張煥宗臺灣大學：化學研究所張柏齡Chang, Po-LingPo-LingChang2007-11-262018-07-102007-11-262018-07-102007http://ntur.lib.ntu.edu.tw//handle/246246/51975本論文主要著重於以毛細管電泳（capillary electrophoresis, CE）為分析工具針對胺基酸、生物鹼做檢測，並結合分子生物技術鑑定分枝桿菌及奴卡氏菌。本文之第一章主要介紹毛細管電泳基本原理和應用；以及分枝桿菌之重要性與臨床檢驗方法。第二章則敘述以發光二極體誘發螢光（light-emitting-diode induced fluorescence, LEDIF）、naphthalene-2,3-dicarboxaldehyde（NDA）作為衍生化試劑，針對胺基酸進行管柱內之衍生化、樣品堆積及分離。和傳統管柱外方法相較，此法呈現出較少的衍生化副產物效應。若以gamma-aminobutyric acid（GABA）為分析物，其可偵測濃度之線性範圍包含10–5到10–8 M，且偵測極限可達4 nM。第三章吾人則組裝發光二極體誘發螢光及電化學發光（electrochemiluminescence, ECL）之裝置並以此同時檢測胺基酸和生物鹼。在此雙重偵測器之毛細管電泳裝置中，ECL偵測器置於毛細管之出口端並與LEDIF差距12公分。一級胺基酸同樣以NDA作為衍生化試劑，二、三級胺則以Ru(bpy)32+作為ECL之電化學試劑。以此經濟裝置對標準樣品作分析時，胺基酸和生物鹼的偵測極限分別為49 nM–0.2 μM和0.66–4.7 μM。第四章的研究內容則著重於以LEDIF檢測肝病病人腹水樣品之分支鏈胺基酸（branched-chain amino acids, BCAAs）。經由聚環氧乙烯（poly(ethylene oxide), PEO）改善白胺酸及異白胺酸的分離效率，吾人間接證明聚環氧乙烯與胺基酸間厭水性作用力的存在。若對腹水之BCAAs作定量分析，其回收率為83.7％–134％。此外，一天之內的精準度為1.7％–5.8％、天與天之間的精準度則為2.2％–7.4％。第五章則是以聚合酶鏈鎖反應（polymerase chain reaction, PCR）及限制片段長度多型性（restriction fragment length polymorphism, RFLP）結合CE對分枝桿菌進行菌種的鑑定。在本章中，總共12個菌種（含52個菌株）皆可在只用一種限制酵素-Hae III的情況下達到鑑定的目的，而最小可測得之DNA片段為12 bp。另外，只要菌種之熱休克蛋白（65-kDa heat shock protein, hsp65）基因有額外的Hae III限制部位或一個Hae III限制部位的差異都可清楚呈現於電泳圖中。第六章則是以同樣方法鑑定少見之分枝桿菌菌種及奴卡氏菌。在此章中，一樣只用一種限制酵素的情況下，12種少見之分枝桿菌及7種奴卡氏菌都能清楚無誤地呈現出完整的RFLP圖譜於電泳圖上。且預估之DNA片段大小與實際之DNA定序結果完全吻合。在第七章中，吾人以蜂巢式聚合酶鏈鎖反應（nested PCR）進行分枝桿菌之鑑定，且證實nested PCR可達到單分子的偵測靈敏度。然而，DNA取樣時受限於波氏分佈（Poisson distribution）的影響，因此會有陽性率不如預期的情況發生。除了靈敏度的改善，吾人亦證實了nested PCR有助於減少非特異性引子-雙體的產生及樣品中PCR抑制物的負面影響。此外，本方法亦證實可以在不經過細菌培養的情況下直接對痰液中的分枝桿菌菌種進行檢測及鑑定。In the dissertation, two major topics focus on amino acids and/or alkaloids determination and identification of Mycobacterium and Nocardia species by capillary electrophoresis (CE). Chapter 1 introduces the principles and applications of capillary electrophoresis along with the importance of Mycobacterium tuberculosis and relative clinical examinations. Chapter 2 states the in-column derivatization, stacking, and separation of amino acids (AA) by capillary electrophoresis in conjunction with light-emitting-diode induced fluorescence (LEDIF) using naphthalene-2,3-dicarboxaldehyde (NDA). In comparison with the off-column approach to the analysis of amino acids, our proposed method provides a lower degree of interference from polymeric NDA compounds and other side products. As a result, the plot of the peak height as a function of γ-aminobutyric acid (GABA) concentration is linear over the range from 10–5 to 10–8 M, with the limit of detection being 4 nM. Chepter 3 describes the determination of alkaloids and amino acids using capillary electrophoresis in conjunction with sequential light-emitting-diode induced fluorescence and electrochemiluminescence (ECL) detections. In the CE-LEDIF-ECL system, the ECL detector was located in the outlet of the capillary, while the LEDIF detector was positioned 12 cm from the outlet. NDA was used to form fluorescent AA–NDA derivatives from amino acids possessing primary amino groups, while Ru(bpy)32+ was used to obtain ECL signals for analytes having secondary and tertiary amino groups. This low-cost CE-LEDIF-ECL system allows the analysis of these AA–NDA derivatives and alkaloids at concentrations in the ranges 49 nM–0.2 μM and 0.66–4.7 μM, respectively. In chapter 4, we have developed a convenient separation method of branched-chain amino acids (BCAAs) from ascites of patients suffer liver diseases. Amino acids was