Model Study On The Method Based On T4 Amplification For Detection Of E. Coil

Detection of bacteria based on bacteriophage technology is a modern inspection method. According to the particular technology being used, this method could be subdivided into phage amplification technology, phage luminescence technology and lysis product ananlysis. Phage amplification technology transforms each target bacterium to a phage plaque and enumerates the bacteria by plaque counting. Phage luminescence technology transforms the target bacteria to luminescent ones and enumerates them directly under epi-fluorescence microscope, or quantifies the target bacteria indirectly by measuring the increased luminescence density. Poduct ananlysis method quantitates the target bacteria by measuring the specific product released from the lysed target bacteria. They are fast, specific, sensitive and economic methods for detection bacteria. Using these methods, but not the most economic first one, for detecting E. coli had been reported.In this model study, with T4 and three E. coli strains being as the model bacteriophage and host bacteria, a method combining phage amplification and real time PCR together were investigated initially to detect bacteria. The study contained five parts. The first subject was to search for the suitable T4 concentration to infect the E. coli of T4 host type in simulate samples quickly and completely. The second subject was to develop a washing skill based on supercentrifigation and millipore filtration to clear away the free phages and other impurities in the samples. The third subject was to find the suitable method for transforming the infected E. colis trapped in millipore filters to progeny T4s and collecting them to form derivative samples. The fourth subject was searching for the quantitive relationship between the number of the alive E. coli of T4 host sype in the simulte samples and progeny T4 number in the derivative samples. The fifth subject was to set up a method such as plaque counting , PCR or real time PCR to quantitate T4s in the derivative samples. Finally, the advancements and the problems in the model study were pointed out.The results of the first part study shown that 109-108PFU/mL of T4 was necessary to infect the E. coli of T4 host type in the liquid simulate sample efficiently and completely within 5 min. The best titer of T4 was 5×108PFU/mL. Too low of T4 concentration caused some E. coli being not infected and too high of T4 concentration increased the burden of washing away free T4 from the sample in the next step of the experiment. When the infection step completed, the washing step should begin immediately to avoid multiplicity of infection.The results of the second part study showed that, for the simulate sample containing only E. coli and media, LB or TS broth was the best washing liquid to clear away free T4s and other impureities from the simulate sample by supercentrifugation and millipore filtration. For the real sample, the E. coli selective liquid medium should be used as the washing liquid to inhibit other microorganisms growing. The washing procedure included two times of cetrifugation-resuspension operations, which separated away large portion of free T4 from the sample firstly. Then, the last suspension including the infected E. coli was transformed to a syringe filter of polysulfone or methyl cellulose with 0.2-0.22μm bore. Next, continuously used 90 mL LB washing through the filter to clean away the remaind free T4 in the sample untile the T4 titer of the filtrate came to 102-103PFU/mL or less, and to trap the infected E. coli in the filter. The procedure of the washing had completed whitin 30min. So that no progeny T4 were lost in the procedure.The results of the third part study indicated that the infected E. coli had better to be kept and incubated in the filter directly for bursting. After washing step, the filter was sealed up by the syringe and the needle cap to provent the sample being contaminated and incubated at 37℃. Most infected E. coli had bursted after 50min. At 90 min of the incubation, certainly quantitative SM buffer was used to elute the progeny T4 out through the filter and this filtrate was collected as derivative sample. The sample was very suitable for quantifying the progeny T4 because no cells and impurities were in it, the solvent of it was SM buffer, and only the concentrations of progeny T4 and soluble residure of lysed E. coli were unknown.According to the above research results and the methods to quantitate the T4 in the derivative sample, the integrated method of sample transformation had been designed carefuly. When T4 would be quantitated by plaque counting method, the sample transformation was done step by step as follows. Add 100μL T4 work solution, its titer of T4 is equal to or little more than 4×109PFU/mL, to 0.9mL liquid simulate sample to form 1mL starting sample.Incubate the sample at 37℃with slightly shaking. Centrifugate the mixture with 12000rpm for 2min and discard the supernatant. Resuspense the sediment in 1mL LB broth. Repeat the centrifugation and resuspension steps twice. Transfer the last resuspension into a syringe filter of polysulfone or methyl cellulose with 0.2-0.22μm bore. Wash the the filter with 10mL LB broth and discard the filtrate. Repeat the washing step 9 times. Seal the filter with the syringe and the needle cap and incubate them at 37℃for 90min. Elute the progeny T4 out through the filter with 10mL SM buffer and collect the filtrate as derivative sample. When the T4 would be measured by PCR or real time PCR, the sample transformation was done by the same method just mentioned above except one change, that