【作者】 陈庆海；

【导师】 府伟灵；

【作者基本信息】 第三军医大学 ， 临床检验诊断学， 2008， 博士

【摘要】 目的:目前对结核分枝杆菌(Bacillus Tuberculosis,TB)的检测,主要靠X线、涂片、培养加药敏、PCR技术等多项传统试验综合分析,方法繁杂、耗时长,敏感度低和特异性差,易导致误诊和漏诊,病人经济负担重。治疗结核的药物种类多、作用原理复杂、长期滥用、临床用药不规范等,造成结核分枝杆菌的耐药基因突变日益复杂,其突变位点范围广,突变形式复杂,从单碱基突变、缺失、插入、错位到多位点、长片段突变等,为准确、全面检测其耐药突变基因造成极大困难,特别是对广泛存在的单碱基突变位点检测,使用传统方法包括传统基因芯片有较大技术难度。其多重耐药问题一直是影响其治疗、传播的主要原因,传统检测方法指标单一,提供的耐药信息量少,因此建立快速、敏感、全面、简便的结核杆菌鉴定及多重耐药检测系统是目前国内外实验诊断学急需解决的问题,也是有效治疗和控制该类致病菌的有效手段。分子信标探针是一种具有很高单碱基突变位点检测能力的核酸探针,但受结构限制,分子信标探针在荧光芯片领域的实际运用目前尚处于空白状态。本研究拟充分利用分子信标探针与靶分子单碱基突变位点检测特异性更强的特性和芯片高通量特点结合,研制构建一种结核杆菌耐药基因多重突变位点分子信标(MB)探针芯片检测技术,可直接对结核杆菌核酸及耐药基因多重靶点进行特异检测,为结核分枝杆菌及耐药基因鉴定、流行病学调查提供新的技术手段。方法:1.根据GenBank找到结核分枝杆菌IS986基因序列,在Primer Premier V5.0软件上设计引物。对反应体系中MgCl2、引物、dNTP浓度以及引物退火温度优化后建立结核杆菌PCR反应扩增体系。通过测序、灵敏性试验以及特异性试验对体系进行综合评估。用beacon designer 2.1软件设计结核分枝杆菌通用分子信标探针。通过对杂交温度、杂交时间以及杂交方式的优化,建立PCR反应中及反应后加入TB分子信标杂交试验。运用凝胶成像分析仪、荧光分光光度计、荧光显微镜等各种途径对MB-PCR杂交产物荧光信号的观测条件进行摸索。进行不同荧光-淬灭分子对标记分子信标的观测。2.通过带单侧延长臂分子信标连接方式和传统生物素(biotin)-亲和素(avidin)连接方式的研究对TB分子信标与芯片固定和杂交方式进行探索。提高分子信标芯片杂交效率优化实验包括:5/寡核苷酸探针浓度;分子信标探针浓度;杂交温度、杂交时间、杂交液配方;点样大小与点样浓度;阴-阳性区分的临界值等。通用TB-MB芯片的临床应用和方法学比较。3.NCBI数据库查找4种结核杆菌耐药基因主要突变区域(多重位点)。针对以上位点设计扩增体系进行扩增体系优化。针对以上位点设计TB分子信标探针及相应单侧延长臂分子信标探针。进行TB耐药突变位点单侧延长臂分子信标探针芯片表面杂交系列实验:杂交温度梯度实验(选择45℃、50℃、55℃、60℃、65℃作杂交温度)。杂交时间梯度实验(分2、4、6、8、10、12、14、16、18h不等时间段杂交)。靶序列浓度梯度杂交实验(分0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09uM等不同浓度杂交)。荧光显微镜的观测。4.针对以上位点进行的相应TB临床耐药分离株PCR扩增。各位点TB临床耐药分离株PCR扩增产物测序。在已摸索出的最佳条件下运用多重MB探针芯片对各位点TB临床耐药分离株PCR产物进行杂交反应并与测序结果对照。主要结果:1.优化后的通用TB-PCR反应体系MgCl2、引物以及dNTP最佳终浓度分别为: 4mmol/l、0.04μmol/l以及0.2 mmol/l;退火温度为55℃。测序结果与GenBank中公布的序列完全一致。体系对其它细菌无交叉反应。检测限度为1拷贝。水浴、PCR仪、恒温摇床的最适杂交温度和适杂时间分别为:55℃,16h;65℃,4h;50℃,7h。荧光显微镜观测MB-PCR杂交阳性产物、MB-PCR杂交阴性产物荧光强度差异明显(p<0.01)。分子信标荧光-淬灭效率较高的有:Cy3-BDH、FAM-DABCLYE;较差的有:Cy3-DABCLYE、FAM–BDH。2.分子信标茎臂生物素-亲和素(biotin-avidin)修饰有相当技术难度。单侧延长臂分子信标采用淬灭分子中间标记,茎结构单侧(淬灭分子一侧)延长臂,能很好的结合在芯片上,且修饰技术难度低、特异性好。oligo探针最适浓度为40μmol/L。三种杂交液的杂交结果比较:0.15%SDS效果好于0.2%SDS,10×SSC效果好于5×SSC。Cy3-BDH探针浓度约15μmol/L、FAM-DABCLYE探针浓度约9μmol/L时杂交反应达到7h以上可检测到较强信号。随着点样直径的缩小,同浓度样本荧光强度不变;随着浓度的倍比减小,点样荧光强度也减小;对较理想区分阴阳性的信号强度:Cy3-BDH浓度约10μmol/L,FAM-DABCLYE浓度约15μmol/L。TB-MB芯片对临床标本总检出率为57.5%(23/40),高于涂片27.5%(11/40)和培养35%(14/40),经统计学处理TB-MB芯片与涂片、培养相比差异有显著性(分别为χ2=7.366,P<0.01;χ2=32.281 , P<0.01)。3.准确定位4种结核杆菌耐药主要突变基因区域:耐链霉素:rpsl(43、88)、rrs;耐异烟肼:katG(315、463);耐乙胺丁醇:embB(285、303、306、330);耐利福平:ropB突变核心区域、扩充的突变区域。针对以上位点设计了扩增体系并成功优化了扩增体系,均顺利扩增出特异性目的片段。针对以上位点成功设计出分子信标探针及相应单侧延长臂分子信标探针。完成了各位点分子信标探针芯片表面杂交系列实验。按rpsl43、rpsl88、rrs、katG315、katG463、embB285、embB303、embB306、embB 330、ropB-MB1-1、ropB-MB1-2、ropB-MB1-3、ropB-MB1-4、ropB-MB2-1、ropB-MB2-2顺序,各位点最佳杂交条件分别是:(杂交温度梯度实验:50℃、50℃、50℃、55℃、65℃、55℃、55℃、45℃、60℃、60℃、50℃、65℃、60℃、55℃、55℃;杂交时间梯度实验:12、14、14、16、12、12、8、14、10、10、10、8、12、10、8h;靶序列浓度梯度杂交实验:0.06、0.05、0.06、0.04、0.05、0.04、0.07、0.05、0.05、0.05、0.06、0.04、0.05、0.04、0.04uM)。荧光显微镜的观测各位点荧光信号阳-阴性区别明显。4.4种TB耐药主要突变基因区域进行的相应临床耐药分离株PCR均顺利扩增。扩增产物测序:耐链霉素rpsL基因43位密码子突变率65%,密码子88为60%,且75%与43codon突变株重叠。Rrs突变率为15%,耐SM突变率多位点重复率较高。耐异烟肼KatG315codon突变率为50%,463位点突变率为37%。耐乙胺丁醇子密码子285密码子突变率为15%;303密码子突变率为6%;306密码子突变率为70%;330密码子突变率为3%;12%未找到突变点,306密码子突变率最多。