| Tuberculosis is the disease of the most morbidity and mortality caused by a singlefactor at present. One-third of worldwide people infected with Mycobacteriumtuberculosis, and there are an increasing of8million patient and2million patientdied from TB every year. World Health Organization declared that: the global state ofTB is in emergency. Some of reasons for TB pandemic again were irrational drugadministration and appearance of multi-resistance strains. In order to effectivelyimplement DORT(Directly Observed Treatment, Short-Course) strategy, it becomecritical for rapid detecting M.tb and correctly selecting antibiotic.The sensitive response characteristics of piezoelectric quartz crystal sensor toenvironmental electrical parameter were exploited, and be applied to monitor thestates of microbial growth to get the information of microorganism growth timely.The piezoelectric sensor was constructed for the detection of M.smegmatis. The effectof the culture ingredient on the microbial growth information was acquired by seriesmulti-channel piezoelectric quartz crystal sensor. The role of the components inM7H9on the growth curve of M.tuberculosis and M.smegmatis were analyzed byunivariate analysis. The function of the ingredients of M7H9was divided into buffer,anabolic composition and growth promoting factors. The piezoelectric frequency shiftcurves are classified as anabolic curve and catabolic curve. The reasons for anaboliccurve of piezoelectric signal were the adsorption of conductance ingredients in theprocessing of anabolism of M. smegatis in M7H9medium, which causes a frequencyshift upward curve. The reasons for catabolic curve were the secretions ofconductance ingredients in the catabolism of M.smegmatis, which causes a frequencyshift downward curve. The analysis of mechanism of piezoelectric signal will help toprepare the identified medium, and enhance the understanding of piezoelectric signalin theory.The conditions of phage stable in the medium were studied based on theefficiency of plating in the plate to enhance the infecting efficiency of phage D29toM.tuberculosis. To reduce the false results, the best method for the inactivating phageD29was studied. The suitable concentration of calcium and magnesium ions on theeffect of adsorption and abortive infection of phage D29to M.semgmatis were studiedto keep the phage D29active. Then, the biological characteristics of the phage D29 interaction with M.smegmatis were evaluated based on the one step growth curve ofthe phage D29in the M.smegmatis, and the effect of environmental factors on the onestep growth curve were analyzed to select the best medium for the detection of M.tuberculosis. In summary, the conditions of phage D29stable and inactivating inmedium, the factors of phage D29multiplication in M.smegmatis were considered toconstruct the phage-piezoelectric sensor.Accoding to the results of previous chapters, novel detection method for M.tuberculosis was constructed based on the phage amplified multichannel seriespiezoelectric crystal sensor. The phage inside M. tuberculosis was protected from theinactivating Ferrous Ammonium Sulfate, and was tranfered to the detection mediumby the carrier M. tuberculosis. Then, the progeny phage D29was released by thecreaking host cell M. tuberculosis in detection medium. The progeny phage D29undergos a rapid cycle process of infection, replication and lysis in M. smegmstis. Thecycles of phage D29constitute the biological chain amplification reaction, and causethe results of growth completely inhibition of M.smegmatis. The number of phageD29cycles completely inhibit the growth of the host cell is linear to the negativelogarithm of M. tuberculosis in the samples. So, the growth state of M.smegmatis isrelated to concentration of M. tuberculosis in the sample. The conductance ingredientsin M7H9medium are utilized by the growth of M.smegmatis, which cause theconductivity change in the medium. The curve of frequency shift against time isobtained by the response of phage-piezoelectric sensor to conductance of the medium.Therefore, the different curves of phage-piezoelectric signal are caused by thedifferent concentrations of M.tuberculosis in sample.The phage-piezoelectric method possesses the advantage of the rapid, sensitivity,security and economy. The biological chain amplification reaction of phage D29in aliquid medium are given a full play to improve the sensitivity of phage method, andthe sensitive response of a piezoelectric sensor to conductivity also enhances thesensitivity of the phage-piezoelectric method. The turnaround time is greatlyimproved by the transformation of detection of M.tuberculosis into that ofM.smegmatis, by phag D29was acting as a bridge. The risk of infection inexprement can be reduced by the phage lysising the M.tuberuclosis. The requirementsof