Enumeration of Vibrio parahaemolyticus in VBNC state by PMA-combined real-time quantitative PCR coupled with confirmation of respiratory activity

Liu, Yufei; Qin, Zhong; Wang, Juan; Lei, Shuwen

doi:10.1016/j.foodcont.2018.03.037

Cited by 44 publications

(34 citation statements)

References 22 publications

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“…Therefore, scientists had utilized EMA/PMA combined with nucleic acid amplification techniques to complete the detection of VBNC cells. Recently, nucleic acid amplification methods combined with EMA/PMA have been widely used for the detection of pathogenic bacteria in the VBNC state, such as PMA-polymerase chain reaction (PCR) (Liu Y. et al, 2018;Zhong and Zhao, 2018).…”

Section: Introductionmentioning

confidence: 99%

Development and Application of a Simple “Easy To Operate” Propidium Monoazide-Crossing Priming Amplification on Detection of Viable and Viable But Non-culturable Cells of O157 Escherichia coli

Zhou

Wang

et al. 2020

Front. Microbiol.

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O157 Escherichia coli is one of the most important foodborne pathogens causing disease even at low cellular numbers. Thus, the early and accurate detection of this pathogen is important. However, due to the formation of viable but non-culturable (VBNC) status, the golden standard culturing methodology fails to identify O157 E. coli once it enters VBNC status. Crossing priming amplification (CPA) is a novel, simple, easy-to-operate detection technology that amplifies DNA with high speed, efficiency, and specificity under isothermal conditions. The objective of this study was to firstly develop and apply a CPA assay with propidium monoazide (PMA) for the rapid detection of the foodborne E. coli O157:H7 in VBNC state. Five primers (2a/1s, 2a, 3a, 4s, and 5a) were specially designed for recognizing three targets, which were rfbE, stx1, and stx2, and evaluated for its effectiveness in detecting VBNC cell of E. coli O157:H7 with detection limits of pure VBNC culture at 10 3 , 10 5 , and 10 5 colony-forming units (CFUs)/ml for rfbE, stx1, and stx2, respectively, whereas those of food samples (frozen pastry and steamed bread) were 10 3 , 10 5 , and 10 5 CFUs/ml. The application of the PMA-CPA assay was successfully used on detecting E. coli O157:H7 in VBNC state from food samples. In conclusion, this is the first development of PMA-CPA assay on the detection of VBNC cell, which was found to be useful and a powerful tool for the rapid detection of E. coli O157:H7 in VBNC state. Undoubtedly, the PMA-CPA method can be of high value to the food industry owing to its various advantages such as speed, specificity, sensitivity, and cost-effectiveness.

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Section: Introductionmentioning

confidence: 99%

Development and Application of a Simple “Easy To Operate” Propidium Monoazide-Crossing Priming Amplification on Detection of Viable and Viable But Non-culturable Cells of O157 Escherichia coli

Zhou

Wang

et al. 2020

Front. Microbiol.

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“…Vibrio parahaemolyticus is an enteric pathogen found in a variety of seafood, especially clams, shrimps, and oysters (Liu et al, 2018;Cao et al, 2019). Although the presence of V. parahaemolyticus is extensive in marine and estuarine environments, not all strains of V. parahaemolyticus result in illness (Colwell et al, 1977).…”

Section: Discussionmentioning

confidence: 99%

A 4-plex Droplet Digital PCR Method for Simultaneous Quantification and Differentiation of Pathogenic and Non-pathogenic Vibrio parahaemolyticus Based on Single Intact Cells

Lei

Wang

et al. 2020

Front. Microbiol.

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Vibrio parahaemolyticus is a significant seafood-borne pathogen, leading to serious acute gastrointestinal diseases worldwide. In this study, a reliable 4-plex droplet digital PCR (ddPCR) was successfully established and evaluated for the simultaneous detection of V. parahaemolyticus based on tlh, tdh, ureR, and orf8 in food samples using single intact cells. The targets tlh and ureR were labeled with 6-Carboxyfluorescein (FAM), and the targets tdh and orf8 were labeled with 5'-Hexachlorofluorescein (HEX). Due to reasonable proration of primers and probes corresponding into the two fluorescence channels of the ddPCR detecting platforms, the clearly separated 16 (2 4) clusters based on fluorescence amplitude were obtained. For better results, the sample hot lysis time and the cycle number were optimized. The results showed that the minimum number of "rain" and maximum fluorescence amplification were presented for precise detection in the condition of 25 min of the sample hot lysis time and 55 cycles. The sensitivity of this 4-plex ddPCR assay was 39 CFU/mL, which was in accordance with that of the conventional plate counting and was 10-fold sensitive than that of qPCR. In conclusion, the 4-plex ddPCR assay presented in this paper was a rapid, specific, sensitive, and accurate tool for the detection of V. parahaemolyticus including pandemic group strains and could be applied in the differentiation of V. parahaemolyticus in a wide variety of samples.

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“…In fact, an integrated stress response through a regulatory web that allowed bacteria to react to environmental changes has been observed (Mathias, Rosenthal, Gaspar, Aragao, & Slongo‐Marcusi, 2013). Such stress responses include the adoption of a viable but nonculturable state (VBNC), which prevents the identification of bacteria using classical microbiology methods (Liu et al., 2018; Liu, Zhong, Wang, & Lei, 2018).…”

Section: Environmental Stressesmentioning

confidence: 99%

Updates on understanding of probiotic lactic acid bacteria responses to environmental stresses and highlights on proteomic analyses

Mbye

Baig

AbuQamar

et al. 2020

Comp Rev Food Sci Food Safe

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Probiotics are defined as live microorganisms that improve the health of the host when administered in adequate quantities. Nonetheless, probiotics encounter extreme environmental conditions during food processing or along the gastrointestinal tract. This review discusses different environmental stresses that affect probiotics during food preparation, storage, and along the alimentary canal, including high temperature, low temperature, low and alkaline pH, oxidative stress, high hydrostatic pressure, osmotic pressure, and starvation. The understanding of how probiotics deal with environmental stress and thrive provides useful information to guide the selection of the strains with enhanced performance in specific situations, in food processing or during gastrointestinal transit. In most cases, multiple biological functions are affected upon exposure of the cell to environmental stress. Sensing of sublethal environmental stress can allow for adaptation processes to occur, which can include alterations in the expression of specific proteins.

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Enumeration of Vibrio parahaemolyticus in VBNC state by PMA-combined real-time quantitative PCR coupled with confirmation of respiratory activity

Cited by 44 publications

References 22 publications

Development and Application of a Simple “Easy To Operate” Propidium Monoazide-Crossing Priming Amplification on Detection of Viable and Viable But Non-culturable Cells of O157 Escherichia coli

Development and Application of a Simple “Easy To Operate” Propidium Monoazide-Crossing Priming Amplification on Detection of Viable and Viable But Non-culturable Cells of O157 Escherichia coli

A 4-plex Droplet Digital PCR Method for Simultaneous Quantification and Differentiation of Pathogenic and Non-pathogenic Vibrio parahaemolyticus Based on Single Intact Cells

Updates on understanding of probiotic lactic acid bacteria responses to environmental stresses and highlights on proteomic analyses

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