耐利福平rpoB全部找到该区段内的突变位点且较复杂,多位点同时突变较多。成功运用多重MB探针芯片对各位点TB临床耐药分离株PCR产物进行杂交反应,与测序结果对比有较好的一致性。结论:1、本研究采用多重分子信标探针成功实现了对4种结核杆菌耐药基因主要突变区域多重靶点的检测。研究摸索了对单碱基靶点突变具高灵敏及特异检测能力的分子信标探针设计模型及其杂交检测技术。2、设计合成了结核杆菌通用高特异性MB探针,并进行了扩增中/扩增后加入通用MB探针系列实验。通过凝胶成像分析仪、荧光分光光度计、荧光显微镜等各种途径对通用MB探针杂交荧光观测条件进行了摸索,为荧光芯片运用作了技术储备。通过与传统方法临床标本进行结核杆菌检测,证实了通用TB-MB探针荧光芯片具有较高的阳性检出率、灵敏性、特异性。3、单侧延长臂分子信标探针的设计发明对解决分子信标探针在芯片表面的固定难题提供了一种简单思路。对解决传统MB探针茎臂修蚀技术难,杂交无特异性、在多分子检测的中高密度芯片领域难以实际应用等难点具积极意义。4、建立了针对4种结核杆菌耐药基因各突变位点的PCR扩增体系,设计、合成、筛选了相应的多重TB分子信标探针及其单侧延长臂分子信标探针,并完成进行MB探针芯片杂交优化系列实验和条件优化。5、成功运用多重MB探针芯片检测各位点TB临床耐药分离株PCR产物的杂交反应。并与测序对照有较好的一致性。说明对分子信标探针对单碱基突变具较高的灵敏和特异检测能力,引入于基因芯片领域,实现了对多靶点碱基突变联检的目标。更多还原

【Abstract】 Objective:The current detecting methods for Bacillus Tuberculosis (TB) include many techniques such as X-ray, sputum smear, cell culture, medicine sensitivity, PCR, and so on. They are tedious or time-consuming methods with low sensitivity and specificity, which may easy lead to misdiagnosis and economic overburden of patients. A great variety of drugs prescribed to patients with Tuberculosis and their overuse lead to the increasingly complicated mutation of drug-resistant genes with wide complex range of mutation sites from the single-base mutation, deletion, insertion, malposition to that with multiple-sites and large fragment which present a barrier to detecting the drug resistant mutation genes accurately and comprehensively. Especially for the test for wide-existing single- base mutation sites, the traditional techniques including the traditional gene chip can hardly address the problems. The multi-drug-resistance is the main factor affecting its treatment and popularity for its simple indicator and scarce information on drug-resistance. Thus to construct a system for identifying the TB and testing it drug resistance in a fast, sensitive, comprehensive and simple way is a issue urgent to addressed in the field of Laboratory Diagnostics both at home and abroad. The molecular beacon probe is a nucleic acid probe with great capacity of testing single-base mutation sites, however, due to its molecular limitation, there still is blank in its practice application in the fluorescence chips.In this study, we attempt to construct a chip technique labelled by MB of TB multiple resistance genes mutation by making advantage of the stronger specificity to detection of the single–base mutation sites of target molecular and the beacon probe, combining with the high throughput of fluorescence, which is capable of directly detecting the specificity of multiple target sites of drug resistant genes and the nucleic acid of TB and provides a new method to identify the drug resistant genes of TB and to make the epidemic investigation.Methods and Materials:1. Based on the IS986 gene sequence of TB found by GenBank, we designed the primers on the software Primer Premier V5.0. After optimizing the MgCl2, primer, dNTR concentration and the annealing temperature, we constructed the TB PCR reaction amplification system and made a comprehensive assessment on the system by sequencing and