labeled by NDA with CN- as nucleophil, the derivatives were then introduced to capillary by hydrodynamic injection and separated by linear polymer under electroosmotic flow. The recovery data range from 83.7% to 134% over three amino acids and five different concentrations. The within-day precisions of BCAAs were range from 1.7% to 5.8%, while between-day precisions were 2.2% to 7.4%. In part of identification of M. tuberculosis, we have demonstrated the separation of DNA or restriction fragments digested from the mycobacterial gene encoding for the 65-kDa heat shock protein (hsp65) by capillary electrophoresis as described in chapter 5. Using a pair of unlabeled primers, Tb11 and Tb12, and only one restriction enzyme, HaeIII, a total of 52 reference and clinical strains encompassing 12 Mycobacterium species were investigated. The electrophoretic separation of high-resolution CE required less than 20 minutes and was capable of identifying fragments as small as 12 bp. A good agreement of measurement was observed between the sizes of restriction fragments resolved by CE and the real sizes deduced from the sequence analysis. Distinct differentiations were also well demonstrated between some species and subspecies by an extra HaeIII digestion site. Furthermore, in chapter 6, additional patterns of 12 less common Mycobacterium and 7 Nocardia species were analyzed and collected for the database of identification purpose. A good agreement of measurement was observed between the sizes of restriction fragments resolved by CE and the real sizes deduced from the sequence analysis. Some closely related species exhibiting similar biochemical characteristics could also be well discriminated by a different or extra HaeIII digestion site. Finally, chapter 7 focus on improve the sensitivity and differentiation in rapidly identifying a small amount of mycobacteria directly from sputum that has significant implications for reducing tuberculosis transmission. In the present study, PCR is replaced with nested PCR (nPCR) in which the primers and other optimizations are redesigned. As the results shown, the implementation of nPCR in PCR-RFLP assay (PRA) with CE (PRACE) is not only able to detect the presence of mycobacterial DNA less than ten copies, but differentiate the species as well. Both Mycobacterium tuberculosis and mycobacteria other than tuberculosis could be identified even without DNA extraction or in the presence of inhibitors. The least interference of primer-dimers improved by nPCR also contributes to the excellent specificity of RFLP patterns.Text contents 1 Introduction 1 1.1 Capillary electrophoresis 1 1.1.1 EOF and zeta potential 3 1.1.2 Electrophoretic mobility (μ) 4 1.1.3 Efficiency and resolution 5 1.1.4 Principle DNA separation by capillary electrophoresis 8 1.1.4.1 Ogston model 10 1.1.4.2 Reptation in a electric field: The biased reptation model 11 1.1.5 Detection systems 13 1.1.6 Applications 16 1.1.6.1 DNA analysis 16 1.1.6.2 Proteins analysis 19 1.1.6.3 Small molecules analysis 20 1.2 Polymerase chain reaction 22 1.2.1 History 22 1.2.2 Principle of PCR 23 1.2.3 Reagents involved in PCR 25 1.2.3.1 Polymerase 25 1.2.3.2 Magnesium and Nucleotides 25 1.2.3.3 Primer 26 1.3 Mycobacterium tuberculosis 28 1.3.1 Epidemiology 30 1.3.2 Pathogenesis 32 1.3.3 Laboratory Diagnosis 35 1.3.3.1 Traditional examinations 35 1.3.3.2 Nucleic acid-based amplification for identification of mycobacteria 37 