RAPID DETECTION OF LISTERIA MONOCYTOGENES IN CHICKEN MEATS WITH COLORIMETRIC LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) METHOD

Abstract

The object of this study is to evaluate the performance of the colorimetric loop-mediated isothermal amplification (LAMP) in detecting Listeria monocytogenes from chicken meats (n: 50; whole wing, breast, drumstick; totally N: 150). For this purpose, the chicken meats were artificially contaminated with 100-104 CFU/25 g (or sample) of L. monocytogenes and 5 others bacteria were used as competitive microbiota. After pre-enrichment, the samples were analyzed using conventional cultural, LAMP and real-time PCR methods. Primer sets of virulence gene hlyA were used for the L. monocytogenes-specific visualized LAMP with hydroxynaphthol blue (HNB) dye and amplified the target gene using specific primers at 65 °C for 45 min. As a result of analysis performed by three methods in the naturally contaminated samples, the presence of L. monocytogenes was detected in 9 of 150 samples (6%). The LAMP, qPCR and conventional method showed similar detection performances for L. monocytogenes in the naturally or artifically contaminated samples. These results demonstrated the use of HNB-LAMP method for, as an alternative to PCR, sensitive and specific to L. monocytogenes under isothermal conditions, could use as a simple, rapid detection technique and has the potential for detection of L. monocytogenes from the chicken meats.

Keywords

• Loop-mediated isothermal amplification (LAMP)
• Real-time PCR
• Listeria monocytogenes
• Chicken meats
• Rapid

Project Number

2476 ID ve PRJ2016/267

KOLORİMETRİK LOOP-MEDIATED İZOTERMAL AMPLİFİKASYON METODU İLE LISTERIA MONOCYTOGENES’İN TAVUK ETLERİNDE HIZLI TESPİTİ

Abstract

Bu çalışmanın amacı tavuk etlerinden (n:50; bütün kanat, göğüs, baget; toplamda N: 150) Listeria monocytogenes’in tespitinde kolorimetrik loop-mediated izotermal amplifikasyon’un (LAMP) performansını değerlendirmektir. Bu amaçla, tavuk etleri 100-104 CFU/25 g (veya bütün tavuk eti) seviyede L. monocytogenes ve rekabetçi mikrobiyota olarak kullanılan 5 diğer bakteri (Salmonella Typhimurium, Campylobacter jejuni, Campylobacter coli, Staphylococcus aureus, Citrobacter freundii) ile inoküle edildi. Ön zenginleştirme sonrası örnekler geleneksel kültürel, gerçek zamanlı PZR ve LAMP kullanılarak analiz edildi. Virulans hlyA gen’in primer setleri (L. monocytogenes-özgü) hidroksinaftol mavisi (HNB) ile görselleştirilmiş LAMP için kullanıldı. Bu hedef gen 65°C 45 dakikada spesifik primerler kullanılarak çoğaltıldı. Üç metot ile gerçekleştirilen analizlerin sonucunda doğal olarak kontamine olmuş 150 örneğin 9’unda (%6) L. monocytogenes varlığı tespit edildi. LAMP, qPCR ve klasik metot doğal ve yapay olarak kontamine olmuş örneklerden L. monocytgones için aynı tespit performansını gösterdi. Bu sonuçlar HNB-LAMP yönteminin, PCR'a alternatif olarak, izotermal koşullar altında L. monocytogenes'e duyarlı, spesifik, basit, hızlı bir tespit tekniği olarak kullanılabileceğini ve L. monocytogenes'in tavuk etlerinden saptanma potansiyeline sahip olduğunu gösterdi.

Keywords

• Loop-mediated izotermal amplifikasyon (LAMP)
• Gerçek zamanlı PZR
• Listeria monocytogenes
• Tavuk etleri
• Hızlı

Supporting Institution

Atatürk Üniversitesi

Project Number

2476 ID ve PRJ2016/267

Thanks

Bu çalışmayı 2476 ID ve PRJ2016/267 kodlu proje ile maddi olarak destekleyen Atatürk Üniversitesi Bilimsel Araştırma Projeleri Koordinasyon Birimi’ne teşekkür ederiz.

