Hostname: page-component-669899f699-rg895 Total loading time: 0 Render date: 2025-04-26T21:41:26.716Z Has data issue: false hasContentIssue false
  • English
  • Français

Application of a multiplex PCR assay for the detection of Shigella, Escherichia coli and Shiga toxin-producing Esch. coli in milk

Published online by Cambridge University Press:  13 March 2009

Syed Riyaz-Ul-Hassan ,
Saima Syed ,
Sarojini Johri ,
Vijeshwar Verma  and
Ghulam Nabi Qazi
Syed Riyaz-Ul-Hassan*
Affiliation:
Biotechnology Division, Indian Institute of Integrative Medicine (Council of Scientific and Industrial Research), Canal Road, Jammu Tawi-180001, India
Saima Syed
Affiliation:
Biotechnology Division, Indian Institute of Integrative Medicine (Council of Scientific and Industrial Research), Canal Road, Jammu Tawi-180001, India
Sarojini Johri
Affiliation:
Biotechnology Division, Indian Institute of Integrative Medicine (Council of Scientific and Industrial Research), Canal Road, Jammu Tawi-180001, India
Vijeshwar Verma
Affiliation:
Biotechnology Division, Indian Institute of Integrative Medicine (Council of Scientific and Industrial Research), Canal Road, Jammu Tawi-180001, India
Ghulam Nabi Qazi
Affiliation:
Biotechnology Division, Indian Institute of Integrative Medicine (Council of Scientific and Industrial Research), Canal Road, Jammu Tawi-180001, India
*
*For correspondence; e-mail: srhassan@iiim.res.in/riyazulhassan@yahoo.com
Get access Rights & Permissions [Opens in a new window]

Abstract

A multiplex PCR (mPCR) assay using previously known genetic markers of Shigella, Escherichia coli and Shiga-toxic Esch. coli was standardized. uidA gene was targeted for the common detection of Esch. coli and Shigella, whereas ipaH and stx1 genes were used as markers for the detection of Shigella and shiga-toxin producing strains, respectively. The standardized assays detected the target organism specifically and selectively. The mPCR developed by combining all the three reactions generated specific products. The inclusivity and exclusivity tests depicted the precise specificity of the mPCR assay. Results were interpreted on the basis of the pattern of amplicons generated: amplifications of the ipaH and uidA gene fragments indicated the presence of Shigella spp., amplification of uidA alone revealed the presence of Esch. coli and additional presence of verotoxin gene amplicon indicated verotoxinogenic nature of the strain. Specific patterns of bands were obtained when different strains of Esch. coli and Shigella spp. were subjected to this assay. The reactions, individually as well as in the mPCR, could detect approximately 1 cell per 20-μl PCR assay. The protocols were validated by analyzing the coded samples of full fat milk spiked with different pathogens. In naturally contaminated raw milk samples (n=100), Esch. coli were detected in all samples and verotoxinogenic Esch. coli in 15 samples. Shigella, however, was not detected in any of the samples. When DNA purified from the samples found positive for Shiga-toxic Esch. coli was directly used as template for the mPCR, the results showed agreement with the enrichment based detection. The mPCR assay, standardized in this study, may be used for rapid microbiological evaluation of milk samples. Further, the study emphasizes the need for better hygienic conditions in dairies.

Keywords

Multiplex PCRShigellaEsch. coliSTECmilk
Type
Research Article
Information
Journal of Dairy Research , Volume 76 , Issue 2 , May 2009 , pp. 188 - 194
Copyright
Copyright © Proprietors of Journal of Dairy Research 2009

