Real-Time TaqMan PCR for Rapid Detection and Quantification of Coliforms in Chilled Meat

Hu, Shuangfang; Yu, Yigang; Li, Rong; Xia, Xingzhou; Xiao, Xi; Li, Xiaofeng

doi:10.1007/s12161-015-0271-y

Cited by 8 publications

(4 citation statements)

References 34 publications

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“…Frequently used culture-based enumeration methods include the (i) classical plate count on violet-red-bile-lactose agar medium [73] or chromogenic media based on the activities of β-D-galactosidase (of coliforms) and β-glucuronidase (of E. coli) [74], (ii) most probable number procedure in lauryl tryptose broth [75] and IDEXX systems, (iii) membrane filtration in selective M-Endo medium [76] and (iv) lateral-flow immune-chromatographic assay using antibodies recognizing coliform genera [77]. Culture-independent methods include (i) real-time PCR targeting the lacZ gene of coliforms [78,79], (ii) fluorescence-based DNA microarray probing E. coli [80], (iii) flow cytometry [81] and (iv) next-generation sequencing or metagenomics that profile all microorganisms, including total/fecal coliforms [82].…”

Section: Escherichia Colimentioning

confidence: 99%

Biological Indicators for Fecal Pollution Detection and Source Tracking: A Review

Saleem

Edge

et al. 2021

Processes

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Fecal pollution, commonly detected in untreated or less treated sewage, is associated with health risks (e.g., waterborne diseases and antibiotic resistance dissemination), ecological issues (e.g., release of harmful gases in fecal sludge composting, proliferative bacterial/algal growth due to high nutrient loads) and economy losses (e.g., reduced aqua farm harvesting). Therefore, the discharge of untreated domestic sewage to the environment and its agricultural reuse are growing concerns. The goals of fecal pollution detection include fecal waste source tracking and identifying the presence of pathogens, therefore assessing potential health risks. This review summarizes available biological fecal indicators focusing on host specificity, degree of association with fecal pollution, environmental persistence, and quantification methods in fecal pollution assessment. The development of practical tools is a crucial requirement for the implementation of mitigation strategies that may help confine the types of host-specific pathogens and determine the source control point, such as sourcing fecal wastes from point sources and nonpoint sources. Emerging multidisciplinary bacterial enumeration platforms are also discussed, including individual working mechanisms, applications, advantages, and limitations.

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Section: Escherichia Colimentioning

confidence: 99%

Biological Indicators for Fecal Pollution Detection and Source Tracking: A Review

Saleem

Edge

et al. 2021

Processes

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“…Numbers of total coliform bacteria were determined by quantitative PCR (qPCR) targeting the lacZ gene (encodes β-galactosidase) in the genera Escherichia, Klebsiella, Citrobacter and Enterobacter (Table 2). The qPCR was carried out according to Hu et al (2016) with slight modification. Briefly, 20 μL PCR mixture containing 2 μL template (DNA extracted from the samples), 10 μL Premix Taq (Ex Taq Version 2.0) (Takara Bio Group, Japan), 1 μL 10 µM primer, 0.5 μL 10 µM probe and 4.5 μL water.…”

Section: Quantification Of Coliforms and E Coli By Quantitative Pcr (Qpcr)mentioning

confidence: 99%

“…Fluorescence (HEX) was measured at 60 °C. For calculation of cell numbers for coliform, one copy of the lacZ gene per cell was assumed (Hu et al, 2016).…”

Section: Quantification Of Coliforms and E Coli By Quantitative Pcr (Qpcr)mentioning

confidence: 99%

Microbial water quality in pangasius and tilapia aquaculture systems in five regions of Bangladesh

Islam¹,

Ahsan²,

Haque³

et al. 2021

MJM

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Aims: To determine abundance of potential pathogenic microorganisms in pangasius and tilapia farms in five major fishproducing areas in Bangladesh by PCR approaches. Methodology and results: Important microbial water quality indicators were studied in water of 38 fish farms producing pangasius (Pangasianodon hypophthalmus) and tilapia (Oreochromis niloticus) in five major fish-producing areas of Bangladesh. The parameters included physicochemical data and PCR detection of total coliforms and E. coli, species of potentially pathogenic Vibrio, and cyanobacterial genes encoding the toxins microcystin and saxitoxin. Quantitative PCR showed that coliform bacteria occurred in all fish farms with densities from one to 2.2 × 10 5 per mL, while E. coli ranged from none to 5.0 × 10 4 per mL. Numbers of total coliforms and E. coli were higher in pangasius farms than in tilapia farms, and when high abundances occurred, coliform bacteria and E. coli bacteria co-varied. Detection of Vibrio-specific genes indicated presence of Vibrio species in 76% of the farms and included V. vulnificus and V. cholerae. The human pathogen type of V. cholerae (carrying the ctxA gene) and the fish pathogen V. parahaemolyticus were not detected. The microcystin-encoding mcyE gene ranged from undetectable to 2.6 × 10 5 copies per mL and tended to be highest in pangasius farms. The saxitoxin-encoding gene sxtA was not found in any of the farms. Conclusion, significance and impact of study: Based on the high abundance of especially coliform bacteria and E. coli, we recommend more efficient water quality monitoring systems to improve detection and control of fecal coliforms and to reduce presence of microcystin-producing cyanobacteria in aquaculture farms in Bangladesh.

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“…Then, a quantitative PCR method for amplification of lacZ (encoding β‐galactosidase) and monitoring of the amplified product has been developed for quantification of coliform number. Since this method does not require a culturing step, quantification can be performed within a short time frame of 2–4 hr (Hu et al, 2016; Martín et al, 2010; Molina et al, 2015), leading to significant reduction of analysis time. However, dedicated equipment and expertise are essential, and therefore, on‐site tests are difficult (Shan et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

Rapid quantification of coliforms in ready‐to‐eat foods using lateral‐flow immunochromatographic assay

Tominaga

2020

Journal of Food Safety

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The degree of coliform contamination in pastries was estimated based on culturing times until positive results were obtained with the lateral-flow immunochromatographic assay (LFIA). Coliform genera Citrobacter, Cronobacter, Enterobacter, Klebsiella, Kluyvera, Pantoea, Raoultella, and Serratia were detected in spoiled pastries, as established with next-generation sequencing. A lateral-flow test strip was constructed using antibodies recognizing the above genera. The culture times required for positive detection with LFIA were 0, 3, 6, and 9 hr at initial inoculation concentrations of 3.8, 2.8, 1.8, and 0.8 log 10 (cfu/ml), respectively. In pastries contaminated with >5.0, 3.0-5.0, 2.0-3.0 log 10 (cfu/g) coliform bacteria, samples became LFIA-positive from 3, 6, and 9 hr culture, respectively. LFIA showed negative for pastries with <2.0 log 10 (cfu/g) coliform contamination. The quantitative category of initial coliform content before culture was predictable with 87% accuracy. This novel method can be applied to monitor food safety of other ready-to-eat consumables.

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Real-Time TaqMan PCR for Rapid Detection and Quantification of Coliforms in Chilled Meat

Cited by 8 publications

References 34 publications

Biological Indicators for Fecal Pollution Detection and Source Tracking: A Review

Biological Indicators for Fecal Pollution Detection and Source Tracking: A Review

Microbial water quality in pangasius and tilapia aquaculture systems in five regions of Bangladesh

Rapid quantification of coliforms in ready‐to‐eat foods using lateral‐flow immunochromatographic assay

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