detecting its sensitivity and specificity. The generally used molecular beacon probe of TB was designed on the software of beacon designer 2.1. After the temperature, time and means in hybridization were optimized, we added into TB beacon probe for hybridization during and after PCR reaction. The fluorescent signal of MB-PCR products and its observing condition were explored by using various instruments such as the Image Acquisition and Analysis System, Fluorospectrophotometer, and Fluorescence Microscope. We made careful observation on the various fluorescence quenching molecular.2. By studying the connection of molecular beacons with one-side extending arm and the connection between the biotin and avidin, we explored on the fixation and hybridization means of TB molecular beacon and the chips. The optimizing experiments on enhancing the hybrid efficiency of molecular beacon chips include: the concentration of 5/oligonucleotide probes and molecular beacon probes, the temperature, duration time and the formula of hybridization, the spotting concentration and the size, the critical value distinguishing the positive and negative. The clinical application of TB-MB chips that were generally used and methodological comparison were also made.3. The main mutation regions of drug resistance in 4 kinds of TB were searched for in the NCBI database. Based on the above sites, we designed the proliferation system and optimized it. The molecular beacon probes and the probes with one-side extending arm were also designed on the basis of the sites. The following series of experiments on the surface of chips molecular beacon probes with one-side extending arm of the mutation sites of TB resistance: the hybridization temperature gradient investigation (45℃、50℃、55℃、60℃、65℃were selected), the hybridization duration gradient investigation( the duration time was made as 2、4、6、8、10、12、14、16、18h), the target sequence hybridization concentration gradient investigation ( the concentration selected were 0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09uM). The observation was made by using Fluorescence Microscope.4. The PCR proliferation of the responsive clinically isolated mycobacterium tuberculosis was made on the basis of above sites. The sequencing of the products was made. The PCR products at each site were hybridized by using the multiple MB chip under the optimized condition we explored and the results of sequencing were made comparison.Results:1. The MgCl2, primers, and the best concentration of dNTP in the generally used TB-PCR reaction system after optimization were: 4mmol/l、0.04μmol/l and 0.2 mmol/l respectively, the annealing temperature was 55℃. The result of sequencing fully agreed with that published by GenBank. No cross-reaction with other bacteria in the system. The testing limitation was 1 copy. The optimized hybridization temperature and duration time for water bath, the PCR instrument, the constant-temperature shaking table were 55℃,16h;65℃,4h;50℃,7h respectively. The significant difference of fluorescence intensity between