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

Article purchase

Temporarily unavailable

References

Aranda, KRS, Fagundes-Neto, U & Scaletsky, ICA 2004 Evaluation of Multiplex PCRs for diagnosis of infection with diarrheagenic Escherichia coli and Shigella spp. Journal of Clinical Microbiology 42 5849–5853CrossRefGoogle ScholarPubMed
Aslam, M, Hogan, J & Smith, KL 2003 Development of a PCR-based assay to detect shiga toxin-producing Escherichia coli, Listeria monocytogenes, and Salmonella in milk. Food Microbiology 20 345–350CrossRefGoogle Scholar
Bej, AK, DiCesare, JL, Haff, L & Atlas, RM 1991 Detection of E. coli and Shigella spp. in water by using the polymerase chain reaction and gene probes for uid. Applied and Environmental Microbiology 57 1013–1017CrossRefGoogle ScholarPubMed
Bettelheim, K 1997 Escherichia coli O157 outbreak in Japan: lessons for Australia. Australian Veterinary Journal 75 108CrossRefGoogle ScholarPubMed
Blanco, M, Blanco, JE, Mora, A, Rey, J, Alonso, JM, Herrmoso, M, Hermoso, J, Alonso, MP et al. 2003 Serotypes, virulence genes, and intimin types of Shiga toxin (verotoxin)-producing Escherichia coli isolated from healthy sheep from Spain. Journal of Clinical Microbiology 41 1351–1356CrossRefGoogle ScholarPubMed
Boerlin, P, McEwen, SA, Boerlin-Petzold, F, Wilson, JB, Johnson, RP & Gyles, CI 1999 Association between virulence factors of Shiga toxin-producing Escherichia coli and disease in humans. Journal of Clinical Microbiology 37 497–503CrossRefGoogle ScholarPubMed
Bradbury, J 1997 Report on Scottish E. coli O157 outbreak released. Lancet 349 1073CrossRefGoogle Scholar
Brett, KN, Ramachandran, V, Hornitzky, MA, Bettelheim, KA, Walker, MJ & Djordjevic, SP 2003 Stx1c is the most common shiga toxin 1 subtype among shiga toxin producing Escherichia coli isolates from sheep but not isolates from cattle. Journal of Clinical Microbiology 41 926–936CrossRefGoogle Scholar
Buysse, JM, Stover, CK, Oaks, EV, Venkatesan, M & Kopecko, DJ 1987 Molecular cloning of invasion plasmid antigen (ipa) genes from Shigella flexneri: analysis of ipa gene products and genetic mapping. Journal of Bacteriology 169 2561–2569CrossRefGoogle ScholarPubMed
Centers for Disease Control 1993 Update: multistate outbreak of Escherichia coli O157:H7 infections from hamburgers western United States, 1992–1993. Morbid and Mortal Weekly Rep 42 258–263Google Scholar
Centers for Disease Control 2005 Bacterial Foodborne and Diarrheal Disease National Case Surveillance. Annual Report. Atlanta: U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, 2007Google Scholar
Centers for Disease Control 2007 Escherichia coli O157:H7 infection associated with drinking raw milk – Washington and Oregan, Nov.–Dec. 2005. Morbidity and Mortality Weekly Report Mar. 2. 56 165–167Google Scholar
Cristancho, L, Johnson, RP, McEwen, SA & Gyles, CL 2008 Escherichia coli O157: H7 and other shiga toxin-producing E. coli in white veal calves. Veterinary Microbiology 126 200–209CrossRefGoogle ScholarPubMed
Ethelberg, S, Olsen, KE, Scheutz, F, Jensen, C, Schiellerup, P, Enberg, J, Petersen, AM, Olesen, B, Gerner-Smidt, P & Molbak, K 2004 Virulence factors for hemolytic uremic syndrome, Denmark. Emerging Infectious Diseases 10 842–847CrossRefGoogle ScholarPubMed
Gerber, A, Karch, H, Allerberger, F, Verweyen, HM & Zimmerhackl, LB 2002 Clinical course and the role of shiga toxin-producing Escherichia coli infection in the hemolytic-uremic syndrome in pediatric patients, 1997–2000, in Germany and Austria: a prospective study. Journal of Infectious Diseases 186 493–500CrossRefGoogle ScholarPubMed
Gracia-Fulgueiras, A, Sanchez, S, Guillen, JJ, Marsilla, B, Aladuena, A & Novarro, C 2001 A large outbreak of Shigella sonnei gastroenterititis associated with consumption of fresh pasteurized milk cheese. European Journal of Epidemiology 17 533–538CrossRefGoogle Scholar
Griffin, PM & Tauxe, RV 1991 The epidemiology of infections caused by Escherichia coli O157:H7, other enterohemorrhagic E. coli, and the associated hemolytic uremic syndrome. Epidemiologic Reviews 13 60–98CrossRefGoogle ScholarPubMed
Hartman, AB, Venkatesan, M, Oaks, EV & Buysee, JM 1990 Sequence and molecular characterization of multicopy invasion plasmid antigen gene, ipaH, of Shigella flexneri. Journal of Bacteriology 172 1905–1915CrossRefGoogle ScholarPubMed
