Taqman® probe based multiplex RT-PCR for simultaneous detection of Listeria monocytogenes, Salmonella spp. and Shiga toxin-producing Escherichia coli in foods

Bundidamorn, Damkerng; Supawasit, Wannakarn; Trevanich, Sudsai

doi:10.1016/j.lwt.2021.111696

Cited by 13 publications

(5 citation statements)

References 33 publications

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“…It has been shown that chromosomally located genes are more stable in comparison to plasmid ones, which are subject to loss from the bacterial cells [30]. The stability of selected target genes of each food-borne pathogen utilized in the study (ssPE, fabZ, rfbE, hylA, invA, ompA, scaD, sat) has previously been reported [31][32][33][34][35]. Other studies that utilized hydrolyses probes targeted different genes for C. jejuni (mapA), L. monocytogenes (prfA), Salmonella spp.…”

Section: Discussionmentioning

confidence: 93%

A novel approach for simultaneous detection of the most common food-borne pathogens by multiplex qPCR

Hodzic¹,

Glavinic²,

Wademan³

2023

Biomol Biomed

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Food contaminated with bacterial pathogens is a great threat to human health and food spoilage, having an impact on public health and the food industry. Research in food safety seeks to develop a practical, rapid, and sensitive detection technique for food-borne pathogens. In the past few decades real-time quantitative polymerase chain reaction (qPCR) has been developed, and multiplex qPCR is a preferred feature. Multiplex qPCR enables simultaneous amplification of many targets of interest in one reaction by using more than one pair of primers. In this study we have developed and evaluated a hydrolysis (TaqMan) probe-based system for simultaneous detection of eight of the most common food-borne pathogens in a single step procedure by multiplex qPCR. A multicolor combinational probe coding (MCPC) strategy was utilized that allows multiple fluorophores to label different probes in combinatorial manner. This strategy enabled simultaneous detection, identification, and quantification of targeted genes. The efficiency of the individual qPCR reactions for each target gene had values comparable to those established for multiplex qPCR, with the detection limits of approximately < 10 copies of DNA per reaction. Pathogen load help predict bacteriological quality status in food products and serve to validate the efficiency of procedures to minimize or eliminate their presence, so newly developed multiplex qPCR was quantitative for each pathogen. During sample preparation, a step to concentrate the target organism from a relatively large sample size, remove all potential PCR inhibitors and yield samples in a volume suitable for qPCR was incorporated.

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Section: Discussionmentioning

confidence: 93%

A novel approach for simultaneous detection of the most common food-borne pathogens by multiplex qPCR

Hodzic¹,

Glavinic²,

Wademan³

2023

Biomol Biomed

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“…in chicken samples at a concentration of 3.2 × 10 5 cfu/mL, even after enriching the samples for 24–48 h. Nevertheless, this method could detect the presence of L. monocytogenes, Salmonella spp. , and E. coli in pork, beef, chicken, and mung bean sprout at a concentration of 1 cfu/25 g after enriching the samples for 18 h ( Bundidamorn et al, 2021 ). In another study, Garrido-Maestu et al (2020) also reported that they could detect the presence of E. coli O157 in ground beef and leafy greens at concentrations of 3.9 cfu/25 g and 3.3 cfu/25 g, respectively, after enriching their samples for 3 h.…”

Section: Recent Advances In Naa-based Methodsmentioning

confidence: 99%

“…Additionally, the use of fluorescent dye-based qPCR yields relatively low specificity, formation of primer-dimer, which could lead to false positives, and limits the multiplex reaction. Employing specific probes, such as carboxyfluorescein (FAM) and Cy5, may improve the sensitivity and specificity, but requires a complex design and high cost ( Bundidamorn et al, 2021 ). For digital PCR, this technology necessitate an adequate number of analytical droplets or chambers to facilitate Poisson distribution, as well as the requirement for appropriate sample dilution to ensure the generation of reliable data ( Lei et al, 2021 ).…”

Section: Remaining Challenges and Possible Opportunitiesmentioning

confidence: 99%

Rapid detection methods for foodborne pathogens based on nucleic acid amplification: Recent advances, remaining challenges, and possible opportunities

Ndraha,

Lin,

Wang

et al. 2023

Food Chemistry: Molecular Sciences

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“…Real-Time PCR reactions were performed using the SLAN-96P real-time PCR detection system (Hongshi, China). The reaction mixture consisted of 10×Taq HS buffer (Mg 2+ plus, 2.5 µL), UNG(1U/µL,0.1µL), dN(U)TP (25mM, 0.2µL), TaqHS DNA polymerase (5U/µL, 0.5µL), invA-F (10µM, 0.5µL), invA-R (10µM, 0.5µL), invA-P (10µM, 0.4µL), along with 2 µL of DNA template, with sterilized double distilled water added to create the nal reaction volume of 25 µL (Bundidamorn, & Trevanich, 2021). Thermocycling conditions included 50°C for 2 min, 95°C for 3 min, with 40 cycles of 95°C for 15 s, followed by 60°C for 45 s and a uorescence read at 60°C at the end of each cycle.…”

Section: Designing Of Primers and Molecular Beacon Probesmentioning

confidence: 99%

A novel fluorescence cross-priming amplification based on universal molecular beacon for rapid and specific detection of Salmonella enterica in food samples

Hou,

Du,

Liu

et al. 2024

Preprint

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A methodology with rapidity and specificity is of great significance for effectively control and management of disease epidemics caused by Salmonella enterica as it has presented an obvious threat to food safety and public health worldwide. One major drawback to the traditional cross-priming amplification (CPA) detection method is the possibility of detecting false-positive signals derived from opening tube lids or non-specific amplification, and molecular beacon was firstly employed to solve aforementioned problems. The reaction system was optimized and the results showed that the MB-CPA method was highly specific for detection of S. enterica. The LOD of established assay was found to be 10 CFU/mL, 40 CFU/mL, 4 CFU/mL in pure culture, chicken sample without and with 6 h enrichment, respectively. And the LOD of MB-CPA was 10 times higher than that of real-time PCR. An application of MB-CPA assay was conducted with 78 naturally contaminated food samples to test its practicality. After an enrichment step at 37℃ for 6h, the results showed 100% sensitivity and 100% specificity compared with standard culture-based method. Considering its rapidity, user-friendliness, cost-effectiveness, this MB-CPA assay will aid in the broader application in food industry for the detection of S. enterica in small or resource-limited food testing laboratories.

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Taqman® probe based multiplex RT-PCR for simultaneous detection of Listeria monocytogenes, Salmonella spp. and Shiga toxin-producing Escherichia coli in foods

Cited by 13 publications

References 33 publications

A novel approach for simultaneous detection of the most common food-borne pathogens by multiplex qPCR

A novel approach for simultaneous detection of the most common food-borne pathogens by multiplex qPCR

Rapid detection methods for foodborne pathogens based on nucleic acid amplification: Recent advances, remaining challenges, and possible opportunities

A novel fluorescence cross-priming amplification based on universal molecular beacon for rapid and specific detection of Salmonella enterica in food samples

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