【作者】 米贤文；

【导师】 何凤姣；

【作者基本信息】 湖南大学 ， 分析化学， 2013， 博士

【摘要】 结核病是目前单因素引起发病率和死亡率最高的疾病。全球有三分之一的人群感染结核分枝杆菌，每年新增800万结核病人，有200万人死于结核病。世界卫生组织于1993年宣布：全球处于结核病紧急状态。引起结核病再次大流行的原因之一是不合理用药导致的耐药菌株的出现。为有效执行DOTS(Directly ObservedTreatment, Short-Course)策略，快速检测结核分枝杆菌和正确选择抗生素治疗成为防治结核病的关键。利用串联式压电石英传感器对环境电参数响应灵敏的特点，将其应用于微生物生长状态监测中，及早得到微生物的生长信息。用串联式压电传感器，分析培养基不同成分对微生物生长压电信号的影响；用单因素分析方法，分析不同成分对耻垢分枝杆菌和结核分枝杆菌生长曲线的影响。将M7H9培养基中成分的作用分为缓冲作用、合成代谢作用、生长促进因子作用，构建分枝杆菌压电检测体系。对压电频移曲线进行分类，将压电频移信号产生的物质基础分为分解代谢所分泌电导成分和合成代谢所吸收电导成分两种类型，分别产生向下的频移曲线和向上的频移曲线，提升对串联式压电微生物传感器频移信号的理论认识。用噬菌体成斑率为指标，研究噬菌体在环境中稳定的条件，保证噬菌体的感染效率；研究噬菌体最佳的灭活方法，减少实验过程出现假阳性。分析钙镁浓度对噬菌体吸附、顿挫感染的影响，得出最佳的钙离子浓度、杀毒剂硫酸亚铁胺浓度。用一步生长曲线研究噬菌体D29与耻垢分枝杆菌作用的特性，分析影响噬菌体生物放大作用的外部环境因素，优化出最适合结核分枝杆菌快速检测的培养基配方。综合考虑噬菌体的稳定条件、灭活条件、一步生长曲线的最优条件和耻垢分枝杆菌在M7H9培养基中压电信号的影响因素，选择适合构建噬菌体生物放大的压电传感器检测培养体系。结核分枝杆菌和耻垢分枝杆菌能被噬菌体D29感染，保护菌体内的噬菌体D29不被硫酸亚铁杀灭。样品中的结核分枝杆菌被作为载体，将加在样品处理液中的噬菌体D29转移到检测池。在检测池中，噬菌体D29裂解结核分枝杆菌释放子代噬菌体，子代噬菌体感染耻垢分枝杆菌，经历快速的感染、复制、裂解的循环过程。这个循环过程构成噬菌体链式生物放大反应，最终得到完全抑制耻垢分枝杆菌生长的结果。样品中结核分枝杆菌的浓度不同，转移到检测池中的噬菌体D29的也浓度不同，完全抑制耻垢分枝杆菌生长所需的循环次数不同，最终导致检测池中耻垢分枝杆菌生长程度不同。当样品中没有结核分枝杆菌充当噬菌体的载体时，则无噬菌体被转移到检测池中，耻垢分枝杆菌的生长不受噬菌体抑制。耻垢分枝杆菌在生长过程中，利用培养基中的电导成分，引起压电传感器产生频移响应信号。不同浓度的结核分枝杆菌引起不同强度的压电频移响应信号。在液体培养基中，充分发挥噬菌体的链式放大作用，提高检测方法灵敏度。应用压电传感器对电参数的灵敏响应，提高检测方法的灵敏度。通过噬菌体D29桥梁作用，将慢速生长的结核分枝杆菌检测，转换成快速生长的耻垢分枝杆菌的检测，大大提高检测速度。噬菌体对结核分枝杆菌的裂解作用，可降低实验风险，安全可靠。耻垢分枝杆菌检测所需要培养基和仪器要求低，成本低廉。用数学模型分析检出时间与溶液中噬菌体浓度的关系，得出检测时间与溶液中1-102pfu/mL噬菌体D29浓度成线性的结果。用双盲对照的原则研究样本处理对检测方法的影响，得出正确的样本处理方法，保证结果的准确度，避免假阳性和假阴性。该方法可以检测102cfu/mL的结核分枝杆菌，检测时间缩短到30小时，具有临床诊断实用价值。噬菌体放大多通道压电传感器法（PA-MSPQC）与Bactec MGIT960法的统计学比较结果，得出两者检测临床样本的灵敏度和特异度一致。但PA-MSPQC法检测时间只需30小时，优于培养法。PA-MSPQC法还可以通过提高噬菌体感染效率，进一步提高方法的灵敏度。防止耐药结核分枝杆菌的出现是控制结核病的关键措施，实现结核分枝杆菌菌株耐药的快速检测，对合理用药和防止耐药菌株的出现有非常重要的意义。我们根据噬菌体只能感染活的结核分枝杆菌的特点，将PA-MSPQC法应用于结核分枝杆菌耐药检测。建立PA-MSPQC法耐药结果的频移标准，根据实验条件和比例法耐药的判别标准，对利福平、异烟肼和乙胺丁醇三种药物，当耻垢分枝杆菌生长引起的频移小于121Hz时，认为菌株对药物耐受；对链霉素药物，当耻垢分枝杆菌引起的压电频移小于92Hz时，判定菌株对链霉素耐药。将双盲实验中，PA-MSPQC法所得的结果与MGIT960法所得结果进行统计学分析，得出PA-MSPQC法与MGIT960法没有统计学差异，能准确检测临床结核分枝杆菌菌株对利福平、异烟肼、乙胺丁醇和链霉素等一线抗结核药物的耐受情况。由于PA-MSPQC法能快速报告实验结果，可以替代传统的培养法用于临床结核分枝杆菌菌株的耐药实验。但是PA-MSPQC同样存在噬菌体感染效率的问题，有可能将未感染噬菌体的结核分枝杆菌当成耐药菌株，影响方法的准确度。因此，优化培养基成分是提高检测方法灵敏度，提高耐药实验准确度的关键。为建立微生物快速传感检测新方法，我们使用石墨烯新材料和核酸序列代替适配体进行初步实验。用化学气相沉积法在铜箔表面制备大面积石墨烯，用拉曼光谱分析在不同气体条件下形成石墨烯的质量，研究氢气流量、温度、基底材料对石墨烯质量的影响，直接制备出可用于电极分析的大面积单层石墨烯。用电化学阻抗谱和循环伏安法研究核酸序列在石墨烯电表面吸附和解离的条件，得出钠的浓度在100mmol/L、镁的浓度在5mmol/L，核酸序列通过π-π堆集作用而吸附，电极表面电子传递阻力增大；在钙、镁离子的存在条件下，吸附的核酸序列与互补序列作用解离，电极表面电子传递阻力减小；电极表面的电子传递阻力的拟合分析得出，电子传递阻力的改变与cDNA浓度成线性关系。适配体是与核酸序列同性质的DNA序列，石墨烯电极和适配体为目标分析物(如病原微生物)的快速传感检测提供可能。更多还原

【Abstract】 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.更多还原