References

1. Ayaz, N.D., Ayaz, Y., Kaplan, Y. Z., Dogru, A. K., & Aksoy, M. H. (2009). Rapid detection of Listeria monocytogenes, in chicken carcasses by IMS-PCR. Annals of Microbiology, 59(4), 741e744.
2. Brescia, P. (2012). Micro-volume purity assessment of nucleic acids using A260/A280 ratio and spectral scanning. BioTek Instruments, Winooski, VT, Tech. Rep. AN060112–12, Rev, 06-04.
3. da Costa, A. , de Lira Nunes, M. , Mendes-Marques, C. , de Almeida, A. and Leal, N. (2014) Loop-Mediated Isothermal Amplification (LAMP) for the Detection of Listeria monocytogenes and Major Pathogenic Serotypes. American Journal of Analytical Chemistry, 5, 1057-1064. doi: 10.4236/ajac.2014.516112.
4. Feng, J., Dai, Z., Tian, X., & Jiang, X. (2018). Detection of Listeria monocytogenes based on combined aptamers magnetic capture and loop-mediated isothermal amplification. Food Control, 85, 443-452.
5. Garrido-Maestu, A., Azinheiro, S., Carvalho, J., Abalde-Cela, S., Carbó-Argibay, E., Diéguez, L., et al. (2017). Combination of microfluidic loop-mediated isothermal amplification with gold nanoparticles for rapid detection of Salmonella spp. in food samples. Front. Microbiol. 8:2159.
6. Goh, S. G., Kuan, C. H., Loo, Y. Y., Chang, W. S., Lye, Y. L., Soopna, P., ... & Son, R. (2012). Listeria monocytogenes in retailed raw chicken meat in Malaysia. Poultry science, 91(10), 2686-2690.
7. Goto, M., Honda, E., Ogura, A., Nomoto, A., & Hanaki, K. I. (2009). Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue. Biotechniques, 46(3), 167-172.
8. Hitchins, A.D., Jinneman, K. and Chen, Y., 2017. BAM: Detection and enumeration of Listeria monocytogenes. Bacteriological Analytical Manual.

