Quantifying viable Vibrio parahaemolyticus and Listeria monocytogenes simultaneously in raw shrimp

Zhang, Zhaohuan; Liu, Haiquan; Lou, Yang; Xiao, Lili; Liao, Chao; Malakar, Pradeep K.; Pan, Yingjie; Yong, Zhao

doi:10.1007/s00253-015-6715-x

Cited by 27 publications

(20 citation statements)

References 51 publications

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“…After heat inactivation at 85°C for 20 min, no genomic DNA was amplified from dead V. parahaemolyticus cells by PMA qPCR (Zhang et al . ), supporting the results. With the exception of V. parahaemolyticus ATCC 33844 in ASW 30 , bacterial cells showed Ct values of 14·88–17·81 until 80 days.…”

Section: Resultssupporting

confidence: 89%

“…The authors determined that heating at ≥70°C for 15 min may disintegrate the membrane structure and permeability of M. avium lethally, and PMA qPCR is effective for the selective signal loss of thermally inactivated cells. After heat inactivation at 85°C for 20 min, no genomic DNA was amplified from dead V. parahaemolyticus cells by PMA qPCR (Zhang et al 2015), supporting the results. With the exception of V. parahaemolyticus ATCC 33844 in ASW 30 , bacterial cells showed Ct values of 14Á88-17Á81 until 80 days.…”

Section: Resultssupporting

confidence: 79%

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Detection of viable but nonculturable Vibrio parahaemolyticus induced by prolonged cold‐starvation using propidium monoazide real‐time polymerase chain reaction

Yoon

Moon

Choi

et al. 2019

Lett Appl Microbiol

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Viable but nonculturable (VBNC) Vibrio parahaemolyticus cannot be detected by the standard cultivation‐based methods. In this study, commonly used viability assessment methods were evaluated for the detection of V. parahaemolyticus in a VBNC state. Vibrio parahaemolyticus cells exposed to nutrient deficiency at cold temperature were used for epifluorescence microscopy with SYTO9 and propidium iodide (PI) staining and real‐time polymerase chain reaction (qPCR) with propidium monoazide (PMA), and its resuscitative ability was determined by a temperature upshift in freshly prepared artificial sea water (ASW; pH 7) fluids. Viable cells with intact membranes always exceeded 5·0 log CFU per ml in ASW microcosms at 4°C. After 80 days, cycle thresholds for V. parahaemolyticus ATCC 27969 were 16·15–16·69. During cold‐starvation, PMA qPCR selectively excluded DNAs from heat‐killed cells. However, there may be some penetration of PMA into undamaged cells that persisted in ASW for 150 days, as evidenced by their ability to resuscitate from a VBNC state after a temperature upshift (25°C); V. parahaemolyticus ATCC 33844 and V. parahaemolyticus ATCC 27969 were successfully reactivated from a VBNC state in ASW microcosms containing <5% NaCl, following enrichment in ASW medium (pH 7). Significance and Impact of the Study Few studies have evaluated the characteristics of and detection methods for viable but nonculturable (VBNC) Vibrio parahaemolyticus induced by cold‐starvation. Currently, VBNC cells are routinely detected by SYTO9 and propidium iodide double staining. However, viable cell counts might be overestimated by this approach, suggesting that the fluorescence dyes may be ineffective for accurately determining the viability of bacterial cells. We demonstrated that quantitative real‐time polymerase chain reaction with propidium monoazide, which selectively permeates damaged cell membranes, can be used to obtain viable cell counts of V. parahaemolyticus after its evolution to a VBNC state under cold‐starvation conditions.

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Section: Resultssupporting

confidence: 89%

Section: Resultssupporting

confidence: 79%

Detection of viable but nonculturable Vibrio parahaemolyticus induced by prolonged cold‐starvation using propidium monoazide real‐time polymerase chain reaction

Yoon

Moon

Choi

et al. 2019

Lett Appl Microbiol

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“…Therefore, EMA/PMA was used to combine qPCR assay to detect viable cells of V. parahaemolyticus. But so far only limited reports have been available reporting detection of viable cells of V. parahaemolyticus using EMA/PMA approach (Zhu et al, 2012; Fakruddin et al, 2013; Zhang et al, 2015a). Zhu et al (2012) used PMA-qPCR to detect viable cells of V. parahaemolyticus from seafood.…”

Section: Ema/pma In Differentiation Of Viable Cells Of Vibrio Parahaementioning

confidence: 99%

Advances and Challenges in Viability Detection of Foodborne Pathogens

et al. 2016

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Foodborne outbreaks are a serious public health and food safety concern worldwide. There is a great demand for rapid, sensitive, specific, and accurate methods to detect microbial pathogens in foods. Conventional methods based on cultivation of pathogens have been the gold standard protocols; however, they take up to a week to complete. Molecular assays such as polymerase chain reaction (PCR), sequencing, microarray technologies have been widely used in detection of foodborne pathogens. Among molecular assays, PCR technology [conventional and real-time PCR (qPCR)] is most commonly used in the foodborne pathogen detection because of its high sensitivity and specificity. However, a major drawback of PCR is its inability to differentiate the DNA from dead and viable cells, and this is a critical factor for the food industry, regulatory agencies and the consumer. To remedy this shortcoming, researchers have used biological dyes such as ethidium monoazide and propidium monoazide (PMA) to pretreat samples before DNA extraction to intercalate the DNA of dead cells in food samples, and then proceed with regular DNA preparation and qPCR. By combining PMA treatment with qPCR (PMA-qPCR), scientists have applied this technology to detect viable cells of various bacterial pathogens in foods. The incorporation of PMA into PCR-based assays for viability detection of pathogens in foods has increased significantly in the last decade. On the other hand, some downsides with this approach have been noted, particularly to achieve complete suppression of signal of DNA from the dead cells present in some particular food matrix. Nowadays, there is a tendency of more and more researchers adapting this approach for viability detection; and a few commercial kits based on PMA are available in the market. As time goes on, more scientists apply this approach to a broader range of pathogen detections, this viability approach (PMA or other chemicals such as platinum compound) may eventually become a common methodology for the rapid, sensitive, and accurate detection of foodborne pathogens. In this review, we summarize the development in the field including progress and challenges and give our perspective in this area.

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“…Vibrio parahaemolyticus PMA-qPCR [62] Raw shrimp Vibrio parahaemolyticus; Listeria monocytogenes PMA-multiplex qPCR [63] Shrimp, pomfret fish, and scallop…”

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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Quantifying viable Vibrio parahaemolyticus and Listeria monocytogenes simultaneously in raw shrimp

Cited by 27 publications

References 51 publications

Detection of viable but nonculturable Vibrio parahaemolyticus induced by prolonged cold‐starvation using propidium monoazide real‐time polymerase chain reaction

Detection of viable but nonculturable Vibrio parahaemolyticus induced by prolonged cold‐starvation using propidium monoazide real‐time polymerase chain reaction

Advances and Challenges in Viability Detection of Foodborne Pathogens

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

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