Journal of Food Hygiene and Safety (한국식품위생안전성학회지)

The Korean Society of Food Hygiene and Safety (한국식품위생안전성학회)
권호 표지
DOI QR코드

DOI QR Code

Rapid Detection for Salmonella spp. by Ultrafast Real-time PCR Assay

Ultrafast Real-time PCR법을 이용한 살모넬라의 신속 검출

  • Kim, Seok Hwan (Food Microbiology Division, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety) ;
  • Lee, Yu-Si (Food Microbiology Division, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety) ;
  • Joo, In-Sun (Food Microbiology Division, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety) ;
  • Kwak, Hyo Sun (Food Microbiology Division, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety) ;
  • Chung, Gyung Tae (Food Microbiology Division, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety) ;
  • Kim, Soon Han (Food Microbiology Division, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety)
  • 김석환 (식품의약품안전처 식품의약품안전평가원 식품위해평가부 미생물과) ;
  • 이유시 (식품의약품안전처 식품의약품안전평가원 식품위해평가부 미생물과) ;
  • 주인선 (식품의약품안전처 식품의약품안전평가원 식품위해평가부 미생물과) ;
  • 곽효선 (식품의약품안전처 식품의약품안전평가원 식품위해평가부 미생물과) ;
  • 정경태 (식품의약품안전처 식품의약품안전평가원 식품위해평가부 미생물과) ;
  • 김순한 (식품의약품안전처 식품의약품안전평가원 식품위해평가부 미생물과)
  • Received : 2017.05.12
  • Accepted : 2017.11.21
  • Published : 2018.02.28
Download PDF
⟨ Previous Next ⟩

Abstract

Salmonella continue to be a major cause of food poisoning worldwide. The rapid detection method of food-borne Salmonella is an important food safety tool. A real-time polymerase chain reaction (PCR) has been used as a rapid method for the detection of pathogens. It has been recently reported that NBS LabChip real-time PCR is a novel, ultrafast, and chip-type-convenient real-time PCR system. We developed the assay method based on NBS LabChip real-time PCR for the rapid detection of Salmonella, which its reaction time was within 20 minutes. Two target genes (invA and stn) were selected to design target specific primers and probes. The new method was validated by checking specificity and sensitivity (limit of detection). This study included forty-two target and twenty-one non-target strains to assess the specificity. This assay was able to identify the 42 Salmonella strains correctly. The limit of detection (LOD) was $10^1copies/{\mu}L$ in Salmonella genomes DNA, while LOD incubated for 4 hr in the inoculated sausage sample ranged from $10^1CFU/g$ to $10^2CFU/g$ as an inoculated cell count. The assay developed in this study could be applied for the investigation of food poisoning pathogens.

Salmonella는 전세계적으로 식중독을 유발하는 주요 원인 균으로서, 식중독을 유발하는 Salmonella를 신속하게 검출하는 방법은 식품 안전을 위한 중요한 도구이다. Real-time PCR은 식중독균을 검출하기 위한 신속검사법으로 널리 사용되어 왔다. 최근에는 NBS LabChip real-time PCR이라는 새로운 시스템이 칩타입으로 조작이 간편하며 초고속의 real-time PCR 시스템이라는 보고가 있었다. 본 연구에서는 살모넬라의 신속한 검출을 위하여 NBS LabChip real-time PCR에 기반하여 real-time PCR 반응 시간이 20분 이내인 검출법을 확인하고자 하였다. 프라이머와 프로브 설계를 위해 두 개의 타겟 유전자(invA, stn)가 선택되었으며, 특이도와 민감도(검출한계)를 평가함으로 개발된 검출법을 검증하고자 하였다. 본 연구에서는 특이도 검증을 위해 Salmonella 균주 42주와 Non-Salmonella 균주 21주를 포함하였으며, 본 방법으로 Salmonella 42주에 대해서만 정확하게 검출이 가능하였다. 검출한계는 살모넬라 genome DNA 기준으로 $10^1copies/{\mu}L$으며, 소시지에서는 4시간 증균 이후 접종균수로서 $10^1CFU/g$에서 $10^2CFU/g$까지 검출이 가능하였다. 본 연구에서 개발된 검출법은 신속한 식중독 원인조사에 활용될 수 있을 것으로 기대된다.