9. Jamali, H., Paydar, M., Ismail, S., Looi, C. Y., Wong, W. F., Radmehr, B., et al. (2015). Prevalence, antimicrobial susceptibility and virulotyping of Listeria species and Listeria monocytogenes isolated from open-air fish markets. BMC Microbiology, 15(1), 144.
10. Jin, Z., Ding, G., Li, G., Yang, G., Han, Y., Hao, N., ... & Li, W. (2020). Rapid detection of foodborne bacterial pathogens using visual high‐throughput microfluidic chip. Journal of Chemical Technology & Biotechnology, 95(5), 1460-1466.
11. Kiddle, G., Hardinge, P., Buttigieg, N. et al. GMO detection using a bioluminescent real time reporter (BART) of loop mediated isothermal amplification (LAMP) suitable for field use. BMC Biotechnol 12, 15 (2012). https://doi.org/10.1186/1472-6750-12-15
12. Kreitlow, A., Becker, A., Ahmed, M., Kittler, S., Schotte, U., Plötz, M., & Abdulmawjood, A. (2021). Combined Loop-Mediated Isothermal Amplification Assays for Rapid Detection and One-Step Differentiation of Campylobacter jejuni and Campylobacter coli in Meat Products. Frontiers in microbiology, 12, 668824. https://doi.org/10.3389/fmicb.2021.668824
13. Ledlod, S., Bunroddith, K., Areekit, S., Santiwatanakul, S., & Chansiri, K. (2020). Development of a duplex lateral flow dipstick test for the detection and differentiation of Listeria spp. and Listeria monocytogenes in meat products based on loop-mediated isothermal amplification. Journal of Chromatography B, 1139, 121834.
14. Lee, S. H., Ahn, J. Y., Lee, K. A., Um, H. J., Sekhon, S. S., Park, T. S., ... & Kim, Y. H. (2015). Analytical bioconjugates, aptamers, enable specific quantitative detection of Listeria monocytogenes. Biosensors and Bioelectronics, 68, 272-280.
15. Mohon, A. N., Elahi, R., Khan, W. A., Haque, R., Sullivan Jr, D. J., & Alam, M. S. (2014). A new visually improved and sensitive loop mediated isothermal amplification (LAMP) for diagnosis of symptomatic falciparum malaria. Acta tropica, 134, 52-57.
16. Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., et al. (2000). Loop-mediated isothermal amplification of DNA. Nucleic Acids Research, 28(12), 63e69.
17. Pang, B., Yao, S., Xu, K., Wang, J., Song, X., Mu, Y., ... & Li, J. (2019). A novel visual-mixed-dye for LAMP and its application in the detection of foodborne pathogens. Analytical biochemistry, 574, 1-6.
18. Parida, M., Sannarangaiah, S., Dash, P. K., Rao, P. V. L., & Morita, K. (2008). Loop mediated isothermal amplification (LAMP): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases. Reviews in medical virology, 18(6), 407-421.
19. Piskata, Z., Servusova, E., Babak, V., Nesvadbova, M., & Borilova, G. (2019). The quality of DNA isolated from processed food and feed via different extraction procedures. Molecules, 24(6), 1188.
20. Rhoades, J. R., Duffy, G., & Koutsoumanis, K. (2009). Prevalence and concentration of verocytotoxigenic Escherichia coli, Salmonella enterica and Listeria monocytogenes in the beef production chain: a review. Food microbiology, 26(4), 357-376.
21. Sagcan, H., & Kara, N. T. (2019). Detection of Potato ring rot Pathogen Clavibacter michiganensis subsp. s epedonicus by Loop-mediated isothermal amplification (LAMP) assay. Scientific reports, 9(1), 1-8.
22. Setiani, B. E., Elegado, F. B., Perez, M. T. M., Mabesa, R. C., Dizon, E. I., & Sevilla, C. C. (2015). API Listeria rapid kit for confirmatory fenotypic conventional biochemical test of the prevalence Listeria monocytogenes in selected meat and meat products. Procedia Food Science, 3, 445-452.
23. Shan, X., Zhang, Y., Zhang, Z., Chen, M., Su, Y., Yuan, Y., ... & Shi, L. (2012). Rapid detection of food-borne Listeria monocytogenes by real-time quantitative loop-mediated isothermal amplification. Food science and biotechnology, 21(1), 101-106.
24. Srisrattakarn, A., Lulitanond, A., Wilailuckana, C., Charoensri, N., Wonglakorn, L., Saenjamla, P., ... & Chanawong, A. (2017). Rapid and simple identification of carbapenemase genes, bla NDM, bla OXA-48, bla VIM, bla IMP-14 and bla KPC groups, in Gram-negative bacilli by in-house loop-mediated isothermal amplification with hydroxynaphthol blue dye. World Journal of Microbiology and Biotechnology, 33(7), 1-10. https://doi.org/10.1007/s11274-017-2295-5
25. Traunsek, U., Toplak, N., Jersek, B., Lapanje, A., Majstorovic, T., & Kovac, M. (2011). Novel cost-efficient real-time PCR assays for detection and quantitation of Listeria monocytogenes. Journal of Microbiological Methods, 85, 40e46.
26. Van Tongeren, S. P., Degener, J. E., & Harmsen, H. J. M. (2011). Comparison of three rapid and easy bacterial DNA extraction methods for use with quantitative real-time PCR. European journal of clinical microbiology & infectious diseases, 30(9), 1053-1061.
27. Wang DG, Huo GC, Ren DX, Li YG (2010) Development and evaluation of a loop-mediated isothermal amplification (LAMP) method for detecting Listeria monocytogenes in raw milk. J Food Saf 30:251-262.
28. Wang, G., Qian, W., Zhang, X., Wang, H., Ye, K., Bai, Y., & Zhou, G. (2015). Prevalence, genetic diversity and antimicrobial resistance of Listeria monocytogenes isolated from ready-to-eat meat products in Nanjing, China. Food Control, 50, 202-208.
29. Wang, L., Li, Y., Chu, J., Xu, Z., & Zhong, Q. (2012). Development and application of a simple loop-mediated isothermal amplification method on rapid detection of Listeria monocytogenes strains. Molecular biology reports, 39(1), 445-449.
30. Wang, T., Kim, S. and An, J.H., 2017. A novel CMOS image sensor system for quantitative loop-mediated isothermal amplification assays to detect food-borne pathogens. Journal of microbiological methods, 133, 1-7.
31. Wieczorek, K., & Osek, J. (2017). Prevalence, genetic diversity and antimicrobial resistance of Listeria monocytogenes isolated from fresh and smoked fish in Poland. Food Microbiology, 64, 164e171.
32. Wu, R., Liu, X., Guo, B., Chen, F., & Wang, X. (2014). Development of double loop-mediated isothermal amplification to detect Listeria monocytogenes in food. Current microbiology, 69(6), 839-845.
33. Xiao, X. L., Zhang, L., Wu, H., Yu, Y. G., Tang, Y. Q., Liu, D. M., et al. (2014). Simultaneous detection of Salmonella, Listeria monocytogenes, and Staphylococcus aureus, by multiplex real-time PCR assays using high-resolution melting. Food Analytical Methods, 7(10), 1960e1972.
34. Yan H, Zhang J, Ma D, Yin J. 2019. qPCR and loop mediated isothermal amplification for rapid detection of Ustilago tritici. PeerJ 7:e7766 https://doi.org/10.7717/peerj.7766
35. Zhao X, Li Y, Wang L, You L, Xu Z, Li L, He X, Liu Y, Wang J, Yang L (2010) Development and application of a loop-mediated isothermal amplification method on rapid detection Escherichia coli O157 strains from food samples. Mol Biol Rep 37:2183–2188.