Standardized multiplex one-step qRT-PCR for hepatitis A virus, norovirus GI and GII quantification in bivalve mollusks and water
Introduction
A wide variety of human enteric viruses may be found as food and water contaminants. However, with few exceptions most well characterized foodborne viral outbreaks are restricted to human norovirus (NoV), and hepatitis A virus (HAV), that thus become the main targets of virus detection in food. Human NoV are shed in extremely high numbers in the feces of infected individuals (up to 1011 genome copies per gram of stool) (Atmar et al., 2008) while patients suffering from hepatitis A may excrete from 106 to 1011 (Costafreda et al., 2006, Pinto et al., 2012). Wastewater treatments do not ensure complete virus removal (Blatchley et al., 2007, Bosch, 2007, El-Senousy et al., 2007), hence viruses become environmental contaminants in numbers high enough to represent a public health threat although in numbers low enough to pose serious difficulties for their detection. Human NoV and HAV infections are mostly transmitted person-to-person but foodborne transmission is increasingly reported. Exposure to contaminated drinking water, to crops irrigated with contaminated waters or to shellfish grown in contaminated waters, eventually results in waterborne and foodborne outbreaks (Kukkula et al., 1999, Costafreda et al., 2006, Le Guyader et al., 2006, Le Guyader et al., 2010, Pintó et al., 2009, Li et al., 2012).
As a consequence of the globalization of food trade, many outbreaks are caused by products imported from third countries (Falkenhorst et al., 2005, Pintó et al., 2009, Sarvikivi et al., 2012). This has been evidenced to be particularly critical in recent outbreaks of hepatitis A (Gillesberg Lassen et al., 2013); (http://www.cdc.gov/hepatitis/Outbreaks/2013/A1b-03-31/). Hepatitis A is highly endemic in developing regions while it is much less frequent in developed regions. This pattern has important implications on the average age of exposure and on the severity of clinical disease which increases with age (Pinto et al., 2012). In young children the infection mostly develops asymptomatically, while in adults the infection presents with symptoms. Since hepatitis A infection induces life-long immunity, severe infections among adults are rare in highly endemic regions where most children are infected early in life. By contrast, in low-endemic areas, the disease occurs mostly in adulthood with a high probability of developing severe symptomatic illness.
In this context, reliable and affordable methodologies for the detection of human NoV and HAV in water and, particularly, in food are needed. In 2004 the European Committee on Standardization (CEN) tasked a technical advisory group (CEN/TC275/WG6/TAG4) with the development of standard methods (quantitative and qualitative) for detection of NoV and HAV in selected foodstuffs that developed as ISO procedures (CEN/ISO TS 15216-1 and CEN/ISO TS 15216-2; April 7, 2013).
In the present study, a multiplex (quadruplex) for the quantitative detection of HAV, NoV GI and NoV GII, including mengovirus as process control, in food and water was developed. The efficiency of the procedures formulated by the CEN/TC275/WG6/TAG4 committee (CEN method), based on four monoplex reactions was compared with the quadruplex format in which all viruses are detected in a single reaction.
Section snippets
Viruses, cells and plasmids
The cell-adapted cytopathogenic pHM175 43c strain of HAV (kindly provided by T. Cromeans, Centers for Disease Control and Prevention, Atlanta, GA) and NoV isolates GI.6 and a GII.4 (New Orleans 2009 type) from stool specimens of patients with gastroenteritis (kindly provided by R. Bartolomé, Hospital Vall d'Hebron, Barcelona) were used as positive controls. The pHM175 43c strain was grown in FRhK-4 cells as previously described (Aragonès et al., 2008).
Infectious mengovirus strain MC0 (ATCC
Quadruplex mastermix under monoplex conditions
A quadruplex reaction, containing all primer sets and probes for the detection of the three targets as well as the process control virus, was first tested using mastermix concentrations of reagents, primers and probes and the RT-PCR program of the monoplex reactions as described in the CEN method. Plasmids containing the cloned amplicons corresponding to each of the three targets were individually tested under these conditions and the corresponding standard curves of ten-log dilution series
Discussion
The European Commission (SANCO 12655/2012) has implemented the monitoring of human NoV and HAV in some food imports in accordance with Art 15 (5) of Regulation (EC) No 882/2004 (http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2009:194:0011:0021:EN:PDF), employing the standardized CEN methodologies (ISO/TS 15216-1 and ISO/TS 15216-2; 2013). These procedures are currently being validated following the principle and technical protocol for the validation of alternative methods in the
Conclusions
A standardized multiplex assay for quantitative NoV GI and GII and HAV detection in water and food, using mengovirus as process control has been developed. The quadruplex assay format has been validated in naturally-contaminated water and food samples in comparison with the four monoplex assay counterparts.
As expected, the quadruplex assay showed a certain loss of sensitivity in comparison with the monoplex counterpart which however was negligible when naturally-contaminated samples were
Acknowledgments
This work was supported in part by grants, CSD2007-00016 and BIO2008-01312 (Ministry of Education and Science, Spain; http://web.micinn.es/), BIO2011-23461 and PS09/02516 (Ministry of Economy and Competitiveness, Spain; http://www.idi.mineco.gob.es/), 2009SGR00024 and XRB-Biotechnolgy Reference Network (Generalitat de Catalunya; www.gencat.cat/agaur) and FP7-KBBE-2012-6 of the European Union (http://cordis.europa.eu/fp7). The funding agencies had no role in study design, data collection and
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2022, Journal of Environmental ManagementCitation Excerpt :This species is quite resistant to environmental pressures, is easy to handle and can be used in laboratory studies or in the field through the caging technique (Bervoets et al., 2005; Capizzi-Banas et al., 2021; Géba et al., 2020; Kerambrun et al., 2016; Le Guernic et al., 2020; Palos Ladeiro et al., 2018). The digestive tissues of bivalve molluscs is generally used to detect the presence of enteric viruses, especially enteroviruses, since it is the main site of contamination within the bivalve maybe due to specific receptors (Fuentes et al., 2014; Le Guyader et al., 2006; Lees and TAG, 2010; Suffredini et al., 2020). Desdouits et al. (2021) have reported accumulation of inactivated SARS-CoV-2 genome in digestive, mantle, and gill tissues of oysters (Crassostrea gigas).
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2018, Journal of Virological MethodsCitation Excerpt :Furthermore, bioaccumulation studies have shown similar behavior and recovery of Mengovirus and NoV GI and GII strains in oysters (Le Guyader et al., 2009). It has also been used as a PCV in the analysis of different types of water (drinking water, wastewater and sewage sludge) (Amdiouni et al., 2013) and various types of food (fresh leafy vegetables, soft and fresh red fruits), products such as tomatoes, cucumbers and fruit salads (Baert et al., 2011; Comelli et al., 2008; Costafreda et al., 2006; da Silva et al., 2007; Fuentes et al., 2014). Ideally, a satisfactory PCV should be used in a way similar to and with effectiveness compared to the viral target; regardless of the nucleic acid extraction method, or RT-PCR kit used.