Multiplex PCR coupled with propidium monoazide for the detection of viable Cronobacter sakazakii, Bacillus cereus, and Salmonella spp. in milk and milk products

Yu, Shuang; Yan, Leina; Wu, Xin; Li, Fan; Wang, Dong; Xu, Hengyi

doi:10.3168/jds.2017-13110

Cited by 51 publications

(20 citation statements)

References 27 publications

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“…Consequently, the PCR signal is strongly reduced even suppressed in membrane-compromised cells compared to intact cells. This methodology has been widely applied to detect viable bacteria (Yu et al 2017), viruses (Fongaro et al 2016), fungi (Vesper et al 2008) in food and environmental samples, and Cryptosporidium spp. oocysts and G. duodenalis cysts in water and wastewater effluents (Brescia et al 2009;Liang and Keeley 2012;Agulló-Barceló et al 2014;Alonso et al 2014;Ma et al 2016;Vande Burgt et al 2018).…”

Section: Introductionmentioning

confidence: 99%

Evaluation of propidium monoazide–based qPCR to detect viable oocysts of Toxoplasma gondii

et al. 2019

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Information on the viability of Toxoplasma gondii oocysts is crucial to establish the public health significance of this environmental transmission stage that can contaminate water and foods. Interest for molecular-based methods to assess viability is growing and the aim of our study was to assess, for the first time, a propidium monoazide (PMA)-qPCR approach to determine the viability of T. gondii oocysts. Untreated and heat-killed (99°C, 5 min) oocysts were incubated with PMA, a photoreactive DNA binding dye, and analyzed by confocal microscopy and flow cytometry to characterize oocysts' dye permeability. Different PMA concentrations (50 to 150 μM), incubation temperatures (22, 37, and 45°C), amplicon length, selected targeted gene, and dyes (PMA, PMAxx™) were evaluated to define optimal conditions to discriminate specifically viable oocysts by PMA-qPCR. In theory, PMA binding to DNA would inhibit PCR amplification in dead but not in viable oocysts. Incubation at 22°C with 100 μM PMA coupled to qPCR targeting a 123-bp sequence of the 529-bp repeat element allowed the distinction between viable and heated oocysts. However, the reduction of viability following heating of oocysts at high temperature was slight and, contrarily to reverse transcriptase-qPCR, the qPCR signal was not totally suppressed in heated suspensions. Therefore, PMA-qPCR is able to assess the impact of heating on T. gondii oocysts' viability but underestimates the efficacy of this treatment. The relevance of this technique to evaluate the efficacy of other inactivation processes and assess exposure of humans to this pathogen requires further investigations.

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Section: Introductionmentioning

confidence: 99%

Evaluation of propidium monoazide–based qPCR to detect viable oocysts of Toxoplasma gondii

et al. 2019

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“…) and milk and milk products (Yu et al . ). In the present study, this pathogen was identified in raw milk and cheese samples and our results may present a different perspective for C. sakazakii .…”

Section: Discussionmentioning

confidence: 97%

“…in vegetables, fruit and environmental samples. In addition, the presence of C. sakazakii was investigated in milk-based ingredients such as milk powder, whey powder and cheese (Heperkan et al 2017) and milk and milk products (Yu et al 2017). In the present study, this pathogen was identified in raw milk and cheese samples and our results may present a different perspective for C. sakazakii.…”

Section: Discussionmentioning

confidence: 99%

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

Tutar

Akıncı

Akyol

2018

Int J of Dairy Tech

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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.

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“…PMA-multiplex qPCR [55] Milk E. coli O157: H7; Salmonella spp. PMA-multiplex qPCR [56] Probiotic yogurt Bifidobacterium EMA-qPCR [57] Lactobacillus paracasei PMA-qPCR [58] Seafood Fish fillets 16S rDNA EMA-qPCR [59,60] PMA-qPCR [61] Raw seafood (oyster, scallop, shrimp, and crab)…”

Section: Gouda Cheesementioning

confidence: 99%

Real-Time Quantitative PCR as a Tool for Monitoring Microbiological Quality of Food

Lopes¹,

Maciel²

2020

Synthetic Biology - New Interdisciplinary Science

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Microbiological parameters of food provide quality information regarding the processing, storage, and distribution conditions, shelf life, as well as whether the food poses a health risk to the population. In this context, the concern with food safety is a competitive advantage, as the pressure of consumers, who are increasingly interested and concerned about what they are consuming, directs the trade to reach the quality of products and services offered. With regard to microbiological analyses, researchers have been developing sensitive techniques to produce rapid results, since traditional methods of microbiological culture are time-consuming and very laborious. Thus, the real-time quantitative PCR technique (qPCR) offers the possibility of quantifying the total bacterial DNA in a food sample without the need of the microbial growth step. That is, the result can be expressed on the same day, and it is possible to perform a simultaneous quantification of more than one pathogen in a single assay. Therefore, it can be a useful tool for monitoring microbiological quality in food industries. In this chapter, we will present the advantages and disadvantages of this methodology for food microbiology emphasizing the challenge of differentiating viable cells from nonviable cells.

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Multiplex PCR coupled with propidium monoazide for the detection of viable Cronobacter sakazakii, Bacillus cereus, and Salmonella spp. in milk and milk products

Cited by 51 publications

References 27 publications

Evaluation of propidium monoazide–based qPCR to detect viable oocysts of Toxoplasma gondii

Evaluation of propidium monoazide–based qPCR to detect viable oocysts of Toxoplasma gondii

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

Real-Time Quantitative PCR as a Tool for Monitoring Microbiological Quality of Food

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