Rapid and simultaneous detection of viable Cronobacter sakazakii, Staphylococcus aureus, and Bacillus cereus in infant food products by PMA-mPCR assay with internal amplification control Fan Li a , Guoyang Xie a , Baoqing Zhou a , Pei Yu a , Shuang Yu a , Zoraida P. Aguilar b , Hua Wei a , Hengyi Xu a, * a State Key Laboratory of Food Science and Technology, Nanchang University, 235 Nanjing East Road, Nanchang, 330047, PR China b Zystein, LLC., Fayetteville, AR, 72704, USA article info Article history: Received 6 March 2016 Received in revised form 30 June 2016 Accepted 17 July 2016 Available online 18 July 2016 Keywords: Cronobacter sakazakii Multiplex PCR Viable cell Infant food abstract Cronobacter sakazakii, Staphylococcus aureus, and Bacillus cereus are important pathogens contaminating infant food products. In this study, we developed a specific, sensitive, and accurate technique for the simultaneous detection of viable C. sakazakii, S. aureus, and B. cereus in infant food products. An internal amplification control (IAC) was added to the multiplex PCR (mPCR) as indicator of false negative results that are mainly caused by the PCR inhibitors in food products. In addition, to detect only the viable bacteria, propidium monoazide (PMA) was applied to selectively suppress the PCR signal from dead cells. The limit of detection (LOD) for the viable cells with or without PMA treatment was 10 4 CFU/mL for C. sakazakii and B. cereus, and 10 2 CFU/mL for S. aureus in pure culture, showing that the PMA treatment did not influence the sensitivity. After 12 h enrichment, the PMA-mPCR-IAC assay could detect as low as 10 1 CFU/g for C. sakazakii and S. aureus, and 10 0 CFU/g for B. cereus in spiked infant food products (infant formula, noodles, milk and rice noodles). This PMA-mPCR-IAC assay we developed hold promise for the simultaneous detection of C. sakazakii, S. aureus, and B. cereus in infant food products. © 2016 Elsevier Ltd. All rights reserved. 1. Introduction The consumption of food products that are important compo- nents of a healthy and balanced diet for infants continues to in- crease in many countries. However, notable outbreaks of illnesses associated with infant food products have been mainly caused by foodborne pathogens (Bowen & Braden, 2006; Chenu & Cox, 2009). Among the foodborne pathogens, Cronobacter sakazakii, Staphylo- coccus aureus, and Bacillus cereus have been frequently reported as agents leading to outbreaks of diseases in infant food products. C. sakazakii is the most commonly reported foodborne pathogen in powdered infant formula associated mainly with neonatal in- fections that can cause life-threatening meningitis, necrotizing enterocolitis, and septicemia with high fatality rates that ranges from 40 to 80% (Huertas et al., 2015; Mohammed, Sallam, & Tamura, 2015). In addition, B. cereus has been involved in diarrhea and emesis. Food poisoning from B. cereus has been mainly linked to cooked rice, noodles, pasta, rice noodles and milk products (Zhang et al., 2014). Furthermore, S. aureus had been suspected to produce enterotoxins and cause food poisoning or staphylococcal infections, such as cellulitis and osteomyelitis. A recent study reported clinical estimates indicating that S. aureus has led to more than 94,000 serious infections and more than 18,000 deaths in the United States since 2005 (Schmelcher et al., 2012). Hence, it is imperative that the detection of these three foodborne pathogens be made available to monitor infant food products and prevent related illnesses. Traditionally, the detection and enumeration of foodborne pathogens rely on selective culture and standard biochemical methods, which include enrichment, selection, biochemical iden- tification, serotype confirmation, and toxin testing. All these methods are time-consuming and laborious (Velusamy, Arshak, Korostynska, Oliwa, & Adley, 2010). Moreover, foodborne patho- gens that normally occur in low numbers tend to lead to large er- rors in sampling and enumeration. Furthermore, culture methods fail to detect viable but non-culturable (VBNC) cells which are capable of causing illness (Jiang, Fu, Chen, Wang, & Liu, 2013). Hence, rapid and accurate methods for the detection of foodborne * Corresponding author. E-mail addresses: kidyxu@163.com, HengyiXu@ncu.edu.cn (H. Xu). Contents lists available at ScienceDirect LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt http://dx.doi.org/10.1016/j.lwt.2016.07.044 0023-6438/© 2016 Elsevier Ltd. All rights reserved. LWT- Food Science and Technology 74 (2016) 176e182