Journal List > Korean J Clin Microbiol > v.13(4) > 1038201

Korean J Clin Microbiol. 2010 Dec;13(4):162-168. Korean.
Published online December 27, 2010.
Copyright © 2010 The Korean Society of Clinical Microbiology
Direct Application of Multiplex PCR on Stool Specimens for Detection of Enteropathogenic Bacteria
Min-Chul Cho,1 Sin-Ae Noh,1 Mi-Na Kim,1 and Kyoung-Mo Kim2
1Department of Laboratory Medicine, Asan Medical Center and University of Ulsan College of Medicine, Seoul, Korea.
2Department of Pediatrics, Asan Medical Center and University of Ulsan College of Medicine, Seoul, Korea.

Correspondence: Mi-Na Kim, Department of Laboratory Medicine, Asan Medical Center and University of Ulsan College of Medicine, 388-1 Pungnap 2-dong, Songpa-gu, Seoul 138-736, Korea. (Tel) 82-2-3010- 4511, (Fax) 82-2-478-0884, Email:
Received May 17, 2010; Revised August 05, 2010; Accepted September 10, 2010.



Causative bacterial agents of infectious diarrheal disease were traditionally diagnosed by stool cultures. Stool culture, however, has a problem because of relatively low sensitivity and long turnaround time. In this study, we evaluated multiplex PCR applied on stool specimens directly to diagnose enteropathogenic bacteria.


From June to September 2009, 173 diarrheal stools submitted for stool cultures were tested by Seeplex® Diarrhea ACE Detection kit (Seegene, Korea) to detect 10 enteropathogenic bacteria. Specimens were cultured for Salmonella, Shigella, Vibrio, and Yersinia. Late 50 specimens were also cultured for Campylobacter. The specimens positive for verotoxin-producing Escherichia coli (VTEC) were further subcultured for detecting enterohaemorrhagic Escherichia coli O157:H7. Electronic medical records were reviewed for clinical and laboratory findings.


Of 173 specimens, multiplex PCR and cultures identified enteropathogens in 36 (20.8%) and 8 specimens (4.6%), respectively. While multiplex PCR detected 5 Salmonella, 15 Campylobacter, 1 Vibrio, 4 Clostridium difficiles toxin B, 5 Clostridium perfringens, 1 Yersinia enterocolitica, 5 Aeromonas, and 2 VTEC, cultures detected 5 Salmonella, 1 Vibrio, 1 Y. enterocolitica, 1 Aeromonas, and 2 E. coli O157:H7.


Multiplex PCR would be useful to detect Campylobacter, VTEC and C. perfringens, as well as have equivalent sensitivity to conventional culture for ordinary enteropathogens such as Salmonella, Shigella, Vibrio, Y. enterocolitica. Direct application of multiplex PCR combined with conventional cultures on stool warrants remarkable improvement of sensitivity to diagnose enteropathogenic bacteria.

Keywords: Multiplex PCR; Infectious diarrheal disease; Enteropathogen


Table 1
Target genes and their product size of multiplex PCR
Click for larger image

Table 2
Distribution of diarrhea pathogens confirmed by multiplex PCR and culture results
Click for larger image

