Development and application of a new multiplex real‐time PCR assay for simultaneous identification of Brucella melitensis, Cronobacter sakazakii and Listeria monocytogenes in raw milk and cheese

Abstract

Brucella melitensis, Cronobacter sakazakii and Listeria monocytogenes are important foodborne pathogens in milk and milk products, which are responsible for a variety of diseases that pose serious hazards to public health and food safety. The objective of this study was to develop a novel multiplex RTi‐PCR for the detection of B. melitensis, C. sakazakii and L. monocytogenes and to characterise the potential risk of these pathogens in raw milk and cheese. The raw milk (n = 25) and cheese samples (n = 20) were analysed by multiplex RTi‐PCR assay and detected for quantification of the three pathogens. In this study, B. melitensis, C. sakazakii and L. monocytogenes were simultaneously identified using BMEII0466, mms operon and hly as target genes, respectively. The multiplex RTi‐PCR assay that was developed showed good sensitivity and selectivity for the pathogenic microorganisms (r2 = 0.986–0.997). Multiplex RTi‐PCR results showed that most of the samples were contaminated with the pathogens screened.

Global and regional prevalence of Cronobacter sakazakii in powdered milk and flour

“…For instance, individual studies from Africa reported Cz prevalence in PMF as 0.00% in PIF 117 , 5.36% 118 , and 11.25% 10 in Egypt; 0.00% 119 (India, PIF), 1.10% 120 (China, PIF), 1.45% 121 (Jordan, PIF), 1.67% 11 (China, PIF), 2.00% 7 (China, PIF), 3.08% 9 (Iraq, PIF), 3.13% 122 (Bangladesh, PIF), 4.07% 123 (China, PIF), 4.12% 124 (China, Ifoods), 4.34% 125 (China, PIF), 5.00% 126 (Iran, PIF), 6.04% 127 (Japan, PIF), 6.86% 127 (Korea, PIF), 7.20% 128 (Iran, PIF), 7.51% 129 (China, IFF), 8.00% 126 (Iran, Ifoods), 10.60% 130 (China, EPIF), 11.94% 40 (China, IFF), 12.28% 131 (China, PIF), 16.53% 11 (China, CPIF), 16.88% 132 (China, PIF), and 47.67% 133 (Korea, IFF) in Asia. In Europe, individuals studies demonstrated Cz prevalence in PMF as 0.00% 134 , Czech Republic, IFF/PIF; 0.00% 135 (Turkey, CPIF), 0.00% 136 (Ireland, PIF), 0.35% 107 (Netherlands, PIF), 0.50% 137 (Slovakia, PIF), 0.74% 138 (UK, FUF), 1.00% 139 (Germany, EPIF), 1.16% 137 (Slovakia, CPIF), 2.50% 136 (Ireland, IFF), 3.33% 140 (Czech Republic, PIF), 4.09% 141 (Austria, PIF), 10.00% 142 ( Turkey, PIF), 11.11% 140 (Czech Republic, IFF), 12.29% 138 (UK, Ifoods), 14.13% 139 (Germany, PIF), 15.00% 142 (Turkey, EPIF), 19.80% 143 (Turkey, IFF), 39.71% 141 (Austria, EPIF), 60.53% 144 (France, EPIF), and 100.00% 145 (Turkey, raw PIF); 4.46% 146 (USA, EPIF) and 62.00% 147 (Mexico, PIF) in North America, and 0.00% 148 (Brazil, EPIF), 4.69% 149 (Chile), 17.91% 148 (Brazil, PIF), 23.33% 150 (Brazil, CPIF), 34.31% 151 (Colombia, IFF), 44.44% 152 (Brazil, CPIF), and 66.67% 150 (Brazil, IFF) from the South America.…”

PCR Based Molecular Diagnostic Assays for Brucellosis: A Review

“…Apart from these, many researchers either have used Brucella with other bacteria or different spp. of Brucella for the development and validation of multiplex real time PCR assays for simultaneous detection of more than two causative agents in a single reaction (Probert et al, 2004;Kumar et al, 2011;Selim et al, 2014 andTutar et al, 2018). Queipo-Ortuno et al, (2009) developed a fluorescent hybridization probe based multiplex real time PCR assay for rapid detection and differential diagnosis of extra pulmonary tuberculosis from brucellosis by targeting bcsp31gene for Brucella genus and senX3-regX3 gene for Mycobacterium tuberculosis for designing of primers and probe.…”

Contamination, persistence and dissemination of Cronobacter during the production of powdered infant formula in China in 2016

“…dublinensis, C. turicensis, C. malonaticus, C. muytjensii, C. condiment, and C. universalis. It is well known that Cronobacter had been classified as pathogenic organisms to a restricted population, especially causing life-threatening infections in neonates and immunocompromised infants (Forsythe, 2018). These organisms are widely distributed in foods including powdered infant formulas (PIF), infant cereal foods, dairy products, fruits, and elements in the environment like soil, water, and dust (Tutar et al, 2018;Ueda, 2017). Cronobacter infections in infants occur mostly through the consumption of contaminated PIF.…”