Reverse Transcription-PCR Analysis of Bottled and Natural Mineral Waters for the Presence of Noroviruses

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: Comparison Of the Performances Of Different Elution Buffersmentioning

confidence: 99%

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

Butot

Putallaz

Sánchez

2007

158

“…Although reverse transcription-PCR (RT-PCR) has become the standard for diagnosis of NoV infection worldwide (1,5,36), many laboratories use multiple sets of primers targeting different regions of the genome because of the difficulty of designing broadly reactive primers that are able to sensitively detect all the different NoV genotypes (36). In addition, confirmatory steps are needed to evaluate the specificity of the amplification product (5,34,35) or to increase the sensitivity for detection of NoV in food and water matrices (6,18). Recently, several SYBR Green and TaqMan-based RT-PCR assays for the detection of NoV have been reported (2,10,12,23,28).…”

mentioning

confidence: 99%

Rapid and Sensitive Detection of Noroviruses by Using TaqMan-Based One-Step Reverse Transcription-PCR Assays and Application to Naturally Contaminated Shellfish Samples

Jothikumar

Lowther

Henshilwood

et al. 2005

323

220

Noroviruses (NoV), which are members of the family Caliciviridae, are the most important cause of outbreaks of acute gastroenteritis worldwide and are commonly found in shellfish grown in polluted waters. In the present study, we developed broadly reactive one-step TaqMan reverse transcription (RT)-PCR assays for the detection of genogroup I (GI) and GII NoV in fecal samples, as well as shellfish samples. The specificity and sensitivity of all steps of the assays were systematically evaluated, and in the final format, the monoplex assays were validated by using RNA extracted from a panel of 84 stool specimens, which included NoV strains representing 19 different genotypes (7 GI, 11 GII, and 1 GIV strains). The assays were further validated with 38 shellfish cDNA extracts previously tested by nested PCR. Comparison with a recently described real-time assay showed that our assay had significantly higher sensitivity and was at least as sensitive as the nested PCR. For stool specimens, a one-step duplex TaqMan RT-PCR assay performed as well as individual genogroup-specific monoplex assays. All other enteric viruses examined were negative, and no cross-reaction between genogroups was observed. These TaqMan RT-PCR assays provide rapid (less than 90 min), sensitive, and reliable detection of NoV and should prove to be useful for routine monitoring of both clinical and shellfish samples.

“…Viral RNAs from water and walls were quantified using real-time RT-PCR. NV GI RNA was quantified using a specific assay for the Valetta strain, as described elsewhere (16), with an ABI Prism 7700 sequence detection system (PE Applied Biosystems, Foster City, CA). The NV RT reaction was performed at 41°C for 60 min using a Sensiscript RT kit (QIAGEN GmbH, Hilden, Germany) consisting of 1ϫ RT buffer, 500 M (each) nucleotides, 1 M 9.4 reverse primer, 1 l of Sensiscript reverse transcriptase, 10 U of RNase inhibitor (Promega, Madison, WI), and 10 l of NV RNA in a final volume of 25 l. NV real-time PCR was performed using a TaqMan Universal PCR Master Mix (Applied Biosystems) consisting of 1ϫ TaqMan buffer, 0.2 M TaqMan probe 9.4, and 0.3 M 9.4 reverse and forward primers in a final volume of 50 l containing 10 l of cDNA.…”

Section: Cells Viruses and Infectionsmentioning

confidence: 99%

“…Water samples were analyzed as described elsewhere (9,16). Briefly, after the bottles were shaken vigorously, the water was filtered through a 0.45-m-poresize positively charged membrane (Zetapor filter membrane; CUNO, Inc., Meriden, CT).…”

Section: Cells Viruses and Infectionsmentioning

confidence: 99%

See 1 more Smart Citation

Attachment of Enteric Viruses to Bottles

Butot

Putallaz

Croquet

et al. 2007

Storage of water that was deliberately contaminated with enteric viruses in polyethylene terephthalate (PET) bottles led to a rapid decrease of the apparent viral load, thereby hampering the development of samples for a collaborative evaluation of viral detection methods for bottled water. To determine if this decrease was due to spontaneous inactivation or to adhesion, an elution protocol was developed and combined with a rapid and sensitive real-time reverse transcription-PCR-based method to quantify adsorbed norovirus (NV), hepatitis A virus (HAV), and rotavirus (RV) on bottle walls. The NV retention on PET bottle walls after 20 and 62 days reached an average level of 85% and 95% of the recovered inoculum, respectively. HAV and RV also showed adsorption onto PET bottles, reaching 90% and 80%, respectively, after 20 days of storage. NV and RV attachment was demonstrated to be dependent on the presence of autochthonous flora, whereas HAV adsorption was independent of it. Application of the elution and viral detection protocol to 294 commercially available water bottles obtained from 25 different countries did not give any positive result, thereby providing further evidence that the sources used for this product are free from enteric viruses and support for the theory that bottled water is not a vehicle for viral diseases.Food-borne and waterborne outbreaks of human enteric viruses, such as norovirus (NV), hepatitis A virus (HAV) and rotavirus (RV), are a matter of serious concern for public health.Although there is no epidemiological evidence that bottled water serves as a vehicle for viral diseases, some doubts were raised concerning its safety due to the reported finding of NV sequences in 33% of commercially available water samples sold in Switzerland (2, 3). However, attempts by other research groups to reproduce these results were always in vain (6,15,16,19,21,27), even though much larger numbers of samples and more-sensitive detection methods were used. Further investigations strongly suggested that the original findings were due to artifacts and systematic mistakes (21). Since these unfounded allegations could have severe economic consequences for the bottled water industry, it is essential to develop internationally accepted virus detection methods for this matrix. An essential step in the validation of such methods is the organization of a collaborative trial to demonstrate its reproducibility. When preparing artificially contaminated samples for the validation of our NV detection method in bottled water based on membrane filtration and real-time reverse transcription-PCR (RT-PCR) (16), we observed a substantial decrease of the viral load after a few days of storage. Since adsorption of human enteric viruses to the walls of different container materials had been reported previously (7,8,20,25,28), we suspected that a similar phenomenon was occurring in our samples. We therefore investigated whether enteric viruses may also adsorb to polyethylene terephthalate (PET) and glass bottles and to what extent ...