The Screening For Salmonella VBNC Mutants And Its Identification

Viable but non-culturable state of becteria received extensive attention from many fields of natural sciences since it was proposed by Huaishu Xu in1980s.Salmonella is an important food-borne pathogens, and in which VBNC states has acquired Important results, especially on the Induction, detection, pathogenic.But now the problem is, VBNC state of bacteria, the existence of its biologicalnature is not stable, and very easy to disappear in the course of the study, the death phase of bacteria into the real, causing the researchers can not do more research.So this study break the convention, obtained by chemical mutagenesis of a stable genetic trait Salmonella VBNC mutant, using amplified fragment length polymorphism(AFLP) and denaturing high perpormance liquid chromatography (DHPLC) to determine the combination of mutation, the purpose is to obtain a stable genetic trait VBNC bacteria model for comprehensive and in-depth study of VBNC mechanism may occur.In this test the standard strain of pig typhoid Salmonella for the study, application of chemical mutagen nitrosoguanidine (NTG), UV, and its combined NTG and UV-induced mutants, and then put the suspected mutant at low temperature (4℃)and oligotrophic(normal LB medium) to induce VBNC state in laboratory conditions, and VBNC state of mutant was detected by the methods of plae counting and fluorescence microscopy, then resuscitation experiments were demonstrated by returning to suitable conditions, reply at room temperature and adding Tween-20and Tween-80.Biological identification was also taken after that with the AFLP technique and Polyacrylamide gel electrophoresis to verify whether get difference segment, and then by the DHPLC technology to access point mutation.The results shows that:With the NTG, UV, and the combined NTG and UV to induce mutant, when use the ultraviolet light, UV irradiation time in the design of the experimental conditions for10s,20,30s,40,50,60s, found a large number of test strains dead, the death rate almost100%.But obtain the desired effect of the mutation with different doses of NTG, especially when the doses of NTG at100μl, the NTG concentration is1mg/ml. Sterile growth plate of the control group, enter the VBNC state, the mutation group there are still bacteria in the growth. Applications of AFLP and DHPLC found that when the amount of NTG in100μl can obtain obvious characterstics of mutations and found a significant mutation.illustrate the applications of a combination of AFLP and DHPLC methods to determine the mutant is a very effective thechnology.