Application of loop-mediated isothermal amplification to detect Salmonella spp. in egg samples
Main Article Content
Abstract
The objective of the study was to evaluate the use of loop-mediated isothermal amplification (LAMP) technique for detection of Salmonella spp. in chicken eggs. Eight Salmonella strains and 26 non-Salmonella enteric strains were used to verify the specificity of the LAMP assay for detection of Salmonella. The Sal4 primer set was used to specifically amplify the target sequence on the invA gene of Salmonella and the hydroxyl naphthol blue was used as the indicator to read the reaction results. Blank egg samples were spiked with serial 10-fold diluted Salmonella suspensions to determine the limit of detection at 50% (LOD50) of the assay. Egg samples collected from multiple locations in Ho Chi Minh City were tested for the presence of Salmonella using both LAMP method and the traditional culture method to compare the effectiveness of these two methods. Among 34 bacterial strains tested by LAMP assay, false-positive or false-negative results were not observed. The assays of spiked egg samples showed that the LOD50 of the LAMP method was less than 2 CFU/25 g sample. These results indicated a high specificity and sensitivity of the LAMP assay in detection of Salmonella in egg samples, even with those of low levels of contamination. Upon testing collected egg samples (n = 42), the LAMP assay produced the same results of Salmonella detection as the culture method. Salmonella was detected in 4 out of 42 samples. This study showed that the LAMP method is highly effective and would be suitable for use in detection of Salmonella in egg samples. The assay has several advantages such as saving time and labor compared to traditional culture methods.
Article Details
References
D’Agostino, M., Robles, S., Hansen, F., Ntafis, V., Ikonomopoulos, J., Kokkinos, P., Alvarez-Ordonez, A., Jordan, K., Delibato, E., Kukier, E., Sieradzki, Z., Kwiatek, K., Milanov, D., Petrović, T., Gonzalez-Garcia, P., Lazaro, D. R., Jackson, E. E., Forsythe, S. J., O’Brien, L., & Cook, N. (2016). Validation of a loop-mediated amplification/ISO 6579-Based method for analysing soya meal for the presence of Salmonella enterica. Food Analytical Methods 9, 2979-2985. https://doi.org/10.1007/s12161-016-0602-7.
Domesle, K. J., Young, S. R., Yang, Q., & Ge, B. (2020). Loop-mediated isothermal amplification for screening Salmonella in animal food and confirming Salmonella from culture isolation. Journal of Visualized Experiments 159. https://doi.org/10.3791/61239.
EFSA & ECDC (European Food Safety Authority & European Centre for Disease Prevention and Control). (2021). The European union one health 2020 zoonoses report. EFS A Journal 19(12), 6971. https://doi.org/10.2903/j.efsa.2021.6971.
FDA (Food and Drug Administration). (2015). Guidelines for the validation of analytical methods for the detection of microbial pathogens in foods and feeds (2nd ed.). Washington DC, USA: FDA Foods Program Regulatory Science Steering Committee.
Ge, B., Domesle, K. J., Yang, Q., Hammack, T. S., Wang, S. S., Deng, X., Hu, L., Zhang, G., Hu, Y., Lai, X., Chou, K. X., Dollete, J. R., Hirneisen, K. A., La, S. P., Richter, R. S., Rai, D. R., Yousefvand, A. A., Park, P. K., Wu, C. H., Eames, T., Kiang, D., Sheng, J., Wu, D., Hahn, L., Ledger, L., Logie, C., You, Q., Slavic, D., Cai, H., Ayers, S. L., Young, S. R., & Pamboukian, R. (2019). Multi-laboratory validation of a loop-mediated isothermal amplification method for screening Salmonella in animal food. Frontiers in Microbiology 10, 562. https://doi.org/10.3389/fmicb.2019.00562.
Ha, T. T., Ngan, T. P., Huong, T. T. C., & Ha, T. T. C. (2017). Antibiotic resistance of E. coli and Salmonella isolated from poultry eggs sold at some markets in Ha Noi city. Vietnam Journal of Agricultural Sciences 15(6), 770-775.
