Evaluation of multiplex SYBR green real-time PCR assay for detection of pathogenic Escherichia coli

 
 
 
  • Abstract
  • Keywords
  • References
  • Untitled
  • PDF
  • Abstract


    Pathogenic Escherichia coli (E. coli) has been implicated in a wide range of disease causing infections. It is essential to generate a method for detecting and differentiating each pathotype of E. coli which is more quickly and efficiently by using less reagent. This study aimed to evaluate a SYBR Green multiplex real-time PCR method for detecting four types of pathogenic E. coli. Two of multiplex real-time PCR system, 6-plex and 3-plex, were set to detect six different virulence factors from ETEC, EPEC, EHEC, and EIEC and evaluate the melting curves and specificity compared to simplex method. The results showed that 3-plex rt-PCR method gave more reliable melting curves than 6-plex. The 3-plex rt-PCR also provided similar melting value (Tm) to simplex system. The results of this specificity assay supported the selection of 3-plex rt-PCR conditions for detection of pathogenic E. coli.


  • Keywords


    Melting Curve; Multiplex Rt-PCR; Pathogenic E. coli; Specificity; SYBR Green.

  • References


      [1] N.A. Yunita, Identification and hazard characterization of pathogenic bacteria in food consumed by school children in Bogor, Master Thesis, IPB University Indonesia (2015)

      [2] R. Anggreini, Contamination analysis of Escherichia coli (E. coli) O157:H7 in beef Makassar city, Undergraduate thesis, Hasanuddin University Indonesia (2015)

      [3] N. Nababan, W.P. Rahayu, D.E. Waturangi, S. Suratmono, R. Puspitasari, N. Indrotristianto, E. Nikastri, S. Yulianingsih, N. Pusparini, Critical points and the presence of pathogenic bacteria in iced beverage processing line, Journal of Infection in Developing Countries 11 (2007) 2017 493-500. https://doi.org/10.3855/jidc.8934.

      [4] FDA. Food and Drug Administration. Bad Bug Book, Foodborne Pathogenic Microorganisms and Natural Toxins, 2nd ed. Silver Spring: FDA (2012).

      [5] T. B. Souza, M. L. Diego, M. S. K. Sonia, C. P. Liliana, P. S. Neusa, C. A. S. Isabel, Real time multiplex assay and melting curve analysis for identifying diarrheagenic Escherichia coli, Journal of Clinical Microbiology 51 (2013) 1031-1033. https://doi.org/10.1128/JCM.02478-12.

      [6] A. Apte, S. Daniel, PCR Primer: A laboratory Manual. 2nd ed. Cold Spring Harbor. USA (2003).

      [7] L. Reyes-Escogido, M. Balam-Chi, I. Rodriguez-Buenfil, J. Valdes, L. Kameyama, F. Martinez-Perez, Purification of bacterial genomic DNA in less than 20 min using chelex-100 microwave: examples from strains of lactic acid bacteria isolated from soil samples, Antonie van Leewenhoek 98 (2010) 456-474. https://doi.org/10.1007/s10482-010-9462-0.

      [8] E. Pestana, S. Belak, A. Diallo, J.R. Crowther, G.J. Viljoen, Early, rapid and sensitive veterinary molecular daignostics real-time PCR application, Springer Netherlands, Netherland, 2010: pp. 252-253. https://doi.org/10.1007/978-90-481-3132-7.

      [9] M.A. Croxen, B.B. Finlay, Molecular mechanism of Escherichia coli pathogenicity. http://www.natute.com/reviw/micro. Accessed January. 2015.

      [10] M.R. Branquinho, R.T.B. Ferreira, P. Cardarelli-Leite, use of real-time PCR to evaluate two DNA extraction methods from food, Ciencia e Tecnologia de Alimentos Campinas 32 (2012) 112-118. https://doi.org/10.1590/S0101-20612012005000012.

      [11] Bio-Rad Laboratories. Chelex 100 and chelex 20 chelating ion exchange resin instruction manual. http://www.biorad.com/webmaster/pdfs/9184_chelelx.PDF. Accessed Maret 2017.