the positive MB-PCR hybridization products and negative ones was observed by using Fluorescence Microscope(p<0.01). The high efficiency of the fluorescence quenching was showed in Cy3-BDH、FAM-DABCLYE, and low efficiency in Cy3-DABCLYE、FAM–BDH.2. It is difficult to modify the stem arm biotin-avidin of molecular beacon. The marking is made in middle of quenching molecular in the molecular beacon with one-side extending arm and the single extending arm with stem structure is capable of fixing well on the chips with high specificity and low difficulty in modifying. The best concentration of oligo probes is 40μmol/L. After comparing the hybridization results of the three fluids, we found that 0.15%SDS showed better result than 0.2%SDS and 10×SSC showed better result than 5×SSC. While the concentration of Cy3-BDH probe was 15μmol/L and that of FAM-DABCLYE probe was about 9μmol/L, the strongest signal was found after 7h hybridization reaction. As the spotting diameter diminished, the fluorescence intensity for the sample with same concentration remained the unchanged. While the concentration reduced, the intensity diminished. The ideal intensities distinguish the positive and negative signals are about 10μmol/L for Cy3-BDH, about 15μmol/L for FAM-DABCLYE. The total detection rate for clinical samples by using the TB-MB chips was 57.5%(23/40), which is 27.5%(11/40) higher than the smear analysis and 35%(14/40) than the method of culture. The statistically significant difference was showed by comparing the three ways of dictation. (χ2=7.366,P<0.01;χ2=32.281 , P<0.01 respectively).3. The main mutation regions of drug resistance in 4 kinds of TB were accurately identified. Resistant streptomycin(SM):rpsl(43、88), rrs;Resistant isoniazid (INH):katG(315、463);Resistant Ethambuto (lEMB):embB(285、303、306、330);Resistant Ethambuto (lEMB):the core area of ropB mutation; the extended mutation area. Based on the above sites, we designed the proliferation system and optimized it. The fragments with specificity were proliferated successfully. The molecular beacon probes and the probes with one-side extending arm were also designed on the basis of the sites. A series of hybridization experiments on the surface of chips molecular beacon probes were successfully conducted. In the order of rpsl43、rpsl88、rrs、katG315、katG463、embB285、embB303、embB306、embB 330、ropB-MB1-1、ropB-MB1-2、ropB-MB1-3、ropB-MB1-4、ropB-MB2-1、ropB-MB2-2, the best hybridization conditions were: 50℃、50℃、50℃、55℃、65℃、55℃、55℃、45℃、60℃、60℃、50℃、65℃、60℃、55℃、55℃(in the hybridization temperature gradient investigation), 12、14、14、16、12、12、8、14、10、10、10、8、12、10、8h(the duration time for the hybridization duration gradient investigation), 0.06、0.05、0.06、0.04、0.05、0.04、0.07、0.05、0.05、0.05、0.06、0.04、0.05、0.04、0.04uM (in the target sequence hybridization concentration gradient investigation). A statistically significant difference between the fluorescence positive signals and negative ones at above sites was observed.4. The PCR proliferation of the responsive clinically isolated mycobacterium tuberculosis