Korean Journal of Veterinary Research (대한수의학회지)

The Korean Society of Veterinary Science (대한수의학회)
권호 표지
DOI QR코드

DOI QR Code

Application of SYBR Green real-time PCR assay for the specific detection of Salmonella spp.

Salmonella spp. 특이적인 검출을 위한 SYBR Green real-time PCR 기법 적용

  • Shin, Seung Won (Department of Infectious Diseases, College of Veterinary Medicine and Brain Korea 21 Program for Veterinary Science, Seoul National University) ;
  • Cha, Seung Bin (Department of Infectious Diseases, College of Veterinary Medicine and Brain Korea 21 Program for Veterinary Science, Seoul National University) ;
  • Lee, Won-Jung (Department of Infectious Diseases, College of Veterinary Medicine and Brain Korea 21 Program for Veterinary Science, Seoul National University) ;
  • Shin, Min-Kyoung (Department of Infectious Diseases, College of Veterinary Medicine and Brain Korea 21 Program for Veterinary Science, Seoul National University) ;
  • Jung, Myunghwan (Department of Infectious Diseases, College of Veterinary Medicine and Brain Korea 21 Program for Veterinary Science, Seoul National University) ;
  • Yoo, Anna (Department of Infectious Diseases, College of Veterinary Medicine and Brain Korea 21 Program for Veterinary Science, Seoul National University) ;
  • Jung, Byeng Yeal (Bacterial Disease Division, Department of Animal and Plant Health Research, Animal, Plant and Fisheries Quarantine and Inspection Agency) ;
  • Yoo, Han Sang (Department of Infectious Diseases, College of Veterinary Medicine and Brain Korea 21 Program for Veterinary Science, Seoul National University)
  • 신승원 (서울대학교 수의과대학 및 BK21 수의과학인력양성 사업단) ;
  • 차승빈 (서울대학교 수의과대학 및 BK21 수의과학인력양성 사업단) ;
  • 이원정 (서울대학교 수의과대학 및 BK21 수의과학인력양성 사업단) ;
  • 신민경 (서울대학교 수의과대학 및 BK21 수의과학인력양성 사업단) ;
  • 정명환 (서울대학교 수의과대학 및 BK21 수의과학인력양성 사업단) ;
  • 유안나 (서울대학교 수의과대학 및 BK21 수의과학인력양성 사업단) ;
  • 정병열 (농림수산검역검사본부 동식물위생연구부 세균질병과) ;
  • 유한상 (서울대학교 수의과대학 및 BK21 수의과학인력양성 사업단)
  • Received : 2012.08.09
  • Accepted : 2012.12.14
  • Published : 2013.03.31
Download PDF
⟨ Previous Next ⟩

Abstract

The aim of this study was to applicate and evaluate a SYBR Green real-time PCR for the specific detection of Salmonella spp. Specificity of the PCR method was confirmed with 48 Salmonella spp. and 5 non-Salmonella strains using invA gene primer. The average threshold cycle ($C_T$) of Salmonella spp. was $11.83{\pm}0.78$ while non-Salmonella spp. was $30.86{\pm}1.19$. Correlation coefficients of standard curves constructed using $C_T$ versus copy number of Salmonella Enteritidis ATCC 13076 showed good linearity ($R^2=0.993$; slope = 3.563). Minimum level of detection with the method was > $10^2$ colony forming units (CFU)/mL. These results suggested that the SYBR Green real-time PCR might be applicable for the specific detection of Salmonella spp. isolates.

