (24). However, large volumes of elution buffers are typically necessary for eluting NoV from food, which requires further concentration and purification methods (8,26). In addition, various PCR inhibitors that interfere with subsequent molecular assays also are present in elution buffers (25).…”

mentioning

confidence: 99%

Immunomagnetic Separation Combined with Real-Time Reverse Transcriptase PCR Assays for Detection of Norovirus in Contaminated Food

Park

Cho

Jee

et al. 2008

We developed an immunomagnetic separation (IMS) technique combined with real-time TaqMan reverse transcriptase PCR (RT-PCR), which allowed detection of norovirus at a level as low as 3 to 7 RT-PCR units from artificially contaminated strawberries. The inoculum recovery rate ranged from 14 to 30%. The data demonstrate that IMS combined with real-time RT-PCR will be useful as a rapid and sensitive method for detecting food-borne microbial contaminants.

“…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

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...