Establishment And Optimum Allocation Of The Test Methods Based On Enterobacteria

The viable but non-culturable state of becteria is the important achievement of Microbiology、Epidemiology and Public health.And the research on viable but non-culturable state already could be distinguish with the state like death state, starvation, and the "L"model.We had hold further realization on the changes of the modality, physiological funcitions, and gene, even now it could be find some study which research the mutant of viable but non-culturable state, and these mutants in culturable state, they think the mutant as the research model to study the bacteria of VBNC.As we know, the tests of induce are not a unique methods, but we can hold the key of the thread, the wredched environment stress is a important facter, we can change the condition of bacteria, so the bacteria must be acclimation.As well, the phenotype would different as the condition is different.Not all bacteria which in the state of viable but non-culturable resuscitation could be success in the condition of laboratory, but we could believe that the mechanism of resuscitation is has a closely correlation with the mechanism of VBNC.It reported that many pathogenic bacterium have the ability into the state of viable but non-culturable, and these bacteria will be the potential etiology and infection sources. What is important, the bacteria which in this state could be anabiosis and multiply in some condition, we can know it must be threaten to human health. But the routine test facility is powerless for these bacteria.So far, the test facility already development vastly, but due to the principle differently, it is very necessary and indispensable to set up an efficient VBNC becteria testing method with more sencitivity and specificity.We all know that mRNA always in a instability state when it within the organism,the half life period is only2-3min, but it can reflect the change of gene level in bacteria,in early study mRNA is one of the standard to judgment the VBNC state of bacteria.In this test:Escherichia coli and Salmonella are we study objection, they induced in hypothermia solution and the solution which has a low-pH with low-temperature, we can take the result that they can get the state of viable but non-culturable, and before the bacteria induced, put them in4℃for cultivate, the long time they sustain, the faster they can be induced in low-pH solution, and keep the VBNC state more longer. Maybe the add the pressure of environment gradually can help it VBNC state keep stealy. VBNC state was detected by LIVE/DEAD kit staining,and take the16SrRNA method to appraisal the inductiong strain, to guarantee that the experimental strain has not been polluted.This test accorded to the data of previous documents, designed the primers, then synthetic the primer.Then set up the PCR and fluorescence quantitative PCR methods.And take the Klebsiella pneumoniae, Enterobacter cloacae, Proteus mirabilis, Serratieae as the matched control, use the optimum allocation of the test methods after some repeated trial.The result show that:the isotype and homologous strain bacteria in the same induced condition may be have different results, Salmonella could be induced in two conditons Escherichia coli could be induced successfully in water contain the glacial acetic acid which the pH=2.0,4℃aerobic culture.We established the RT-PCR and RT-qPCR test methods of Salmonella and Escherichia coli successfully, and result showed that those methods showed favourable specificity, sensitivity and repetitiveness.