Duplex PCR for specific detection of Escherichia coli and its differentiation from other Enterobacteriaceae

SUSWETA DAS MITRA; WILFRED ANTHONY DAS; RITUPARNA TEWARI; NIMITA VENUGOPAL C; BHUVANA MANI; KRITHIGA NATESAN; BIBEK RANJAN SHOME; HABIBUR RAHMAN

doi:10.56093/ijans.v85i8.50931

Authors

SUSWETA DAS MITRA Research Associate, ICAR-National Institute of Veterinary Epidemiology and Disease Informatics (NIVEDI), Ramagondanahalli, Yelahanka, Bengaluru, Karnataka 560 064 India
WILFRED ANTHONY DAS Senior Research Fellow, ICAR-National Institute of Veterinary Epidemiology and Disease Informatics (NIVEDI), Ramagondanahalli, Yelahanka, Bengaluru, Karnataka 560 064 India
RITUPARNA TEWARI Junior Research Fellow, ICAR-National Institute of Veterinary Epidemiology and Disease Informatics (NIVEDI), Ramagondanahalli, Yelahanka, Bengaluru, Karnataka 560 064 India
NIMITA VENUGOPAL C Young Technical Professional, ICAR-National Institute of Veterinary Epidemiology and Disease Informatics (NIVEDI), Ramagondanahalli, Yelahanka, Bengaluru, Karnataka 560 064 India
BHUVANA MANI Senior Research Fellow, ICAR-National Institute of Veterinary Epidemiology and Disease Informatics (NIVEDI), Ramagondanahalli, Yelahanka, Bengaluru, Karnataka 560 064 India
KRITHIGA NATESAN Senior Research Fellow, ICAR-National Institute of Veterinary Epidemiology and Disease Informatics (NIVEDI), Ramagondanahalli, Yelahanka, Bengaluru, Karnataka 560 064 India
BIBEK RANJAN SHOME Principal Scientist, ICAR-National Institute of Veterinary Epidemiology and Disease Informatics (NIVEDI), Ramagondanahalli, Yelahanka, Bengaluru, Karnataka 560 064 India
HABIBUR RAHMAN Director, ICAR-National Institute of Veterinary Epidemiology and Disease Informatics (NIVEDI), Ramagondanahalli, Yelahanka, Bengaluru, Karnataka 560 064 India

https://doi.org/10.56093/ijans.v85i8.50931

Keywords:

Duplex polymerase chain reaction, Enterobacteriaceae, Escherichia coli

Abstract

Escherichia coli is a normal inhabitant of gastrointestinal tract of humans and animals and one of the most important causes of bovine mastitis. Definitive identification of E. coli from other members of Enterobacteriaceae remains ambiguous. The present study describes a duplex PCR, targeting 2 housekeeping genes, the lacy (lactose permease) and phoA (alkaline phosphatase) for the reliable detection of E. coli that enables its differentiation from biochemically and phylogenetically related bacteria. The assay was evaluated employing 19 ATCC (American type culture collection) reference strains of Enterobacteriaceae family. Validation of the assay with E. coli (154) isolated from milk and faeces rendered the assay to be specific. The results suggest that the technique can be used for accurate detection of E. coli and thus can be adapted for testing bacteriological safety of milk, for field applications, and in laboratories handling clinical samples. This PCR (polymerase chain reaction) can successfully distinguish E. coli including E. coli O157 from Shigella spp and other related enterobacteria, emphasizing its relevance and utility in studies related to E. coli infection.

Downloads

Download data is not yet available.

References

Chakravorty S, Helb B D, Connell M and Alland N. 2007. A detailed analysis of 16S ribosomal RNA gene segments for the diagnosis of pathogenic bacteria. Journal of Microbiological Methods 69: 330–39. DOI: https://doi.org/10.1016/j.mimet.2007.02.005

Gautam S K, Kumar S S, Batish V K, Grover S and Mohanty A K. 2012. Rapid and sensitive detection of Escherichia coli in milk by 16S rRNA gene targeted PCR. Indian Journal of Animal Sciences 82: 204–08.

Griesbeck-Zilch B, Meyer H H D, Schwerin C H K A and Wellnitz O. 2008. Staphylococcus aureus and Escherichia coli cause deviating expression profiles of cytokines and lactoferrin messenger ribonucleic acid in mammary epithelial cells. Journal of Dairy Science 91: 2215–24. DOI: https://doi.org/10.3168/jds.2007-0752

Horakova K, Mlejnkova H and Mlejnek P. 2008. Specific detection of Escherichia coli isolated from water samples using polymerase chain reaction targeting four genes: cytochrome bd complex, lactose permease, beta-D-glucuronidase, and beta- D-galactosidase. Journal of Applied Microbiology 105: 970– 76. DOI: https://doi.org/10.1111/j.1365-2672.2008.03838.x

Lan R, Alleles M C, Donohoe K, Martinez M B and Reeves P R. 2004. Molecular evolutionary relationships of entero-invasive Escherichia coli and Shigella spp. Infection and Immunity 72: 5080–88. DOI: https://doi.org/10.1128/IAI.72.9.5080-5088.2004

Maheux A F, Picard F J, Boissinot M, Bissonnenette L, Paradis S and Bergeron M G. 2009. Analytical comparison of nine PCR primer sets designed to detect the presence of Escherichia coli/ Shigella in water samples. Water Research 43: 3019–28. DOI: https://doi.org/10.1016/j.watres.2009.04.017

Ochoa T J, Ruiz J, Molina M, Del Valle L J, Vargas M, Gil A I, Ecker L, Barletta F, Hall E, Cleary T G and Lanata C F. 2009. High frequency of antimicrobial drug resistance of diarrheagenic Escherichia coli in infants in Peru. American Journal of Tropical Medicine and Hygiene 81: 296–301. DOI: https://doi.org/10.4269/ajtmh.2009.81.296

Pavlovic M, Luze A, Konrad R, Berger A, Sing A, Busch U and Huber I. 2011. Development of a duplex real-time PCR for differentiation between E. coli and Shigella spp. Journal of Applied Microbiology 110: 1245–51. DOI: https://doi.org/10.1111/j.1365-2672.2011.04973.x

Rozen S and Skaletsky H J. 2000. Primer3 on the WWW for general users and for biologist programmers. In: Krawetz. S., Misener, S. (Eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, NJ, pp. 365–86. DOI: https://doi.org/10.1385/1-59259-192-2:365

References and further reading may be available for this article. To view references and further reading you must purchase this article.

Sandhya S, Chen W and Mulchandani A. 2008. Molecular beacons: A real-time polymerase chain reaction assay for detecting Escherichia coli from fresh produce and water. Analytical Chimica Acta 614: 208–21. DOI: https://doi.org/10.1016/j.aca.2008.03.026

WHO. 2011. Outbreak of E. coli O104:H4 infection in Germany, France and Sweden http://www.euro.who.int/en/what-we-do/health-topics/emergencies/international-health-regulations/news/news/2011/07/outbreaks-of-e.-coli-o104h4-infection. Accessed on 21 July, 2011.