摘要
Objective:To analyse molecular detection of coliforms and shorten the time of PCR.Methods:Rapid detection of coliforms by amplification of lacZ and uidA genes in a multiplex PCR reaction was designed and performed in comparison with most probably number(MPN)method for 16 artificial and 101 field samples.The molecular method was also conducted on isolated coliforms from positive MPN samples;standard sample for verification of microbial method certificated reference material;isolated strains from certificated reference material and standard bacteria.The PCR and electrophoresis parameters were changed for reducing the operation time.Results:Results of PCR for lacZ and uidA genes were similar in all of standard,operational and artificial samples and showed the 876 bp and 147 bp bands of lacZ and uidA genes by multiplex PCR.PCR results were confirmed by MPN culture method by sensitivity 86%(95%CI:0.71-0.93).Also the total execution time,with a successful change of factors,was reduced to less than two and a half hour.Conclusions:Multiplex PCR method with shortened operation time was used for the simultaneous detection of total coliforms and Escherichia coli in distribution system of Arak city.It's recommended to be used at least as an initial screening test,and then the positive samples could be randomly tested by MPN.
Objective:To analyse molecular detection of coliforms and shorten the time of PCR.Methods:Rapid detection of coliforms by amplification of lacZ and uidA genes in a multiplex PCR reaction was designed and performed in comparison with most probably number(MPN)method for 16 artificial and 101 field samples.The molecular method was also conducted on isolated coliforms from positive MPN samples;standard sample for verification of microbial method certificated reference material;isolated strains from certificated reference material and standard bacteria.The PCR and electrophoresis parameters were changed for reducing the operation time.Results:Results of PCR for lacZ and uidA genes were similar in all of standard,operational and artificial samples and showed the 876 bp and 147 bp bands of lacZ and uidA genes by multiplex PCR.PCR results were confirmed by MPN culture method by sensitivity 86%(95%CI:0.71-0.93).Also the total execution time,with a successful change of factors,was reduced to less than two and a half hour.Conclusions:Multiplex PCR method with shortened operation time was used for the simultaneous detection of total coliforms and Escherichia coli in distribution system of Arak city.It’s recommended to be used at least as an initial screening test,and then the positive samples could be randomly tested by MPN.
基金
Supported by the Ministry of Power of I.R.Iran(Grant No.201)
