Detection of Shigella dysenteriae Type 1 in Milk by PCR

  • Pallavi Gupta Biotechnology Division, Defence Research and Development Establishment, Gwalior
  • Ram Kumar Dhaked Biotechnology Division, Defence Research and Development Establishment, Gwalior
Keywords: Shigella dysenteriae, Shiga toxin, detection, spiked milk, PCR

Abstract

Shigellosis is severest form of bacillary dysentery, a disease limited to humans and certain other primates. Conventional microbiological techniques used for the isolation and biochemical identification of microbes are time-consuming as well as labour intensive. Due to the fastidious nature of Shigella and the lengthy culture time, a rapid and sensitive system for the detection of bacteria is highly desirable. Nucleic acid-based techniques have enormous potential in the detection of biological weapons because of their specificity, sensitivity, and the speed with which results can be obtained. The application of polymerase chain reaction in sensitive detection of bacterial pathogens in direct food samples is largely affected by the quality of the template DNA. To overcome the shortcomings of conventional methods, we have defined simple method of sample preparation that facilitates PCR based detection of Shigella dysenteriae in milk. In present study, gene specific primers were designed to detect stxA gene. The genomic DNA extraction from the spiked milk was carried out by proteinase K, lysozyme, chloroform-isoamyl alcohol and heat treatments. The optimized extraction of DNA by addition of chloroform-isoamyl alcohol proved to be the most satisfactory  amongst all, with limit of detection of 2.0 x 103 cfu/ reaction and was completed in 4h.

 

Author Biographies

Pallavi Gupta, Biotechnology Division, Defence Research and Development Establishment, Gwalior

Dr Pallavi Gupta received her PhD (Biotechnology) from Jiwaji University, Gwalior and is presently working as Scientist ‘D’ in Biotechnology at DRDE, Gwalior. She is involved in development of detection systems for bacterial toxin. Development of immunological detection assays i.e., sandwich ELISA, dot ELISA and flow-through assay. Protection studies regarding evaluation of the immuno-modulatory potential of recombinant bacterial toxin.

Ram Kumar Dhaked, Biotechnology Division, Defence Research and Development Establishment, Gwalior

Dr Ram Kumar Dhaked received his PhD (Biochemistry) from Jiwaji University, Gwalior and is presently working as Scientist ‘F’ in Biotechnology at DRDE, Gwalior. He is involved in development of detection systems for bacterial toxin. He also has expertise in post-exposure therapeutics of Botulinum toxin and Shiga toxin.

Published
2017-08-03
How to Cite
Gupta, P., & Dhaked, R. (2017). Detection of Shigella dysenteriae Type 1 in Milk by PCR. Defence Life Science Journal, 2(3), 370-374. https://doi.org/10.14429/dlsj.2.11028