Food Environ Virol. 2015 Sep;7(3):239-48.

Development of a Practical Method to Detect Noroviruses Contamination in Composite Meals.

Saito H1, Toho M, Tanaka T, Noda M.
  • 1Akita Prefectural Research Center for Public Health and Environment, 6-6 Senshu-Kubotamachi, Akita, 010-0874, Japan, hrsaito@akita.office.ne.jp.

 

Abstract

Various methods to detect foodborne viruses including norovirus (NoV) in contaminated food have been developed. However, a practical method suitable for routine examination that can be applied for the detection of NoVs in oily, fatty, or emulsive food has not been established. In this study, we developed a new extraction and concentration method for detecting NoVs in contaminated composite meals. We spiked NoV-GI.4 or -GII.4 stool suspension into potato salad and stir-fried noodles. The food samples were suspended in homogenizing buffer and centrifuged to obtain a food emulsion. Then, anti-NoV-GI.4 or anti-NoV-GII.4 rabbit serum raised against recombinant virus-like particles or commercially available human gamma globulin and Staphylococcus aureus fixed with formalin as a source of protein A were added to the food emulsion. NoV-IgG-protein A-containing bacterial complexes were collected by centrifugation, and viral RNA was extracted. The detection limits of NoV RNA were 10-35 copies/g food for spiked NoVs in potato salad and stir-fried noodles. Human gamma globulin could also concentrate other NoV genotypes as well as other foodborne viruses, including sapovirus, hepatitis A virus, and adenovirus. This newly developed method can be used as to identify NoV contamination in composite foods and is also possibly applicable to other foodborne viruses.

PMID: 25796206

 

Supplements:

Food safety measures, including prevention of viral contamination, have become necessary in recent years due to expansion of the food industry and distribution system. Noroviruses (NoVs) are major causes of food poisoning and food-related outbreaks of gastroenteritis worldwide. However, identification of the responsible etiological agents responsible has been restricted due to the difficulty in detecting agents in food samples. It is necessary to concentrate and purify virus particles in a large volume of food emersion into a small volume of suspension to extract viral RNA for genetic assays such as RT-PCR. We have devised an application of immune precipitation by Staphylococcus aureus to recover virus particles from a food emersion. In this study, we developed a “PANtrap method” for the detection and quantification of foodborne viruses in different food samples including samples in solid, liquid, paste and oily states.

 

Figure 1

As contaminated food models, we used stir fried noodles and potato salad representing fat-rich food and carbohydrate-rich food. Initially, these food samples spiked with NoV-GII.4 were suspended in homogenizing buffer After centrifugation at 1,870×g for 30 minutes, the supernatant which is still turbid was transferred into a new 50 ml test tube. Slide1

 

Figure 2

Antiboby against NoV was added to make an NoV-IgG complex. Subsequently, PANSORBIN®, Staphylococcus aureus fixed with formalin (Merk KGaA.), was added. At the beginning of this study, we developed the PANtrap method using NoV-specific rabbit antisera according to the theological backbone. However, the antisera against NoV were prepared by virus-like particles consisting of the capsid protein derived from an expression system using recombinant bacurovirus. Therefore antisera against NoV are available in restricted laboratories. So, we attempted to use a commercially available gamma globulin agent instead of antisera.

Slide2

Figure 3

NoV-IgG complexes adsorbed by PANSORBIN® were collected by centrifugation at 1,870×g for 20 minutes. This PANSORBIN® pellet was resuspended and was transferred into a 1.5 ml micro-tube. Nucleic acid was extracted with phenol / chloroform. After centrifugation, the aqueous solution was transferred into a new 1.5 ml micro-tube. This mixture was applied to an appropriate RNA extraction kit to collect 50 to 60 μl of concentrated and cleared solution containing NoV viral nucleic acid.

Slide3

Figure 4A and 4B

As shown in Figure 4A and 4B, recovery rate using anti NoV-GII.4 rabbit serum was significantly higher than non-specific adsorption without an antibody in both of the foods. Similarly, NoV-GII.4 could be recovered using gamma globulin, although the rate was about 1/3 of that using a specific antiserum (within 3 cycles of the Ct value).

Slide4

In conclusion, we have developed a method for detecting NoV in contaminated foods including composite meals. The general protocol has many advantages compared with other methods. As shown in Figure 1 to Figure 3, the protocol does not involve time-consuming processes such as overnight incubation or expensive apparatus. Additionally, all reagents in the protocol are commercially available. Even if the food emersion is turbid, virus particles are recovered effectively independent of food type. Using gamma globulin, many foodborne viruses are recovered at the same time. Gamma globulin for research use has been put on the market (Advy Japan Co., Ltd.). Reagent set consist of PANSORBIN® and gamma globulin was also released (Merk KGaA.). There have been two reported food poisoning cases of NoV detection from a food samples using this method with a gamma globulin preparation (1,2). As for an alternative application, convalescent-phase serum is effective in food poisoning cases caused by an unknown or emerging virus. Thus, the PANtrap method will be a valuable asset for further studies in food safety.

 

Slide5

 

References:

  1. Miyoshi T, Uchino K, Nishiguchi T, Okayama F, Yoshida E, Tanaka T, Seo M, Tsujimoto Y, Ohashi G, Fujii F, Noda M, Saito H 2011 Food poisoning outbreak at a kindergarten due to consumption of boxed lunch, from which norovirus genome was detected. Infectious Agents Surveillance Report 32 (12): 364-365
  2. Tsuchiya Y, Sahara A, Jinbo T, Nakano T, Kato K, Ogai T, Kosugi K 2015 A foodborne outbreak of Norovirus caused by bread. Jpn. J. Food. Microbiol. 32: 153-158

 

Acknowledgements:

This study was supported by grants for Research on Food Safety from the Ministry of Health, Labour, and Welfare of Japan.

 

Contact:fig6

Hiroyuki Saito, Ph.D.

Chief

Virus Research Laboratory, Department of Microbiology 

Akita Prefectural Research Center for Public Health and Environment

Akita 010-0874, Japan 

hrsaito@akita.office.ne.jp

 

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