Table 3
Clinical characteristics of PCR-positive patients
Click for larger image

1. Baldi F, Bianco MA, Nardone G, Pilotto A, Zamparo E. Focus on acute diarrhoeal disease. World J Gastroenterol 2009;15:3341–3348.
2. Vernacchio L, Vezina RM, Mitchell AA, Lesko SM, Plaut AG, Acheson DW. Diarrhea in American infants and young children in the community setting: incidence, clinical presentation and microbiology. Pediatr Infect Dis J 2006;25:2–7.
3. Farthing MJ. Diarrhoea: a significant worldwide problem. Int J Antimicrob Agents 2000;14:65–69.
4. Tarr PI, Gordon CA, Chandler WL. Shiga-toxin-producing Escherichia coli and haemolytic uraemic syndrome. Lancet 2005;365:1073–1086.
5. Thielman NM, Guerrant RL. Clinical practice. Acute infectious diarrhea. N Engl J Med 2004;350:38–47.
6. Guerrant RL, Van Gilder T, Steiner TS, Thielman NM, Slutsker L, Tauxe RV, et al. Practice guidelines for the management of infectious diarrhea. Clin Infect Dis 2001;32:331–351.
7. Cheng AC, McDonald JR, Thielman NM. Infectious diarrhea in developed and developing countries. J Clin Gastroenterol 2005;39:757–773.
8. Thapar N, Sanderson IR. Diarrhoea in children: an interface between developing and developed countries. Lancet 2004;363:641–653.
9. Gadewar S, Fasano A. Current concepts in the evaluation, diagnosis and management of acute infectious diarrhea. Curr Opin Pharmacol 2005;5:559–565.
10. Shin HB, Jeong SH, Kim M, Kim WH, Lee K, Chong Y. Isolation trend of enteropathogenic bacteria in 1969-1998. Korean J Clin Microbiol 2001;4:87–95.
11. Korea Center for Disease Control & Prevention; The prevalence and characteristics of bacteria causing acute diarrhea in Korea, 2008.
12. Iijima Y, Asako NT, Aihara M, Hayashi K. Improvement in the detection rate of diarrhoeagenic bacteria in human stool specimens by a rapid real-time PCR assay. J Med Microbiol 2004;53:617–622.
13. Reller ME, Lema CA, Perl TM, Cai M, Ross TL, Speck KA, et al. Yield of stool culture with isolate toxin testing versus a two-step algorithm including stool toxin testing for detection of toxigenic Clostridium difficile. J Clin Microbiol 2007;45:3601–3605.
14. Chapela MJ, Fajardo P, Garrido A, Cabado AG, Ferreira M, Lago J, et al. Comparison between a TaqMan polymerase chain reaction assay and a culture method for ctx-positive Vibrio cholerae detection. J Agric Food Chem 2010;58:4051–4055.
15. Nhung PH, Ohkusu K, Miyasaka J, Sun XS, Ezaki T. Rapid and specific identification of 5 human pathogenic vibrio species by multiplex polymerase chain reaction targeted to dnaJ gene. Diagn Microbiol Infect Dis 2007;59:271–275.
16. Daube G, Simon P, Limbourg B, Manteca C, Mainil J, Kaeckenbeeck A. Hybridization of 2,659 Clostridium perfringens isolates with gene probes for seven toxins (alpha, beta, epsilon, iota, theta, mu, and enterotoxin) and for sialidase. Am J Vet Res 1996;57:496–501.
17. Jang YH, Chung J, Baek S, Park S, Sung H, Kim MN. Implementation of multiplex PCR for species identification and toxin typing in toxigenic Clostridium difficile culture. Korean J Clin Microbiol 2009;12:11–16.
18. Paton AW, Paton JC. Multiplex PCR for direct detection of Shiga toxigenic Escherichia coli strains producing the novel subtilase cytotoxin. J Clin Microbiol 2005;43:2944–2947.
19. Kim JS, Lee GG, Park JS, Jung YH, Kwak HS, Kim SB, et al. A novel multiplex PCR assay for rapid and simultaneous detection of five pathogenic bacteria: Escherichia coli O157:H7, Salmonella, Staphylococcus aureus, Listeria monocytogenes, and Vibrio parahaemolyticus. J Food Prot 2007;70:1656–1662.
20. O'Leary J, Corcoran D, Lucey B. Comparison of the EntericBio multiplex PCR system with routine culture for detection of bacterial enteric pathogens. J Clin Microbiol 2009;47:3449–3453.
21. Butzler JP. Campylobacter, from obscurity to celebrity. Clin Microbiol Infect 2004;10:868–876.
22. Thorney JP, Shaw JG, Gryllos IA, Eley A. Virulence properties of clinically significant Aeromonas species: evidence for pathogenicity. Rev Med Microbiol 1997;8:61–72.
23. Choi JP, Lee SO, Kwon HH, Kwak YG, Choi SH, Lim SK, et al. Clinical significance of spontaneous Aeromonas bacterial peritonitis in cirrhotic patients: a matched case-control study. Clin Infect Dis 2008;47:66–72.
24. Kang JM, Kim BN, Choi SH, Kim NJ, Woo JH, Ryu J, et al. Clinical features and prognostic factors of Aeromonas bacteremia. Infect Chemother 2005;37:161–166.
25. Labbe R. Clostridium perfringens. In: Doyle MP, editor. Foodborne Bacterial Pathogens. New York: Marcel Dekker, Inc.; 1989. pp. 191-234.
26. Kokai-Kun JF, Songer JG, Czeczulin JR, Chen F, McClane BA. Comparison of western immunoblots and gene detection assays for identification of potentially enterotoxigenic isolates of Clostridium perfringens. J Clin Microbiol 1994;32:2533–2539.
27. Lee SW, Lee BK, Lee YJ, Lee HS, Jung SC, Sun KH, et al. A study on epidemiological characteristics and control methods of EHEC infection in Korea. Korean J Epidemiol 2005;27:37–52.
28. Oh YH, Kim YB. Use of polymerase chain reaction and serum antibodies for diagnosis of enterohemorrhagic Escherichia coli. J Korean Soc Microbiol 1998;33:99–110.