In comparison to the traditional biochemical tests for identification of bacteria, ribotyping is a high throughput method that enables rapid identification of microorganism. Ribotyping involves isolation of genomic DNA, amplification of 16S rRNA and finally sequencing of the variable region to establish the identity of the organisms. In this project bead beating method for the extraction of DNA is used and the products directly used for PCR amplification without any purification step. One set of universal primer is used for the amplification. The product of amplification was visualized in 0.8% agarose gel. Amplicons obtained were 1500bp. Initially the optimization of the DNA etraction and PCR reaction were carried out employing standard strains of Escherichia coli and Bacillus subtilis. Subsequently effluents were collected asceptically from and the organisms were isolated. Morphologically distinct organisms selected and subjected for ribotyping. During the project bead beating method and PCR conditions were standardized.
Amplification of DNA of Bead beater optimised samples by
PCR with amplicon size 1500bp where M- 1kb DNA ladder
C- Control DNA , lane 1 – IS 1.4, 2 –IS 1.5