1.4 References 40 2 Stacking, Derivatization, and Separation by Capillary Electrophoresis of Amino Acids from Cerebrospinal Fluids 71 2.1 Abstract 71 2.2 Introduction 73 2.3 Materials and Methods 77 2.3.1 Chemicals 77 2.3.2 CSF 77 2.3.3 CE-LEDIF 78 2.3.4 Off-column derivatization 79 8.1.5 Electrophoresis procedure 80 2.3.6 In-column derivatization, stacking, and separation procedure 81 2.4 Results and Discussion 83 2.4.1 Effect of polymer solution on resolution 83 2.4.2 In-column derivatization, stacking, and separation under discontinuous conditions 84 2.4.3 Reaction time 87 2.4.4 Analysis of standard samples 89 2.4.5 Analysis of CSF samples 91 2.5 References 94 3 Capillary Electrophoresis with Sequential LED-Induced Fluorescence and Electrochemiluminescence Detection for the Determination of Amino Acids and Alkaloids 110 3.1 Abstract 110 3.2 Introduction 112 3.3 Materials and Methods 115 3.3.1 Chemicals 115 3.3.2 Fabrication of ECL detection cell 115 3.3.3 ECL detection of alkaloids and proline 116 3.3.4 CE-LEDIF-ECL system 116 3.3.5 Sample preparation 118 3.3.6 Amino acid derivatization with NDA 118 3.3.7 Electrophoresis procedures 119 3.4 Results and Discussion 120 3.4.1 CE-LEDIF-ECL system 120 3.4.2 Separation of amino acids and alkaloids 122 3.4.3 Analysis of urine 125 3.4.4 Analysis of tobacco extracts 127 3.5 References 130 4 Determine the Branched-Chain Amino Acids of Ascites with Liver Diseases Patients by Capillary Electrophoresis with Light-Emitted-Diode Induced Fluorescence 142 4.1 Abstract 142 4.2 Introduction 144 4.3 Materials and Methods 147 4.3.1 Chemicals 147 4.3.2 Specimens collection 147 4.3.3 Instrumentation 148 4.3.4 Fluorescent-dye derivatization 149 4.3.5 Electrophoresis procedure 150 4.3.6 Linearity, limit of detection and limit of quantitative 150 4.3.7 Quantitative and calibration assay 151 4.3.8 Recovery and precision 151 4.4 Results and Discussion 153 4.4.1 Linearity, limit of detection and limit of quantitative 153 4.4.2 Matrix effect on separation performance 154 4.4.3 Calibration assay 154 4.4.4 Recovery and precision 155 4.5 References 157 5 Capillary Electrophoretic-Restriction Fragment Length Polymorphism Patterns of Mycobacterial hsp65 Gene 169 5.1 Abstract 169 5.2 Introduction 171 5.3 Materials and Methods 174 5.3.1 Mycobacterium strains 174 5.3.2 DNA extraction, amplification and restriction enzyme digestion 174 5.3.3 Capillary electrophoresis 175 5.3.4 Analysis of amplified hsp65 gene sequence 176 5.4 Results and Discussion 178 5.5 References 185 6 The hsp65 Gene Patterns of Less Common Mycobacterium and Nocardia Species by PCR-Restriction Fragment Length Polymorphism Analyses with Capillary Electrophoresis 196 6.1 Abstract 196 6.2 Introduction 198 6.3 Materials and methods 201 6.3.1 Study strains 201 6.3.2 DNA extraction, PCR amplification, and restriction enzyme digestion 201 6.3.3 Separation of restriction fragments by capillary electrophoresis 202 6.3.4 Analysis of amplified hsp65 gene sequences 204 6.4 Results and Discussion 205 6.5 References 214 7 Ultra-sensitive Rapid Identification of Mycobacteria by Nested PCR and High-resolution Capillary Electrophoresis 228 7.1 Abstract 228 7.2 Introduction 230 7.3 Materials and Methods 232 7.3.1 Specimens and DNA extraction 232 7.3.2 PCR amplification and enzymatic digestion 233 7.3.3 Separation of PCR products by slab gel electrophoresis 234 7.3.4 Separation of PCR products by capillary electrophoresis 235 7.4 Results and Discussion 236 7.5 References 244 8 Conclusions 258 9 Appendix 262 Figure and Table contents Figure 1 1 (A) Structure of double layer and (B) ζ potential. 