Abstract:

PCR-based molecular biotechnologies are ineffective in discriminating dead cells from viable ones,which may result in inaccurate estimation of cell number.In order to solve this problem,propidium monoazide（PMA） treatment was used as a pretreatment for the genome extraction of environmental samples,in which PMA covalently crosslinks with DNA molecules in dead cells and inhibits the PCR amplification of DNA molecules.The results indicate that the PCR amplification of DNA from heat-killed E.coli cells can be inhibited by PMA with a mass concentration of 3μg/mL after an exposure for more than 3min,while the PCR amplification of DNA from viable cells is slightly inhibited by PMA with a mass concentration of more than 50μg/mL,and that PMA treatment is effective when the turbidity is less than 10 NTU and is ineffective when the turbidity is greater than 100NTU.

Key words: propidium monoazide, deoxyribonucleic acid, polymerase chain reaction, viable cell detection