Abstract
Listeria contamination in foods of animal origin is one of the most concerning food safety issues. A duplex, SYBR green-based, real-time PCR assay was developed with high-resolution melting analysis-based differentiation of the genus Listeria and Listeria monocytogenes. The primers were designed and tested against other related foodborne pathogens. The assay was optimized for standard parameters in a non-orthogonal fashion and validated following international standards. The LODabs and LOQ of the assay were calculated to be 0.78 and 1.56 ng of the target DNA. The LODrel of the assay was found to be 1% Listeria DNA in background DNA. The assay was evaluated for applicability in artificially spiked samples, providing a 120 CFU/ml detection. The assay was validated with proficiency test samples and also with samples collected for surveillance analysis. This well-established and validated assay can be utilized as a qualitative and quantitative tool for addressing the Listeria contamination in the food safety contexts.
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Data availability
The datasets generated during the research study are available from the corresponding author upon reasonable request.
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Acknowledgements
We are grateful to the Director, ICAR - National Meat Research Institute, Hyderabad, India for providing necessary facilities to carry out this experiment.
Funding
Funding Agency: Department of Biotechnology (DBT), Ministry of Science and Technology, India. Award Number: BT/PR39032/ADV/90/285/2020.
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M.R. Vishnuraj: Conceptualization, Methodology, Investigation, Formal analysis, Visualization, Writing—original draft, Writing—review & editing, Project administration. G Ajay: Methodology, Investigation, Formal analysis, Visualization, writing—original draft, Writing—reviewing & editing. N. Aravind Kumar: Methodology, Investigation, Formal analysis, Visualization, writing—original draft, Writing—reviewing & editing. J Renuka: Methodology, Investigation, Formal analysis. Niveditha Pollumahanti: Methodology, Investigation, Formal analysis. Anusha Chauhan: Methodology, Investigation, Formal analysis. Vaithiyanathan, S: Conceptualization, Formal analysis, Visualization, Resources. Deepak B. Rawool: Conceptualization, Formal analysis, Visualization, Resources S.B. Barbuddhe: Conceptualization, Visualization, Resources, Writing—review & editing.
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Vishnuraj, M.R., Ajay, G., Aravind Kumar, N. et al. Duplex real-time PCR assay with high-resolution melt analysis for the detection and quantification of Listeria species and Listeria monocytogenes in meat products. J Food Sci Technol 60, 1541–1550 (2023). https://doi.org/10.1007/s13197-023-05695-2
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DOI: https://doi.org/10.1007/s13197-023-05695-2