Comparative Analysis of Viral Concentration Methods for Detecting the HAV Genome Using Real-Time RT-PCR Amplification

Department of Food and Nutrition, School of Food Science and Technology, Chung-Ang University, 72-1, Nae-ri, Daeduck-myun, Ansung-si, Kyounggi-do, 456-756, South Korea.
Food and Environmental Virology (Impact Factor: 2.36). 06/2012; 4(2):68-72. DOI: 10.1007/s12560-012-9077-x
Source: PubMed


Hepatitis A is a major infectious disease epidemiologically associated with foodborne and waterborne outbreaks. Molecular detection using real-time RT-PCR to detect the hepatitis A virus (HAV) in contaminated vegetables can be hindered by low-virus recoveries during the concentration process and by natural PCR inhibitors in vegetables. This study evaluated three virus concentration methods from vegetables: polyethylene glycol (PEG) precipitation, ultrafiltration (UF), and immunomagnetic separation (IMS). UF was the most efficient concentration method, while PEG and IMS were very low for the recovery rate of HAV. These results demonstrate that UF is the most appropriate method for recovering HAV from contaminated vegetables and that this method combined with the real-time RT-PCR assay may be suitable for routine laboratory use.

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    • "c o m / l o c a t e / i j f o o d m i c r o CEN ISO/TS 15216-parts 1 & 2 (ISO/TS, 15216-1, 2013; ISO/TS, 15216-2, 2013). The detection of enteric viruses in food is difficult due to the low level of viral contamination in food products and the presence of substances that can inhibit PCR amplification (Lee et al., 2012; Maunula et al., 2013; Suffredini et al., 2014). The use of appropriate controls (process control virus and external control RNA) should be consistently included whenever testing foods for viral contamination. "
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    ABSTRACT: Noroviruses (genogroup I (NoV GI) and genogroup II (NoV GII)) and the hepatitis A virus (HAV) are frequently involved in foodborne infections worldwide. They are mainly transmitted via the fecal-oral route, direct person-to-person contact or consumption of contaminated water and foods. In food virology, detection methods are currently based on identifying viral genomes using real-time reverse transcriptase PCR (RT-qPCR). One of the general requirements for detecting these viruses in food involves the use of a process control virus to monitor the quality of the entire viral extraction procedure as described in the ISO/TS 15216-1 and 15216-2 standards published in 2013. The selected process control virus should have similar morphological and physicochemical properties as the screened pathogenic virus and thus have the potential to provide comparable extraction efficiency. The aim of this study was to determine which virus should be used for process control, murine norovirus (MNV-1) or Mengovirus, when testing for the presence of HAV, NoV GI and NoV GII in bottled water, lettuce and semi-dried tomatoes. Food samples were spiked with HAV, NoV GI or NoV GII alone or in the presence of MNV-1 or Mengovirus. Recovery rates of each pathogenic virus were compared to those of both process control viruses using a multiple comparison procedure. Neither process control virus influenced the recovery of pathogenic virus regardless of the type of food matrix. MNV-1 was the most appropriate virus for validating the detection of HAV and NoV GII in all three food matrices as well as NoV GI in lettuce. Mengovirus proved to be the most appropriate control for NoV GI detection in bottled water and semi-dried tomatoes. The process control virus is essential for validating viral detection in food and the choice of virus depends on food type and the screened pathogenic virus. Copyright © 2015 Elsevier B.V. All rights reserved.
    International Journal of Food Microbiology 02/2015; 202:57-65. DOI:10.1016/j.ijfoodmicro.2015.02.029 · 3.08 Impact Factor
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    • "According to Scherer et al. (2010), the average recovery rates of NoV from lettuce and raspberry were 23 and 24 %, respectively. Lee et al. (2012) reported that the recovery rates for HAV were 0.65 and 0.7 % from inoculated lettuce and perilla leaf, respectively, as calculated using the IMS/RT-PCR method to detect HAV. HAV recovery rates from lettuce and perilla leaf were similar to those from oyster and mussel. "
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    ABSTRACT: Outbreaks of viral diseases are frequently associated with the consumption of minimally processed shellfish. Among the viruses in these outbreaks, hepatitis A virus (HAV) and human norovirus (NoV) have been increasingly reported as the most common food-borne pathogens. These viruses must be concentrated in tested samples in order to be detected. In this study, a method for the detection of NoV and HAV in shellfish using an immuno-magnetic separation (IMS) procedure combined with reverse transcriptase (RT)-PCR was developed. The IMS/RT-PCR method was applied to investigate the recovery rates of HAV, NoV GI.1, and GII.4 from oyster and mussel. Based on IMS/RT-PCR results, recovery rates for HAV from oyster and mussel test samples were 2.4 and 1.1 %, respectively. The NoV GI.1 recovery rates from oyster and mussel samples were 4.9-9.2 % (mean 6.9 %) and 4.3-8.6 % (mean 6.2 %), respectively, and the NoV GII.4 recovery rates were 8.8 and 8.5 %, respectively. These results verified that HAV, NoV GI.1, and GII.4 can be detected in all the test samples using the IMS/RT-PCR method, although the three inoculated viruses were recovered with low efficiency. In conclusion, the IMS/RT-PCR method can be used to efficiently and rapidly detect viruses such as HAV and NoV in shellfish such as oyster and mussel.
    Food and Environmental Virology 06/2014; 6(4). DOI:10.1007/s12560-014-9156-2 · 2.36 Impact Factor
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    • "Also, Arenavirus has been detected in patients using a nested RT-PCR assay (Park et al., 1997). RT-PCR and real-time PCR methods were reported to detect hepatitis A (Hutson et al., 2004; Lee et al., 2012; Stals et al., 2013). Molecular biology techniques have been used for detection of the most common foodborne viruses like Norovirus and hepatitis A in shellfish, but none are usually available for other foods virus. "
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    ABSTRACT: Foodborne pathogens comprise microorganisms such as viruses, bacteria and parasites that can be transmitted by food and affect public health worldwide. The most common viruses transmitted via food are hepatitis A virus and Norwalk-like caliciviruses. Also, the most common bacteria involved in foodborne illnesses are Campylobacter jejuni, Clostridium perfringens, Salmonella spp, Escherichia coli O157:H7; and the most important parasites that can cause these conditions are Giardia duodenalis, Cryptosporidium parvum, Cyclospora cayetanensis, Toxoplasma gondii, Trichinella spiralis, Taenia saginata and/or solium, Entamoeba histolytica, Anisakis spp. and Diphyllobothrium spp. Because of their eventual small number in the sample, their detection and identification is not always easy. On the other hand, conventional methods like cultures are almost labor intensive, time consuming and costly. Recently, molecular techniques have been developed for rapid, sensitive and specific identification. The most common molecular methods are polymerase chain reaction (PCR)-based techniques. In this article, the sensitive and specific molecular tests for routine detection and identification of foodborne pathogens are reviewed.
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