was using 1mL instead of 10mL SM buffer to elute the progeny T4 out through the filter to get the derivative sample. The results of the fourth part study showed us the follows.With Escherichia coli B,Escherichia coli K 12 or Escherichia coli AMC 198 bing as representative of E. coli of T4 host type respectively, the logarithm of the number of this kind E. colies in the simulate sample was linear correlation with the logarithm of the number of progeny T4s in the derivative sample. A equation of linear regression, which expressed this kind relathionship, had set up by regression analysis of the experimental data. Theoretical analysis indicated that the key factors, which decided the slope rate and the intercept of the equation of linear regression, included the nutrition supply to the infected E. colies during sample transform, the filter and the washing liquid being used, the strain of E. colies being examed, and the operations of the sample transformation.Pseudomonas fluorescens or Citrobacter Froundii, being as representative of any microorganism except T4 host bacteria respectively and appearing in simulate sample, could not produce any progeney T4 during the sample transformation procedure, but might decrease burst size of the infected E. colis by competing nutrituion with the infected E. colis. Supplying enough nutrition to the infected E. colis and using the liquid selective medium of E. coli as the washing liquid in the sample transformation procedure could eliminate the interference of these microorganisms completely.When the sample transformation procedure uses E. coli selective medium as washing liquid, always uses the same kind of millipor filter,supplys enough nutrition to the infected E. colies, and always keeps the transformation operations constant, the equation of linear regression will only depend on the kind of the E. coli strain being tested. Therefor, using the E. coli collected from one kind of sample as the testing strains to do the simulative experiments, the equation of linear regression set up by regression analysis of the data getting in the experiments, can be used for quantitative detecting the alive E. coli of T4 host type in other samples belong to the same kind.The results of the fifth part study were as follows.Plaque counting for quantitating T4 in the derivative sample was a reliable method, The combination of the sample transformation method and plaque counting method together made a simple and practicable method for detection of E. coli of T4 host type. The detection limit of it was about 20CFU/mL. The whole process of it needed 8-9h to be completed.PCR could be used for roughly quantitating T4 in the derivative sample. A pair of primers correlating with the lignase gene of T4 was better than others in the aspects of sensitivity and speciality. When the primers being used, the optimized annealing temperature was 58℃. Boiling method was satisfactory for preparing the template sample. PCR analysis, with the optimized reaction conditions and the template samples made by boiling the derivative samples, could distinguish roughly the T4 concentrations in deferent derivative samples. The detecting limit of the method was 500PFU/mL.Real time PCR could be a fast and precise method for quantitating T4 in the derivative sample. Using Light Cycler Fast Start DNA Master plus SYBR Green I had much higher amplification efficency than using common real time PCR reagents. The optimized reaction condition was as follows. The first pair of primers (Lig F and Lig R) and the master regents were used. The concentration of each primer was 0.5mM. The voluem of the template sample was 5μL. The total volum of reaction solution was 20μL. The annealing temperature and time were 61℃and 5sec. The elongating temperature and time were 72℃and 10sec. The denaturing temperature and time of the first cycle were 95℃and 5min. And the denaturing temperature and time of other cycles were 95℃and 8sec. When using the serial template samples made by diluting a purified T4 DNA stock and using the optimized reaction conditions, real time PCR amplification curves were lined up with equal interval, the different Cts being separated evenly and clearly, and the melting curves all peaked at about 82℃which was the melting point of the specific amplification product. When using the serial template samples made from the serial derivative samples by 7 min boiling and using the optimized reaction conditions, real time PCR amplification curves were lined up one by one, the serial Cts increased in proper order, and the detection limit was less than 35PFU/mL. Therefor, real time PCR was a fast, sensitive and precise method for measuring progeny T4 in the derivative simple. Combining the sample transforming method, boiling method for preparing template and real time PCR for quantifing progeny T4 together, a fast method for detection of E. coli of T4 host type had been set up initially. The whole procedure of it could be finished in about 3.5h.To sum up, the study had found a fast method for detecting E. coli of T4 host type. It transforms the E. colies quantitativly to progeney T4 within 2h. It quantitates the number of progeny T4 in the next 1h by real time PCR. Then the concentration of the alive E. coli of T4 host type in the sample can be calculated out immediately. This method can not be used for detecting the E. coli of non-T4 host type so that another bacteriophage, which can infect all kind of E. coli, should be used instead of T4 to solve the problem. Although the defect exists, it is predicted that the method, as a modle of young technology with wide utilizing space, has good perspection of development and utilization.