detection of Shigella dysenteriae type 1 in Milk by Pcr

Shigellosis is the 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 timeconsuming 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, simple method of sample preparation that facilitates PCR based detection of Shigella dysenteriae in milk is defined. In present study, gene specific primers were designed to detect stxA gene. The genomic DNA extraction from the spiked milk was carried out using proteinase K, lysozyme, chloroform-isoamyl alcohol and heat treatments. For detection 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 4 h.