Hu, L., Ma, L. M., Zheng, S., He, X., Hammack, T. S., Brown, E. W., & Zhang, G. (2018). Development of a novel loop-mediated isothermal amplification (LAMP) assay for the detection of Salmonella ser. enteritidis from egg products. Food Control 88, 190-197. https://doi.org/10.1016/j.foodcont.2018.01.006.
ISO (International Organization for Standardization). (2017). Microbiology of the food chain - Horizontal method for the detection, enumeration and serotyping of Salmonella - Part 1: Detection of Salmonella spp. (ISO 6579-1:2017). Retrieved from February 20, 2022, from https://www.iso.org/standard/56712.html.
ISO (International Organization for Standardization). (2016). Microbiology of the food chain - Method validation - Part 2: Protocol for the validation of alternative (proprietary) methods against a reference method (ISO 16140-2:2016). Retrieved from February 20, 2022, from https://www.iso.org/standard/54870.html.
Le, P. H., Vo, C. M., Nguyen, H. M., Nguyen, T. T., Nguyen, C. T. K., & Bui, H. T. D. (2019). Survey on Escherichia coli, Salmonella contamination and residues of some antibiotics in pork and chicken meat in some provinces of Southwest region. Journal of Veterinary Science and Technology XXVI(7), 47-54.
Lin, L., Zheng, Q., Lin, J., Yuk, H. G., & Guo, L. (2020). Immuno- and nucleic acid-based current technique for Salmonella detection in food. European Food Research and Technology 246(8), 373-395. https://doi.org/10.1007/s00217-019-03423-9.
Luu, T. Q., Bui, H. M., & Nguyen, H. V. (2013). Evaluation of the risk of Salmonella contamination of pork in Hanoi. Vietnam Journal of Preventative Medicine XXIII (4), 10-17.
Nagamine, K., Hase, T., & Notomi, T. (2002). Accelerated reaction by loop-mediated isothermal amplification using loop primers. Molecular and Cellular Probes 16(3), 223-229. https://doi.org/10.1006/mcpr.2002.0415.
Nguyen, N. T., Nguyen, V. T. B., Nguyen, C. V., Truong, D. T. Q., Tran, N. T., Tran, H. T. T., Nguyen, N. T. H., Bach, K. T., Vo, H. B., Pham, N. T., Campbell, J., Thwaites, G., & Carrique-Mas, J. (2018). Antimicrobial residues and resistance against critically important antimicrobials in non-typhoidal Salmonella from meat sold at wet markets and supermarkets in Vietnam. International Journal of Food Microbiology 266, 301-309. https://doi.org/10.1016/j.ijfoodmicro.2017.12.015.
Notomi, T., Mori, Y., Tomita, N., & Kanda, H. (2015). Loop-mediated isothermal amplification (LAMP): principle, features, and future prospects. Journal of Microbiology 53(1), 1-5. https://doi.org/10.1007/s12275-015-4656-9.
Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., & Hase, T. (2000). Loop-mediated isothermal amplification of DNA. Nucleic Acids Research 28(12), 63e. https://doi.org/10.1093/nar/28.12.e63.
Park, S. H., Aydin, M., Khatiwara, A., Dolan, M. C., Gilmore, D. F., Bouldin, J. L., Ahn, S., & Ricke, S. C. (2014). Current and emerging technologies for rapid detection and characterization of Salmonella in poultry and poultry products. Food Microbiology 38, 250-262. https://doi.org/10.1016/j.fm.2013.10.002.
