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A Loop-Mediated Isothermal Amplification-Based Method for Analysing Animal Feed for the Presence of Salmonella

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Abstract

A loop-mediated isothermal amplification (LAMP)-based method has been developed for the analysis of animal feed for the presence of Salmonella spp. The method is compatible with the standard culture-based method ISO 6579:2002. Samples comprising 100 g feed are pre-enriched in 900 ml buffered peptone water, and then 2× 100-μl aliquots are taken, one for secondary enrichment in a LAMP-friendly broth and the other for secondary enrichment according to ISO 6579:2002. A 3-μl aliquot of the LAMP-friendly culture is added directly into a LAMP reaction. LAMP-positive signals are considered presumptive of the presence of Salmonella spp.; confirmation is obtained by continuation of ISO 6579:2002. Negative LAMP reactions can be considered as signifying that the sample does not contain viable Salmonella. The LAMP assay incorporates an internal amplification control (IAC); the target and IAC signals are distinguished by the difference in annealing temperatures of the amplicons. The method provides simple and reliable screening of feedstuffs for Salmonella spp., with uncontaminated samples being identified in 24 h less than using the current ISO standard method alone. The method was used to analyse 79 samples of animal feed and identified 13 Salmonella-positive samples.

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Notes

  1. This IAC approach will not be applicable if using turbidity as an end-point detection system (Chen et al. 2011; Soli et al. 2013). However, users of turbidity-based detection should recognise its limitations and the potential ambiguity of its results; in that, false-negative result interpretation cannot be totally avoided.

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Acknowledgments

The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007‐2013) under grant agreement no. 265702. MD-V was supported by a grant from the Spanish Ministry. MD and NC were supported by a grant Q-SAFFE. We thank Q-SAFFE project partners, particularly John Thompson and Sons Ltd., for the supply of feed samples. We thank Almudena Perez and Zohartze Monteagudo for their technical assistance.

Conflict of Interest

Marta Diez-Valcarce has no conflict of interest. Nigel Cook has no conflict of interest. Martin D’Agostino has no conflict of interest. Susana Robles has no conflict of interest. Begoña Losilla-Garcia has no conflict of interest. This article does not contain any studies with human or animal subjects.

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Authors and Affiliations

  1. Food and Environment Research Agency (FERA), Sand Hutton, York, UK

    Martin D’Agostino, Susana Robles, Begoña Losilla-Garcia & Nigel Cook

  2. Instituto Tecnológico Agrario de Castilla y León (ITACyL), Junta de Castilla y León, Valladolid, Spain

    Marta Diez-Valcarce

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  1. Martin D’Agostino
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  2. Marta Diez-Valcarce
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  3. Susana Robles
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  4. Begoña Losilla-Garcia
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  5. Nigel Cook
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Correspondence to Martin D’Agostino.

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D’Agostino, M., Diez-Valcarce, M., Robles, S. et al. A Loop-Mediated Isothermal Amplification-Based Method for Analysing Animal Feed for the Presence of Salmonella . Food Anal. Methods 8, 2409–2416 (2015). https://doi.org/10.1007/s12161-015-0148-0

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  • DOI: https://doi.org/10.1007/s12161-015-0148-0

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