Abstract
Recently, foodborne pathogen is a common and distressing disease around world to cause a threat to life and economic damages and it is urgent to develop a tools to diagnosis of such pathogens in the early stage to prevent potential outbreak. Although conventional cell extraction and recovery of DNA from pathogen and PCR method have been widely used, the methods require complex steps, experts, and expensive chemicals and instruments to improve the PCR performance. Herein, we report a droplet-based polymerase chain reaction (ddPCR) system which allows identifying single-cell level of foodborne pathogens. E. coli O157:H7 and Salmonella cells were selected as model bacterial foodborne pathogens. The ddPCR system could be a useful platform for the quantitative detection of foodborne pathogens without any pretreatment process.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Scallan, E. et al. Foodborne illness acquired in the United States-major pathogens. Emerg. Infect. Dis. 17, 7 (2011).
Scallan, E. et al. Foodborne illness acquired in the United States-unspecified agents. Emerg. Infect. Dis. 17, 16 (2011).
Morens, D.M., Folkers, G.K. & Fauci, A.S. The challenge of emerging and re-emerging infectious diseases. Nature 430, 242–249 (2004).
Khan, R. et al. Antimicrobial activity of five herbal extracts against multi drug resistant (MDR) strains of bacteria and fungus of clinical origin. Molecules 14, 586–597 (2009).
Velusamy, V. et al. An overview of foodborne pathogen detection: In the perspective of biosensors. Biotechnol. Adv. 28, 232–254 (2010).
Weinstein, M.P. et al. Controlled evaluation of BacT/Alert standard aerobic and FAN aerobic blood culture bottles for detection of bacteremia and fungemia. J. Clin. Microbiol. 33, 978–981 (1995).
Gebert, S., Siegel, D. & Wellinghausen, N. Rapid detection of pathogens in blood culture bottles by realtime PCR in conjunction with the pre-analytic tool MolYsis. J. Infect. 57, 307–316 (2008).
Rangel, J.M. et al. Epidemiology of Escherichia coli O157:H7 outbreaks, United States, 1982-2002 (2005).
Louie, M., Louie, L. & Simor, A.E. The role of DNA amplification technology in the diagnosis of infectious diseases. Can. Med. Assoc. J. 163, 301–309 (2000).
Zhu, Z. et al. Highly sensitive and quantitative detection of rare pathogens through agarose droplet microfluidic emulsion PCR at the single-cell level. Lab Chip 12, 3907–3913 (2012).
Miller, O.J. et al. Directed evolution by in vitro compartmentalization. Nat. Methods 3, 561–570 (2006).
Hindson, C.M. et al. Absolute quantification by droplet digital PCR versus analog real-time PCR. Nature Methods 10, 1003–1005 (2013).
Huggett, J.F., Cowen, S. & Foy, C.A. Considerations for digital PCR as an accurate molecular diagnostic tool. Clin. Chem. 61, 79–88 (2015).
Cao, L. et al. Advances in digital polymerase chain reaction (ddPCR) and its emerging biomedical applications. Biosens. Bioelectron. 90, 459–474 (2017).
Tsao, S.C.-H. et al. Monitoring response to therapy in melanoma by quantifying circulating tumour DNA with droplet digital PCR for BRAF and NRAS mutations. Sci. Rep. 5, 11198 (2015).
Sedlak, R.H. et al. Identification of chromosomally integrated human herpesvirus 6 by droplet digital PCR. Clin. Chem. 60, 765–772 (2014).
Mashaghi, S., Abbaspourrad, A., Weitz, D.A. & van Oijen, A.M. Droplet microfluidics: A tool for biology, chemistry and nanotechnology. Trends Analyt. Chem. 82, 118–125 (2016).
Cámara, E., Albiol, J. & Ferrer, P. Droplet digital PCRaided screening and characterization of Pichia pastoris multiple gene copy strains. Biotechnol. Bioeng. 113, 1542 (2016).
Rakszewska, A., Tel, J., Chokkalingam, V. & Huck, W.T. One drop at a time: toward droplet microfluidics as a versatile tool for single-cell analysis. NPG Asia Mater. 6, e133 (2014).
Geng, T., Novak, R. & Mathies, R.A. Single-cell forensic short tandem repeat typing within microfluidic droplets. Anal. Chem. 86, 703–712 (2013).
Chokkalingam, V. et al. Probing cellular heterogeneity in cytokine-secreting immune cells using droplet-based microfluidics. Lab Chip 13, 4740–4744 (2013).
Leng, X. et al. Agarose droplet microfluidics for highly parallel and efficient single molecule emulsion PCR. Lab Chip 10, 2841–2843 (2010).
Geng, T. & Mathies, R.A. Minimizing inhibition of PCR-STR typing using digital agarose droplet microfluidics. Forensic Science International: Genetics 14, 203–209 (2015).
Park, K.J. et al. Micropillar arrays enabling single microbial cell encapsulation in hydrogels. Lab Chip 14, 1873–1879 (2014).
Author information
Authors and Affiliations
Corresponding authors
Rights and permissions
About this article
Cite this article
Jang, M., Jeong, S.W., Bae, N.H. et al. Droplet-based digital PCR system for detection of single-cell level of foodborne pathogens. BioChip J 11, 329–337 (2017). https://doi.org/10.1007/s13206-017-1410-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s13206-017-1410-x