Keywords

References

  1. Park N.B., Song E.J., Kim K.B.W.R., Lee C.J., Jung J.Y., Kwak J.H., Choi M.K., Kim M.J., Nam K.W., Ahn D.H.: Antimicrobial activity of Myagropsis yendoi extract. Kor. J. Fish Aquat. Sci., 43, 642-647 (2010).
  2. Kim G.Y., Yang G.M., Park S.B., Kim Y.H., Lee K.J., Son J.Y., Kim H.J., Lee S.R.: Rapid detection kit for Salmonella typhimurium. J. Biosystems Eng., 36, 140-146 (2011). https://doi.org/10.5307/JBE.2011.36.2.140
  3. Trong T.A, Nicholas A.F., Melita A.G., Karen H.K., Frederick J.A., Jon A.C.: Global burden of invasive nontyphoida salmonella disease, 2010. Emerging Infectious Diseases, 21, 941-949 (2015). https://doi.org/10.3201/eid2106.140999
  4. Coburn B., Grassl G.A., Finlay, B.B.: Salmonella, the host and disease: a brief review. Immunol. Cell Biol., 85, 112-118 (2007). https://doi.org/10.1038/sj.icb.7100007
  5. Yang B., Qu D., Zhang X., Shen J., Cui S., Shi Y., Xi M., Sheng M., Zhi S., Meng J.: Prevalence and characterization of Salmonella serovars in retail meats of marketplace in Shaanxi, China. Int. J. Food Microbiol., 141, 63-72 (2010). https://doi.org/10.1016/j.ijfoodmicro.2010.04.015
  6. Ministry of Food and Drug Safety (MFDS): www.foodsafetykorea.go.kr (2017).
  7. Na D.H., Hong Y.H., Yoon M.Y., Park E.J., Lim T.H., Jang J.H., Kim B.Y., Lee D.H., Song C.S.: Prevalence of Salmonella species isolated from old hen delivery trucks in Korea and application of disinfectant for the reduction of Salmonella contamination. Korean J. Poult. Sci., 40, 11-16 (2013).
  8. Cho A.R., Dong H.J., Cho S.B.: Rapid and sensitive detection of Salmonella spp. by using a Loop-Mediated isothermal amplification assay in duck carcass sample. Korean J. Food Sci., 33, 655-663 (2013).
  9. Cowden J.M., Lynch D., Joseph C.A., OMahony M., Mawer S.L., Rowe B., Bartlett C.L.: Case-control study of infections with Salmonella enteritidis phage type 4 in England. BMJ, 299, 771-773 (1989). https://doi.org/10.1136/bmj.299.6702.771
  10. Nastasi A., Mammina C., Piersante G.P., Robertazzo M., Caruso P.: A foodborne outbreak of Salmonella enteritidis vehicled by duck and hen eggs in southern Italy. New Microbiol., 21, 93-96 (1998).
  11. Tansel O., Ekuklu G., Otkun M., Otkun M.T., Akata F., Tugrul M.: A food-borne outbreak caused by Salmonella enteritidis. J. Yonsei Med., 44, 198-202 (2003). https://doi.org/10.3349/ymj.2003.44.2.198
  12. Humphrey T.J.: Contamination of eggs and poultry meat with Salmonella enterica serovar Enteritidis, In: Salmonella enteric serovar Enteritidis in humans and animals. Epidemiology, Pathogenesis, and Control. Iowa State University Press, Ames, IA, pp. 183-192 (1999).
  13. Lu S., Manges A.R., Xu Y., Fang F.C., Riley L.W.: Analysis of virulence of clinical isolates of Salmonella enteritidis in vivo and in vitro. Infect. Immun., 67, 5651-5657 (1999).
  14. Meyer H.: Animals as sources of infections in humans-salmonellosis. Dtsch. Tierarztl. Wochenschr., 106, 344-351 (1999).
  15. Gast, R.K. and Beard, C.W.: Isolation of Salmonella Enteritids from internal organs of experimentally infected hens. Avian. Dis., 34, 991-993 (1990). https://doi.org/10.2307/1591394
  16. Cover, T.L. and Aber, R.C.: Medical progress (Yersinia enterocolitica). N. Engl. J. Med., 321, 16-24 (1989). https://doi.org/10.1056/NEJM198907063210104
  17. Tassinari A., Franco B.D.G., Landgraf M.: Incidence of Yersinia spp. in food in Sao Paulo, Brazil. Int. J. Food Microbiol., 21, 262-270 (1994).
  18. Park H.J., Kim H.J., Park S.H., Shin E.G., Kim J.H., Kim H.Y.: Direct and quantitative analysis of Salmonella enterica Serovar Typhimurium using real-time PCR from artificially contaminated chicken meat. J. Microbiol. Biotechnol., 18, 1453-1458 (2008).
  19. Kim H.J., Kim Y.S., Chung M.S., Oh D.H., Bahk G.J., Chun H.S., Ha S.D.: Advances in rapid detection methods for foodborne pathogens. J. Fd. Hyg. Safety, 25, 376-387 (2010).
  20. Mandal P.K., Biswas A.K., Choi K., Pal U.K.: Methods for rapid detection of foodborne pathogens: An overview. American J. Food Technology, 6, 87-102 (2011).
  21. Guillermo L.C., Joaquin V.M.S., Monica A.U., Victoria L.A.: Microarray detection and characterization of bacterial foodborne pathogens. Springer Briefs in Food, Health, and Nutrition, USA, pp. 13-114 (2012).