the medium for the M.smegmatis culture is relatively low and the instrument for thedetection is cost-effective.The detection limit of the proposed method is102cfu/mL of M.tuberculosis, andthe turnaround time is30h with practical value in clinical diagnosis. The mathmatical model was established to analyze the relationship among the turnaround time,M.tuberculosis and M.smegmatis, and obtained that there is a linear relationshipbetween the turnaround time and concentration of M.smegmatis at range of1-102pfu/mL. The effects of sample processing methods on the results are studied bydouble blind principle, so we can get the accurate results to avoid the false negativeand false positive. The comparasion with the statistic method indicates that there is nosignificant difference between the results of PA-MSPQC and MGIT. The sensitivityand specificity of the two methods are consistant with each other. However, theturnaround time of PA-MSPQC method is less than30h which is superior to110h ofMGIT.The detection of multi-resistant strains and rational administration are verycritical for the prevention of T.B epidemic. Considering the advantages ofphage-piezoelectic method and the characteristics of phage D29, it is ideal for thedrug susceptibility testing. Due to it only infects viable, not infects dead bacterium,novel method for rapid drug susceptibility testing of M. tuberculosis is constituted bycombining the phage-piezoelectric method with the drug treatment technology. Itsbasic idea is that the clinical isolates treated with drugs at a critical concentrationprescribled by CLSI, and the resistant strains can survive from the killing of drug andkeep alive, and the sensitive strains be inactivated. The resistant strains can be rapidlydetected by the previous mentioned method. The principle of the phage-piezoelectricmethod for the detection of resistant strains of M. tuberculosis is divided into5steps:First, the suspension of M. tuberculosis(105cfu/mL) were exposed to the environmentin the presence of the anti-mycobacterial agents for48h (a，a’). The resistant strainscan survive from the antibiotic killing and keep alive. Second, the viableM.tuberculosis can be infected by thephage D29, thus causing that phage D29isprotected from inactivation by FAS (b). Third, sample solution is transferred todetection medium after10-fold dilution to eliminate the role of FAS (c), and phageD29protected within viable resistant M.tuberculosis replicates and ultimately lysistheir host cells. Forth, the released phage infected the rapidly growing M.smegmatishost in which they undergo a rapid cycle process of infection, replication and lysis (d);Finally, the phage-piezoelectric signal of resistant strains was obtained because alarge number of phage D29was proliferated, and M.smegmatis growth was inhibited(e). On the contrary, the sensitive strains of M.tuberculosis are killed by antibiotic.Neither phage survived (b’), nor phage is transferred to the detection medium (c’). Nophage is amplified (d’), and therefore no phage-piezoelectric signal of sensitive isolates is detected (e’). The detection of resistant M.tuberculosis was transferred intothe detection of M.smegmatis by the resistant M. tuberculosis as carrier of phage D29.The turnaround time of drug susceptibility testing was sharply shortened by thePA-MSPQC method.To establish the rapid sensening detection method for microorganism, novelmethod was constructed based on the new type graphene and aptamers. The large-areaof high quality and uniform graphene films are synthsised on the copper foil by thechemistry vapor deposition. Then, the conditions of different gases on the quality ofgraphene were investigated by Raman spectroscopy, such as the effect of gas flow rate,temperature and substrate material on the quality. The large area single graphene wasprepared. The conditions of adsorption and dissociation of a nucleic acid sequence onthe surface of graphene are deterimined that: the nucleic acid sequences adsorbed onthe graphene surface by the π-π stacking in the presence of100mmol/L calcium ion,5mmol/L magnesium, which results in the increasing of electron transfer resistance onthe graphene surface; the nucleic acid sequence are dissociated from the graphenesurface causing by the complementary senquence which results in the reducing ofelectron transfer resistance on the graphene surface. The results of fitting analysis ofelectron transfer resistance on the surface are obtained that the changes of electrontransfer resistnace is linear to the concentration of complementary DNA. The natureof aptamer is similar to DNA senquence. So, the changes of aptamer of adsorption anddissociation on the graphene electrode offer the possibility for the rapid sensingdetection of microorganism. |
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