Stanaway, J. D., Parisi, A., Sarkar, K., Blacker, B. F., Reiner, R. C., Hay, S. I., Nixon, M. R., Dolecek, C., James, S. L., Mokdad, A., Abebe, G., Ahmadian, A., Alahdab, F., Alemnew, B., Alipour V., Bakeshei, F. A., Debalkie, M., Ansari, F., Arabloo, J., Asfaw, E. T., Bagherzadeh, M., Bassat, Q., Belayneh, Y. M. M., Carvalho, F., Daryani, A., Mekonnen, F., Demis, A., Dubey, M., Ejeta, E., Dunachie, S., Eftekhari, A., Fernandes, E., Fard, R. F., Gedefaw, G., Meharie, B. G., Gibney, K. B., Hasanzadeh, A., Hong, L. C., Kasaeian, A., Teklemariam, Z., Molla, A., Malekzadeh, R., Melese, A., Mengistu, D. T., Meštrović, T., Miazgowski, B., Mohammad, K. A., Mohammadian, M., Hafshejani, A. M., Nguyen, C. T., Hoang, L., Nguyen, S. H., Legesse, Y., Olagunju, A. T., Olagunju, T. O., Pourjafar, H., Qorbani, M., Rabiee, M., Rabiee, N., Rafay, A., Rezapour, A., Samy, A., Sepanlou, S., Shaikh, M. A., Sharif, M., Shigematsu, M., Tessema, B., Tran, B., Ullah, I., Yimer, E. M., Zaidi, Z., Murray, C. J. L., & Crump, J. (2019). The global burden of non-typhoidal Salmonella invasive disease: a systematic analysis for the global burden of disease study 2017. The Lancet Infectious Diseases 19(12), 1312-1324. https://doi.org/10.1016/S1473-3099(19)30418-9.
Techathuvanan, C., & D’Souza, D. H. (2012). Reverse-transcriptase loop-mediated isothermal amplification as a rapid screening/monitoring tool for Salmonella enterica detection in liquid whole eggs. Journal of Food Science 77(4), M200-M205. https://doi.org/10.1111/j.1750-3841.2011.02601.x.
Trampel, D. W., Holder, T. G., & Gast, R. K. (2014). Integrated farm management to prevent Salmonella enteritidis contamination of eggs. The Journal of Applied Poultry Research 23(2), 353-365. https://doi.org/10.3382/japr.2014-00944.
Tran, B. N. (2012). The prevalence of Salmonella infection in waterfowl and waterfowl products in Hau Giang province. Can Tho University Journal of Science 23a, 235-242.
Velusamy, V., Arshak, K., Korostynska, O., Oliwa, K., & Adley, C. (2010). An overview of foodborne pathogen detection: In the perspective of biosensors. Biotechnology Advances 28(2), 232-254. https://doi.org/10.1016/j.biotechadv.2009.12.004.
VS (Vietnam Standards). (2017). Microbiology of the food chain - Horizontal method for the detection, enumeration and serotyping of Salmonella - Part 1: Detection of Salmonella spp (TCVN 10780-1:2017). Retrieved from February 20, 2022, from https://luattrongtay.vn/ViewFullText/Id/2bbf2643-3fc2-4724-83d5-823e5f3baeef.
Wang, D. G., Brewster, J. D., Paul, M., & Tomasula, P. M. (2015). Two methods for increased specificity and sensitivity in loop-mediated isothermal amplification. Molecules 20(4), 6048-6059. https://doi.org/10.3390/molecules20046048.
Yang, Q., Chen, S., & Ge, B. (2013). Detecting Salmonella serovars in shell eggs by loop-mediated isothermal amplification. Journal of Food Protection 76(10), 1790-1796.
https://doi.org/10.4315/0362-028X.JFP-13-140.
Yang, Q., Domesle, K. J., & Ge, B. (2018). Loop-mediated isothermal amplification for Salmonella detection in food and feed: Current applications and future directions. Foodborne Pathogens and Disease 15(6), 309-331. https://doi.org/10.1089/fpd.2018.2445.
Yang, Q., Domesle, K. J., Wang, F., & Ge, B. (2016). Rapid detection of Salmonella in food and feed by coupling loop-mediated isothermal amplification with bioluminescent assay in real-time. BMC Microbiology 16, 112. https://doi.org/10.1186/s12866-016-0730-7.
Zhang, G., Brown, E.W., & Gonzalez-Escalona, N. (2011). Comparison of realtime PCR, reverse transcriptase real-time PCR, loop-mediated isothermal amplification, and the FDA conventional microbiological method for the detection of Salmonella spp. in produce. Applied and Environmental Microbiology 77(18), 6495-6501. https://doi.org/10.1128/AEM.00520-11.