Heijnen, L & Medema, G 2006 Quantitative detection of E. coli, E. coli O157:H7 and other shiga toxin producing E. coli in water samples using culture method combined with real-time PCR. Journal of Water Health 4 487–498CrossRefGoogle ScholarPubMed
Jochimsen, B, Nygaard, P & Vestergaard, T 1975 Location on the chromosome of Escherichia coli of genes governing purine metabolism. Molecular and General Genetics 143 85–91CrossRefGoogle ScholarPubMed
Karmali, MA 1989 Infections by verocytotoxin-producing Escherichia coli. Clinical Microbiology Reviews 2 15–38CrossRefGoogle ScholarPubMed
Koneman, EW, Allen, SD, Janda, WM, Schreckenberger, PC & Winn, WC 1997 The enterobacteraiceae. In Color Atlas and Diagnostic Microbiology, V Edn. pp. 171–241 (Ed. Allen, A.). Lippincott Williams and Wilkins, Philadelphia, USAGoogle Scholar
Lampel, KA, Orlandi, PA & Kornegay, L 2000 Improved template preparation for PCR-based assay for detection of food-borne bacterial pathogens. Applied and Environmental Microbiology 66 4539–4542CrossRefGoogle ScholarPubMed
Novel, G & Novel, M 1973 Mutants d' Escherichia coli K-12 affectes pour leur croissance sur methyl β-D-glucuronide: localisation du gene de structure de la P-Dglucuronidase (uidA). Molecular and General Genetics 120 319–335CrossRefGoogle Scholar
Olsen, JE, Aabo, S, Hill, W, Notermans, S, Wernars, K, Granum, PE, Popovic, T, Rasmussen, HN & Olsvik, O 1995 Probes and polymerase chain reaction for detection of food-borne bacterial pathogens. International Journal Food Microbiology 28 1–78CrossRefGoogle ScholarPubMed
Paton, JC & Paton, AW 1998 Pathogenesis and diagnosis of Shiga toxin-producing Escherichia coli infections. Clinical Microbiology Reviews 11 450–479CrossRefGoogle ScholarPubMed
Perelle, S, Dilasser, F, Grout, J & Fach, P 2007 Screening food raw material for the presence of the world's frequent clinical cases of shiga toxin-encoding Escherichia coli O26, O103, O111, O145 and O157. International Journal of Food Microbiology 113 284–288CrossRefGoogle ScholarPubMed
Rozen, S & Skaletsky, HJ 2000 Primer3 on the WWW for general users and for biologist programmers. In Bioinformatics Methods and Protocols: Methods in Molecular Biology, pp. 365–386 (Eds Krawetz, S & Misener, S). Totowa, NJ: Humana PressGoogle Scholar
Saiki, RK 1989 The design and optimization of the polymerase chain reaction. In PCR Technology: Principles and Applications for DNA Amplification, pp. 7–22 (Ed. Ehrlich, HA), New York, StocktonCrossRefGoogle Scholar
Stefan, A, Scaramagli, S, Bergami, R, Mazzini, C, Barnanera, M, Perelle, S & Fach, P 2007 Real-time PCR and enzyme-linked fluorescent assay methods for detecting Shiga-toxin-producing Escherichia coli in mincemeat samples. Canadian Journal of Microbiology 53 337–342CrossRefGoogle ScholarPubMed
Versalovic, J & Lupski, R 2002 Molecular detection and genotyping of pathogens: more accurate and rapid answers. Trends in Microbiology 10 S15–S21CrossRefGoogle ScholarPubMed
Villalobo, E & Torres, A 1998 PCR for the detection of Shigella spp. in Mayonnaise. Applied and Environmental Microbiology 64 1242–1245CrossRefGoogle ScholarPubMed
Wang, L, Li, Y & Mustaphai, A 2007 Rapid and simultaneous quantification of Escherichia coli O157:H7, Salmonella, and Shigella in ground beef by multiplex real-time PCR and immunomagnetic separation. Journal Food Protection 70 1366–1372CrossRefGoogle Scholar
Wani, SA, Pandit, F, Samanta, I, Bhat, MA & Buchh, AS 2004 Molecular epidemiology of Shiga toxin – producing Escherichia coli in India. Current Science 87 1345–1353Google Scholar
Werber, D, Fruth, A, Buchholz, U, Prager, R, Kramer, MH, Ammon, A & Tschape, H 2003 Strong association between shiga toxin-producing Escherichia coli O157 and virulence genes stx2 and eae as possible explanation for predominance of serogroup O157 in patients with haemolytic uraemic syndrome. European Journal of Clinical Microbiology and Infectious Diseases 22 726–730CrossRefGoogle ScholarPubMed
Yang, YG, Song, MK, Park, SJ & Kim, SW 2007 Direct detection of Shigella flexneri and Salmonella typhimurium in human feces by real-time PCR. Journal of Microbiology and Biotechnology 17 1616–1621Google ScholarPubMed
Zagrebneviene, G, Jasulaitiene, V, Morkunas, B, Tarbunas, S & Ladygaite, J 2005 Shigella sonnei outbreak due to consumption of unpasteurized milk curds in Vilnius, Lithuania, 2004. Euro Surveillance 2005;10(12):E051201·3. Available from: http://www.eurosurveillance.org/ew/2005/051201.asp#3CrossRefGoogle Scholar