Clonging And Analyzing Of MRNA Suppresion Subtractive Hybridization Gene Of VBNC Listeria Monocytogenes

Since 1982,the viable but non-culturable state is reported,more and more species of bacteria can decline culturable cell count less then0.1CFU/L while the total cell count usually remains constant at the initial level under prolonged starvation in a sterile laboratory microcosm.these bacterias can escape from traditional detection,and threaten public safety.Nowaday, the study of microbiology,epidemiology and public health all focus on VBNC bacterias.Although the VBNC state had been reported a lots of years,while the VBNC hypothesis has frequently generated sharp debate.mRNA as a universally acknowledged active substance. It half life period is only 2-3 minuts,very short.But the genetic information within the mRNA can authentically reflect the change of gene level,during baterias enter viable but non-culturable state,and then it reveals physiological mechanism of the state.With the development of science,molecular biotechnology continually suplies new tools and minds for genetic engineering.Up to now, at least 70 species bacteria had been reported to enter VBNC states. Suppression subtractive hybridization defferential display compare to the past detecting technology,it is high sensitive,low false positive and easy operating. We rapidly analysis listeria monocytogenes gene in the mRNA take use of the technology,it layes foundations for researching the formation mechanism and establishing listeria monocytogenes detection method.In our study,we observe the bacteria through plate culture, LIVE/DEAD backlight bacterial viability kit,the result of these researches show that listeria monocytogenes can enter viable but non-cultural state and huddle thallus under acetic acid or hydrochloric acid (PH 2.0),4℃,5 to 7 days.The homology of the two states is higher than 99%,it illustrates that bateria is not pollute.In our study we extract listeria monocytogenes'mRNA through magneticaction,adopt suppression subtractive hybridization method to clone and anlysis the differential display of the two states.Results show that,these 33 differential fragments all belong to VBNC listeria monocytogenes through reverse hybrid.The homology of these two frament is higher than 98% compare to normal bacteria through Blast,it illustrates that the fragments also belong to listeria monocytogenes,the result reliable.