Improvement of PCR in detection of coliform in water pollution

ABSTRACT

In molecular diagnosis of microbial agent, purification of chromosome is very important step. In this study, after cell destruction, DNA replication was done by increasing the denaturation time, without DNA purification. In this experimental study eight different dilution of E.coli [8/100, 4/100, 2/100, 1/100, 1/200, 1/400, 1/800 and 1/1600] solution were madce in D.W, Bacteria were separated by filtration. Polymerase chain reaction method was used to propagate 162 rRNA gene by design primers without DNA Purification. In order to confirme sensitivity of PCR, contamination of 15 different sources of Arak well water wafer was compared by MPN method. For confirmed sensitivity of PCR, 15 sources of water in Arak were examined and compared with MPN method. Present of bacteria in diution soution were confirmed by culture. Polymerale Chain reaction [PCR] data were shown this method is able to recognize bacteria in above dilutions after filtration. This study showed high sensitivity of PCR method in compare to MPN method. Results were shown without stages of extraction of DNA, PCR were done without losing chromosome. Therefore false negative results were decrease and avoided from difficult phases