  22. Lee J.H., Song K.Y., Hyeon J.Y., Hwang I.G., Kwak H.S., Han J.A., Chung Y.H., Seo K.H.: Comparison of standard culture method and real-time PCR assay for detection of Staphylococcus aureus in processed and unprocessed foods. Korean J. Food Sci. Ani. Resour., 30, 410-418 (2010). https://doi.org/10.5851/kosfa.2010.30.3.410
  23. Lee S.H., Kim S.W., Lee SH., Kim E.S., Kim D.J., Park S.H., Lee E.J., Lee S.Y., Lee J.S., Lim C.S., Kim W.K., In K.H.: Rapid detection of mycobacterium tuberculosis using a novel ultrafast chip-type real-time polymerase chain reaction system. Chest., 146, 1319-1326 (2014). https://doi.org/10.1378/chest.14-0626
  24. Song H.O., Kim J.H., Ryu H.S., Lee D.H., Kim S.J., Kim D.J., Suh I.B., Choi D.Y., In K.H., Kim S.W., Park H.P.: Polymeric labchip real-time PCR as a point-of-care-potential diagnostic tool for rapid detection of influenza A/H1N1 virus in human clinical specimens. Plos one, 12, 1-8 (2012).
  25. Johnson G., Nolan T., Bustin S.A.: Real-time quantitative PCR, pathogen detection and MIQE. Methods Mol. Biol., 943, 1-16 (2013).
  26. Moore, M.M. and Feist, M.D.: Real-time PCR method for Salmonella spp. targeting the stn gene. J. Appl. Microbiol., 102, 516-530 (2007).
  27. Joo J.W., Hong K.P., Kim Y.H., Cho S.B.: Selective detection of Salmonella sp. and Salmonella Typhimurium in meat by polymerase chain reaction. J. East Asian Soc. Dietary Life, 19, 295-300 (2009).
  28. Kumar R., Surendran P.K., Thampuran N.: Rapid quantification of Salmonella in seafood using real-time PCR assay. J. Microbiol. Biotechnol., 20, 569-573 (2010).
  29. Niu P.H., Zhang C., Wang J., Tan W.J., Ma X.J.: Detection and identification of six foodborne bacteria by two-tube multiplex real time PCR and melting curve analysis. Biomed. Environ. Sci., 27, 770-778 (2014).
  30. Malorny B., Paccassoni E., Fach P., Bunge C., Martin A., Helmuth R.: Diagnostic real-time PCR for detection of Salmonella in food. Appl. Environ. Microbiol., 70, 7064-7052 (2004).
  31. Jeong Y.S., Jung H.K., Jeon W.B., Seo H.J., Hong J.H.: Simultaneous detection of Staphylococcus aureus, Salmonella enterica subsp. Vibrio parahaemolyticus by multiplex polymerase chain Reaction. J. Korean Soc. Food Sci. Nutr., 39, 595-601 (2010). https://doi.org/10.3746/jkfn.2010.39.4.595
  32. Wegmuller B., Luthy J., Candrian U.: Direct polymerase chain reaction detection of Campylobacter jejuni and Campylobacter coli in raw milk and dairy products. Appl. Environ. Microbiol., 59, 2161-2165 (1993).
  33. Heo E.J., Song B.R., Park H.J., Kim Y.J., Moon J.S., Wee S.H., Kim J.S., Yoon Y.H.: Rapid detection of Listeria monocytogenes by real-time PCR in processed meat and dairy products, J. Food Protection, 77, 453-458 (2014). https://doi.org/10.4315/0362-028X.JFP-13-318
  34. Nam H.M., Srinivasan V., Gillespie B.E., Murinda S.E., OliverT S.P.: Application of SYBR green real-time PCR assay for specific detection of Salmonella spp. in dairy farm environmental samples. Int. J. Food Microbiology, 102, 161-171 (2005). https://doi.org/10.1016/j.ijfoodmicro.2004.12.020
  35. Sharma, V.K. and Carlson, S.A.: Simultaneous detection of Salmonella strains and Escherichia coli O157:H7 with fluorogenic PCR and single-enrichment-broth culture. Appl. Environ. Microbiol. 66, 5472-5476 (2000). https://doi.org/10.1128/AEM.66.12.5472-5476.2000
  36. Rodriguez-Lazaro, D. and Hernandez, M.: Isolation of Listeria monocytogenes DNA from meat products for quantitative detection by real-time PCR. J. Rapid Method Automat Microbiol., 14, 395-404 (2006). https://doi.org/10.1111/j.1745-4581.2006.00058.x
  37. Navas J., Ortiz S., Lopez P., Jantzen M.M., Lopez V., Martinez-Suarez J.V.: Evaluation of effects of primary and secondary enrichment for the detection of Listeria monocytogenes by real-time PCR in retail ground chicken meat. Foodborne Pathog. Dis., 3, 347-354 (2006). https://doi.org/10.1089/fpd.2006.3.347
  38. Jasson V., Jacxsens L., Luning P., Rajkovic A., Uyttendaele M.: Review. Alternative microbial methods: An overview and selection criteria. Food Microbiol., 27, 710-730 (2010). https://doi.org/10.1016/j.fm.2010.04.008
  39. Kim J.H., Koo E.J., Seong J.Y., Myeong H.K., Oh S.W.: Rapid methods for the detection of foodborne pathogens from seafoods. Safe Food, 11, 16-24 (2016).