was made successively on the basis of above sites. The results for the proliferation products sequencing were: the mutation rate of the resistant streptomycin(SM)rpsL genes codon 43 was 65%, codon 88 was 60% with 75% mutant strains overlapping with codon 43; the mutation rate of Rrs was 15% and the multiple sites repetition rate of resistant SM was considerably high; the mutation rate of the resistant Resistant isoniazid (INH) KatG315codon was 50% and that 463 was 37%; the mutation rate of the Resistant Ethambuto (lEMB) genes codon 285 was 15% and those of 303,306, and 330 were 6%, 70% and 3% respectively, and that of 306 was highest. All the mutation sites of resistant rifampicin rpoB were found and showed completed and more simultaneous mutation in multiple sites. The PCR products at each site were hybridized successfully by using the multiple MB chip under the optimized condition we explored. The results of good consistency in sequencing comparison were showed.Conclusion 1.In this study, we successfully made detection on the multiple targets of the main mutation genes with drug resistance of 4 TB by using the multiple molecular beacon probes. The design model and its hybridization techniques of the molecular beacon probes with high specificity and sensitivity to the single base targets were explored.2. We designed the MBs with high specificity generally used in detection of TB and used them in a series of experiment of proliferation and after proliferation. We also used the various instruments such as the Image Acquisition and Analysis System, Fluorosp ectrophotometer, and Fluorescence Microscope to explore the fluorescent observation condition of hybridization by using MBs, which reserved a technique for application of fluorescent chips. By comparing with the traditional ways to detest the clinical sample of TB, we proved that the TB-MB fluorescent chips have high detection rate, specificity and sensitivity.3. The design of molecular beacon probes with one-side extending arm has provided a simple idea to solve the problem of fixation of probes on the surface of chips, which is of significance of application of traditional MBs into the high density chips which can not be achieved for its difficulty in stem arm modification and the absence of specificity.4.The PCR proliferation system of the mutation genes on the basis of sites of 4 types of TB was successfully made. The responsive multiple TB molecular probes and its probes with one-side extending arm were synthesized, designed and screened. A series of experiments on the hybridization of MB probe chips and its optimization was also made.5. The PCR products at each site were hybridized successfully by using the multiple MB chip under the optimized condition we explored. The results of good consistency in sequencing comparison were showed. It indicated that the molecular beacon probes have high specificity and sensitivity to the single base mutation. If used in the genes chip fields, the combined detection for the multiple targeted single base mutation could be achieved.更多还原