Keywords

References

  1. Aarts HJM, Joosten RG, Henkens MHC, Stegeman H, van Hoek AHAM. Rapid duplex PCR assay for the detection of pathogenic Yersinia enterocolitica strains. J Microbiol Methods 2001, 47, 209-217. https://doi.org/10.1016/S0167-7012(01)00305-0
  2. Bhagwat AA. Rapid detection of Salmonella from vegetable rinse-water using real-time PCR. Food Microbiol 2004, 21, 73-78. https://doi.org/10.1016/S0740-0020(03)00020-0
  3. Carli KT, Unal CB, Caner V, Eyigor A. Detection of Salmonellae in chicken feces by a combination of tetrathionate broth enrichment, capillary PCR, and capillary gel electrophoresis. J Clin Microbiol 2001, 39, 1871-1876. https://doi.org/10.1128/JCM.39.5.1871-1876.2001
  4. Chen S, Yee A, Griffiths M, Larkin C, Yamashiro CT, Behari R, Paszko-Kolva C, Rahn K, De Grandis SA. The evaluation of a fluorogenic polymerase chain reaction assay for the detection of Salmonella species in food commodities. Int J Food Microbiol 1997, 35, 239-250. https://doi.org/10.1016/S0168-1605(97)01241-5
  5. Chen W, Martinez G, Mulchandani A. Molecular beacons: a real-time polymerase chain reaction assay for detecting Salmonella. Anal Biochem 2000, 280, 166-172. https://doi.org/10.1006/abio.2000.4518
  6. Chiu TH, Chen TR, Hwang WZ, Tsen HY. Sequencing of an internal transcribed spacer region of 16S-23S rRNA gene and designing of PCR primers for the detection of Salmonella spp. in food. Int J Food Microbiol 2005, 97, 259-265. https://doi.org/10.1016/j.ijfoodmicro.2004.04.005
  7. Cogan TA, Humphrey TJ. The rise and fall of Salmonella Enteritidis in the UK. J Appl Microbiol 2003, 94 (Suppl), 114S-119S. https://doi.org/10.1046/j.1365-2672.94.s1.13.x
  8. Crump JA, Luby SP, Mintz ED. The global burden of typhoid fever. Bull World Health Organ 2004, 82, 346-353.
  9. Daum LT, Barnes WJ, McAvin JC, Neidert MS, Cooper LA, Huff WB, Gaul L, Riggins WS, Morris S, Salmen A, Lohman KL. Real-time PCR detection of Salmonella in suspect foods from a gastroenteritis outbreak in Kerr County, Texas. J Clin Microbiol 2002, 40, 3050-3052. https://doi.org/10.1128/JCM.40.8.3050-3052.2002
  10. De Medici D, Croci L, Delibato E, Di Pasquale S, Filetici E, Toti T. Evaluation of DNA extraction methods for use in combination with SYBR Green I real-time PCR to detect Salmonella enterica serotype Enteritidis in poultry. Appl Environ Microbiol 2003, 69, 3456-3461. https://doi.org/10.1128/AEM.69.6.3456-3461.2003
  11. Eijkelkamp JM, Aarts HJM, van der Fels-Klerx HJ. Suitability of rapid detection methods for Salmonella in poultry slaughterhouses. Food Anal Methods 2009, 2, 1-13. https://doi.org/10.1007/s12161-008-9040-5
  12. Eriksson E, Aspan A. Comparison of culture, ELISA and PCR techniques for salmonella detection in faecal samples for cattle, pig and poultry. BMC Vet Res 2007, 3, 21. https://doi.org/10.1186/1746-6148-3-21
  13. Foley B, McKeown P, de Lappe N, Cormican M. Salmonellosis in Ireland, 2006. EPI Insight 2007, 8, 2-3.
  14. Fukushima H, Tsunomori Y, Seki R. Duplex real-time SYBR Green PCR assays for detection of 17 Species of foodor waterborne pathogens in stools. J Clin Microbiol 2003, 41, 5134-5146. https://doi.org/10.1128/JCM.41.11.5134-5146.2003
  15. Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res 1996, 6, 986-994. https://doi.org/10.1101/gr.6.10.986
  16. Hoorfar J, Ahrens P, Radstrom P. Automated 5' nuclease PCR assay for identification of Salmonella enterica. J Clin Microbiol 2000, 38, 3429-3435.
  17. Jothikumar N, Griffiths MW. Rapid detection of Escherichia coli O157:H7 with multiplex real-time PCR assays. Appl Environ Microbiol 2002, 68, 3169-3171. https://doi.org/10.1128/AEM.68.6.3169-3171.2002
  18. Jothikumar N, Wang X, Griffiths MW. Real-time multiplex SYBR Green I-based PCR assay for simultaneous detection of Salmonella serovars and Listeria monocytogenes. J Food Prot 2003, 66, 2141-2145. https://doi.org/10.4315/0362-028X-66.11.2141
  19. Knutsson R, Lofstrom C, Grage H, Hoorfar J, Radstrom P. Modeling of 5' nuclease real-time responses for optimization of a high-throughput enrichment PCR procedure for Salmonella enterica. J Clin Microbiol 2002, 40, 52-60. https://doi.org/10.1128/JCM.40.1.52-60.2002
  20. Liming SH, Bhagwat AA. Application of a molecular beacon-real-time PCR technology to detect Salmonella species contaminating fruits and vegetables. Int J Food Microbiol 2004, 95, 177-187. https://doi.org/10.1016/j.ijfoodmicro.2004.02.013
  21. Nam HM, Srinivasan V, Gillespie BE, Murinda SE, Oliver SP. Application of SYBR Green real-time PCR assay for specific detection of Salmonella spp. in dairy farm environmental samples. Int J Food Microbiol 2005, 102, 161-171. https://doi.org/10.1016/j.ijfoodmicro.2004.12.020
  22. Newton-Clarke M. Principles of prevention and control of salmonellosis. Equine Vet Educ 1995, 7, 67-69. https://doi.org/10.1111/j.2042-3292.1995.tb01192.x
  23. Nogva HK, Lillehaug D. Detection and quantification of Salmonella in pure cultures using 5'-nuclease polymerase chain reaction. Int J Food Microbiol 1999, 51, 191-196. https://doi.org/10.1016/S0168-1605(99)00122-1
  24. Oosterom J. Epidemiological studies and proposed preventive measures in the fight against human salmonellosis. Int J Food Microbiol 1991, 12, 41-51. https://doi.org/10.1016/0168-1605(91)90046-R
  25. Perry JD, Freydiere AM. The application of chromogenic media in clinical microbiology. J Appl Microbiol 2007, 103, 2046-2055. https://doi.org/10.1111/j.1365-2672.2007.03442.x
  26. Rahn K, De Grandis SA, Clarke RC, McEwen SA, Galan JE, Ginocchio C, Curtiss R 3rd, Gyles CL. Amplification of an invA gene sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella. Mol Cell Probes 1992, 6, 271-279. https://doi.org/10.1016/0890-8508(92)90002-F
  27. Rodriguez-Lazaro D, Hernandez M, Esteve T, Hoorfar J, Pla M. A rapid and direct real time PCR-based method for identification of Salmonella spp. J Microbiol Methods 2003, 54, 381-390. https://doi.org/10.1016/S0167-7012(03)00071-X
  28. Tirado C, Schmidt K. WHO surveillance programme for control of foodborne infections and intoxications: preliminary results and trends across greater Europe J Infect 2001, 43, 80-84. https://doi.org/10.1016/S0163-4453(01)90861-8
  29. Waltman WD, Mallinson ET. Isolation of Salmonella from poultry tissue and environmental samples: a nationwide survey. Avian Dis 1995, 39, 45-54. https://doi.org/10.2307/1591981

Cited by

  1. Oesophageal dysmotility in a dog following surgical correction of an oesophageal perforation vol.9, pp.2, 2013, https://doi.org/10.1002/vrc2.57