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DNase treated DNA multiplex polymerase chain reaction assay for rapid detection of viable food borne pathogen

Authors:

L. Mahesha N. Sigera Nadugala ,

Asian Institute of Technology, LK
About L. Mahesha N. Sigera

Food Engineering and Bioprocess Technology Programme, Asian Institute of Technology, SERD, P.O. Box 4 Klong Luang,

Pathumthani  12120, Bangkok, Thailand.

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Sudip K. Rakshit

Asian Institute of Technology, TH
About Sudip K.

Food Engineering and Bioprocess Technology Programme, Asian Institute of Technology, SERD, P.O. Box 4 Klong Luang,

Pathumthani  12120, Bangkok, Thailand.
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Abstract

The main objective of this work was to develop methods to overcome the problems associated with rapid detection of food borne pathogens using PCR based techniques. A multiplex PCR method was developed as a solution to the problem of having to test for one organism at a time. DNaseI enzyme treatment followed by PCR [DNase Treated DNA (DTD) PCR] was experimented with to find a solution to the problem of false positive results obtained by amplification of DNA from dead cells. Four sets of primers were used for detection of eaeA, hly, invA and gryB genes of frequently occurring food borne pathogens Escherichia coli O157:H7, Listeria monocytogene, Salmonella enterica and Vibrio parahaemolyticus respectively. Experiments proved that DNaseI has the ability to remove DNA from dead cells without causing any damage to the DNA present inside live cells. DNaseI at a level of 10U/100 μL was found to remove DNA sourced from 5 x 107 dead cells in food systems within one hour of incubation. In the specificity test no interferences or non-specific amplification was observed when the multiplex protocol was tested with 89 strains of bacteria. The method developed was found to be sensitive to a minimum cell count of 102 cells in both pure cultures and in artificially spiked food systems. There was no interference or inhibitory actions when the protocol was applied to shrimps. Thus, this DTD multiplex PCR assay can be practically applied for simultaneous identification of viable cells of four important pathogens namely E. coli O157:H7, S. enterica, L. monocytogenes and V. parahaemolyticus.

 

Keywords: detection, DNaseI, DTD multiplex PCR, food borne pathogens.

doi :10.4038/jnsfsr.v35i4.1310

J.Nat. Sci.Foundation Sri Lanka 2007 35(4) 225-233

 

How to Cite: Nadugala, L.M.N.S. and Rakshit, S.K., 2007. DNase treated DNA multiplex polymerase chain reaction assay for rapid detection of viable food borne pathogen. Journal of the National Science Foundation of Sri Lanka, 35(4), pp.225–233. DOI: http://doi.org/10.4038/jnsfsr.v35i4.1310
Published on 28 Dec 2007.
Peer Reviewed

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