Int J Food Microbiol. 2015 May 18;201:1-6. doi: 10.1016/j.ijfoodmicro.2015.02.012.

Application of viability PCR to discriminate the infectivity of hepatitis A virus in food samples.


Morenoa, R. Aznara, b, G. Sáncheza,b

aDepartment of Microbiology and Ecology, University of Valencia, Av. Dr. Moliner, 50, 46100 Burjassot, Valencia, Spain

bDepartment of Biotechnology, Institute of Agrochemistry and Food Technology (IATA-CSIC), Av. Agustín Escardino, 7, 46980 Paterna, Valencia, Spain



Transmitted through the fecal–oral route, the hepatitis A virus (HAV) is acquired primarily through close personal contact and foodborne transmission. HAV detection in food is mainly carried out by quantitative RT-PCR (RT-qPCR). The discrimination of infectious and inactivated viruses remains a key obstacle when using RTqPCR to quantify enteric viruses in food samples. Initially, viability dyes, propidium monoazide (PMA) and ethidium monoazide (EMA), were evaluated for the detection and quantification of infectious HAV in lettuce wash water. Results showed that PMA combined with 0.5% Triton X-100 (Triton) was the best pretreatment to assess HAV infectivity and completely eliminated the signal of thermally inactivated HAV in lettuce wash water. This procedure was further evaluated in artificially inoculated foods (at concentrations of ca. 6 × 104, 6 × 103 and 6 × 102 TCID50) including lettuce, parsley, spinach, cockles and coquina clams. The PMA–0.5% Triton pretreatment reduced the signal of thermally inactivated HAV between 0.5 and 2 logs, in lettuce and spinach concentrates. Moreover, this pretreatment reduced the signal of inactivated HAV by more than 1.5 logs, in parsley and ten-fold diluted shellfish samples inoculated at the lowest concentration. Overall, this pretreatment (50 μM PMA–0.5% Triton) significantly reduced the detection of thermally inactivated HAV, depending on the initial virus concentration and the food matrix.

KEYWORDS: Ethidium monoazide; Hepatitis A virus; Propidium monoazide; Quantitative PCR; Viability dyes

PMID: 25720326



Hepatitis A infection, caused by hepatitis A virus (HAV), is the leading cause of human viral hepatitis throughout the world and is mainly propagated via the fecal-oral route. Foodborne outbreaks caused by HAV are mainly associated with shellfish, produce (soft fruits and leafy greens), and ready-to-eat meals. Nowadays, current methods for the detection of HAV naturally present in foods are based on real-time reverse transcription PCR (RT-qPCR) however the inability to differentiate between infections and inactivated viruses is considered a major limitation of RT-qPCR. Hence, there is an emerging attention toward the use of viability PCR which combines the use of photo-reactive dyes (e.g. ethidium monoazide, EMA, or propidium monoazide, PMA) with a high affinity for DNA/RNA and a photo-chemical reaction which leads to covalent binding of the dye to DNA/RNA thus preventing its amplification by PCR (1). The main challenge of this approach requires eliminating PCR signal of inactivated microorganism following EMA/PMA treatment.

During a previous study we optimized a PMA pretreatment for assessing HAV infectivity (2). Moreover, a French team reported that viability pretreatments are enhanced by the use of surfactants (3). The purpose of this study was to assess the applicability of viability PCR for the detection and quantification of infectious HAV in lettuce wash water, leafy greens and shellfish.

We first tested various viability dyes/surfactants combinations. The treatment of thermally-inactivated HAV with 50 μM PMA and 0.5% Triton precluded its detection in lettuce wash water (Figure 1A). Moreover, using this approach a significant improvement in the discrimination of thermally-inactivated HAV in vegetables and shellfish was achieved (Figure 2B).



Figure 1: Schematic representation of viability RT-qPCR detection results for infectious and thermally inactivated HAV suspensions inoculated in lettuce wash water (A) and spinach concentrates (B).


Acknowledgements: This study was supported by grants AGL2009-08603 from the Spanish Ministry of Science and Innovation, and ACOMP/2010/279 and ACOMP/2012/199 from the Generalitat Valenciana. G. Sánchez was supported by the “Ramón y Cajal” Young Investigator program of the Spanish Ministry of Economy and Competitiveness (RYC-2012-09950).



  1. Elizaquível P, Aznar R, Sánchez G (2014). Recent developments in the use of viability dyes and quantitative PCR in the food microbiology field J Appl Microbiol 116:1-13.
  2. Sánchez G, Elizaquível P, Aznar R (2012). Discrimination of infectious hepatitis A viruses by propidium monoazide real-time RT-PCR. Food Environ Virol 4:21–25.
  3. Coudray-Meunier C, Fraisse A, Martin-Latil S, Guiller L, Perelle S (2013). Discrimination of infectious hepatitis A virus and rotavirus by combining dyes and surfactants with RT-qPCR. BMC Microbiol 13:216–231.



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