Contents lists available at ScienceDirect Food Control journal homepage: www.elsevier.com/locate/foodcont Specific detection of viable Salmonella Enteritidis by phage amplification combined with qPCR (PAA-qPCR) in spiked chicken meat samples Alejandro Garrido-Maestu a,* , Pablo Fuciños b , Sarah Azinheiro a , Carla Carvalho c,d , Joana Carvalho a , Marta Prado a a Department of Life Sciences, Nano4Food Unit, Food Quality and Safety Research Group, Av. Mestre José Veiga s/n, 4715-330, Braga, Portugal b Department of Life Sciences, Nano4Food Unit, Food Processing Research Group, Av. Mestre José Veiga s/n, 4715-330, Braga, Portugal c Department of Nanoelectronics Engineering, Nanodevices Research Group, International Iberian Nanotechnology Laboratory, Av. Mestre José Veiga s/n, 4715-330, Braga, Portugal d Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal ARTICLE INFO Keywords: Bacteriophage vB_SenS_PVP-SE2 qPCR Phage amplification Salmonella enteritidis Chicken ABSTRACT Serovar Enteritidis represents 45.7% of all Salmonella reported human cases identified in Europe. Additionally, “minced meat and meat preparations from poultry” have a high level of non-compliance, regarding Salmonella regulation. In the current study, a novel method based on the amplification of the Salmonella bacteriophage vB_SenS_PVP- SE2, coupled with real-time PCR (qPCR), was developed and evaluated, for the rapid detection of viable Salmonella Enteritidis in chicken samples. The results obtained indicated that the qPCR method could detect down to 0.22 fg/μL of pure virus DNA and a concentration of viral particles of 10 3 pfu/mL. After a short bacterial recovery step, the addition of bacteriophages to spiked chicken samples indicated that 8 cfu/25 g could be de- tected within 10 h, including the time for DNA extraction and qPCR analysis. Additionally, the evaluation of the performance parameters: relative sensitivity, specificity, accuracy, positive and negative predictive values, and index kappa of concordance, obtained values higher than 92%, and the acceptability limit values were within the limits. All these results demonstrate that the proposed methodology is a powerful tool for the rapid detection of viable Salmonella Enteritidis. 1. Introduction The genus Salmonella comprises two species (Salmonella enterica and Salmonella bongori) and more than 2500 different serovars (Grimont & Weill, 2007). This genus is one of the most common foodborne patho- gens worldwide, as highlighted by the fact that in 2015, 94 625 cases of salmonellosis were reported in Europe, representing about 28% of all reported foodborne diseases in Europe, and a 1.9% increase with re- spect to 2014; furthermore, ten member states reported 126 fatal cases (EFSA and ECDC, 2017). It has been extensively reported that the traditional methods for the detection of foodborne pathogens are lengthy and laborious. For in- stance, those described by the International Organization for Standardization (ISO) and the Bacteriological Analytical Manual (BAM) from the U.S. Food and Drug Administration (Andrews, Jacobson, & Hammack, 2011; ISO, 2003) require several hands-on steps over several days (from three to six with confirmation). Against the classical approaches, molecular methods have arisen as fast and reliable alter- natives. Focus has been put mainly on those based on nucleic acids amplification, such as the Polymerase Chain Reaction or the real-time PCR (PCR/qPCR, (Chapela, Garrido-Maestu, & Cabado, 2015)), and more recently those based on isothermal amplification such as Loop- mediated isothermal amplification (LAMP, (D'Agostino, Diez-Valcarce, Robles, Losilla-Garcia, & Cook, 2015)), Ligase Chain Reaction (LCR (Jang et al., 2003),), or Recombinase Polymerase Amplification (RPA, (Kim & Lee, 2016)). However, a drawback commonly attributed to these techniques is their incapacity to differentiate between viable and non-viable microorganisms. Efforts to overcome this limitation have resulted in the development of alternative approaches that lead to the specific detection of viable bacteria, such as amplification coupled with propidium monoazide (PMA) treatment, RNA amplification, or specific sample treatments to eliminate non-viable microorganisms, thus al- lowing the direct application of conventional nucleic acid amplification techniques (D'Urso et al., 2009; Feng et al., 2016; Zhang, Brown, & https://doi.org/10.1016/j.foodcont.2018.12.038 Received 15 October 2018; Received in revised form 26 December 2018; Accepted 27 December 2018 * Corresponding author. E-mail address: alejandro.garrido@inl.int (A. Garrido-Maestu). Food Control 99 (2019) 79–83 0956-7135/ © 2018 Elsevier Ltd. All rights reserved. T