      [12] L.D. Moja, Artifacts in QPCR investigating unspecific product, master’s degree Project, University of Skovde, Sweden (2014).

      [13] IDT. Integrated DNA Technologies. Performing intercalating dye PCR/qPCR assays: Review examples of PCR melt curve data with our scientists to determine what it can/cannot tell us about resulting PCR amplicons. https://sg.idtdna.com. Accessed April 2, 2019.

      [14] M.K. Gunnel, C.D. Lovelace, B.A. Satterfield, E.A. Moore, L.O. Kim, R.A.A. Robison, Multiplex real-time PCR assay for the detection and differentiation of Francisella tularensis subspecies, Journal of Medical Microbiology 61 (2012) 1525-1531. https://doi.org/10.1099/jmm.0.046631-0.

      [15] Z. Chai, W. Ma, F. Fu, Y. Lang, W. Wang, G. Tong, Q. Liu, X. Cai, X. Li, A SYBR green-based real-time rt-PCR assay for simple and rapid detection and differentiation of highly pathogenic and classical type 2 porcine reproductive and respiratory syndrome virus circulating in China, Archives Virology 158 (2013) 407-415. https://doi.org/10.1007/s00705-012-1504-7.

      [16] L. Chassagne, P. Nathalie, R. Frederic, L. Valerie, B. Richard, D. Julien, Detection of stx1, stx2, and eae genes of enterohemorrhagic Escherichia coli using SYBR green in a real-time polymerase chain reaction, Diagnostic Microbiology, and Infectious Disease 64 (2009) 98-101. https://doi.org/10.1016/j.diagmicrobio.2009.01.031.

      [17] W. Zhang, M. Bielaszewska, A. Bauwens, A. Fruth, A. Mellmann, H. Karch, Real-time multiplex PCR for detecting shiga toxin 2-producing Escherichia coli O104:H4 in human stolls, Journal of Clinical Microbiology 50 (2012) 1752-1754. https://doi.org/10.1128/JCM.06817-11.

      [18] K.H. Kim, J.I. Cho, C.Y. Cheung, J.M. Lim, S. Cho, D.H. Cho, C. S. Kang, D.H. Kim, Development of multipleks PCR assays to identify Escherichia coli pathogenic genes in food, Journal of Food Science and Biotechnology 19 (2010) 1205-1210. https://doi.org/10.1007/s10068-010-0172-1.

      [19] A. Kagambega, O. Martikainen, T. Lienemann, A. Siitonen, A.S Traore, N. Barro, K. Haukka, Diarrheagenic Escherichia coli detected by 16-plex PCR in raw meat and beaf intestines sold at local markets in Ougadougou, Burkina Faso, International Journal of Food Microbiology 153 (2012) 154-158. https://doi.org/10.1016/j.ijfoodmicro.2011.10.032.

      [20] M.A.M. Mohammed, Molecular characterization of diarrheagenic Escherichia coli isolated from meat products sold at Mansoura city, Egypt, Food Control 25 (2012) 159-164. https://doi.org/10.1016/j.foodcont.2011.10.026.

      [21] J. Tobias, S.R. Vutukuru, Simple and rapid multilex PCR for identification of the main human diarrheagenic Escherichia coli, Microbiological Research 176 (2012) 564-570. https://doi.org/10.1016/j.micres.2011.11.006.

      [22] A. Sjoling, L. Sadeghipoorjahromi, D. Novak, J. Tobias, Detection of major diarrheagenic bacterial pathogens by multiplex PCR panel. Microbiological Research 172 (2014) 34-40. https://doi.org/10.1016/j.micres.2014.12.003.

      [23] B. C. Satterfield, Cooperative primers, 2.5 million-fold improvment in the reduction of nonspecific amplification, Journal of Molecular Diagnostic 16 (2014) 163-173. https://doi.org/10.1016/j.jmoldx.2013.10.004.


 

HTML

View

Download

Article ID: 30389
 
DOI: 10.14419/ijet.v9i2.30389




Copyright © 2012-2015 Science Publishing Corporation Inc. All rights reserved.