63 Figure 1 2 Comparison of (A) electroosmotic flow and (B) Laminar flow. 64 Figure 1 3 Schematic representation of flexible polymers in solution.. 65 Figure 1 4 Idealized representation of the different mechanism of migration of DNA in an array of fixed obstacles.. 66 Figure 1 5 DNA separation by using capillary gel electrophoresis under electroosmotic flow. 67 Figure 1 6 Schematic drawing of the PCR cycles. 68 Figure 1 7 Pathogenesis of tuberculosis in the early phase of infection. 69 Figure 2 1 Separation of six amino acids in the absence and presence of PEO. 102 Figure 2 2 A representative mechanism of the process of in-column derivatization, stacking, and separation of amino acids. 103 Figure 2 3 The impact of reaction time on in-column derivatization and separation of five amino acids by CE-LEDIF without a low-pH plug. 104 Figure 2 4 The impact of the GABA concentration on the formation of side products in off-column and in-column derivatization modes. 105 Figure 2 5 In-column derivatization, stacking, and separation of five amino acids by CE-LEDIF.. 106 Figure 2 6 Electropherograms of a CSF sample when conducting in-column and off-column derivatization.. 107 Figure 3 1 Schematic illustration of the CE-LEDIF-ECL system used for the analysis of AA–NDA derivatives, proline, nicotine, and anabasine. 137 Figure 3 2 Structures of proline and the alkaloids identified using ECL. 138 Figure 3 3 Electropherograms, obtained using the CE-LEFID-ECL system, of a mixture containing standard AA–NDA derivatives, proline, nicotine, and anabasine.. 139 Figure 3 4 Separation of urine samples using the CE-LEDIF-ECL system. 140 Figure 3 5 Separation of tobacco extracts using the CE-LEDIF-ECL system. 141 Figure 4 1 Structure, molecular weight and pI value of branched-chain amino acids. 161 Figure 4 2 Separation improvements of BCAAs with increase PEO concentration. 162 Figure 4 3 The matrix effect of ascites on separation efficiency.. 163 Figure 4 4 Linear plots constructed by standard addition for BCAAs quantification from ascites. 164 Figure 5 1 CE of 10-bp DNA ladder showing 33 10-bp repeats plus a fragment of 1,668 bp. 189 Figure 5 2 Relationship between the sizes of DNA ladders (10-200 bp) and corresponding electrophoretic migration time. 190 Figure 5 3 Electropherograms of mycobacterial hsp65 genes with HaeIII digestion.. 191 Figure 6 1 Electropherograms of mycobacterial hsp65 genes with HaeIII digestion. 222 Figure 6 2 Electropherograms of nocardial hsp65 genes with HaeIII digestion. 224 Figure 7 1 Capillary electrophoregrams of conventional PCR products using the primers reported by Telenti et al... 250 Figure 7 2 Capillary electrophoregrams of final products of nPCR using MTB DNA as initial templates 251 Figure 7 3 Probabilities of nPCR gene production are affected by the initial templates of MTB DNA 252 Figure 7 4 For capillary electrophoregrams of M. heckashorne and M. celatum 253 Figure 7 5 Agarose electrophoretic gels demonstrate hsp65 gene bands produced from traditional PCR and nPCR of mycobacteria from sputum. 254 Figure 7 6 For capillary electrophoregrams of sputum specimen #5 (A-D), traditional PCR products are exhibited in A. 255 Table 1 1 Examining and reporting acid-fast smears………………………………70 Table 2 1 Effect of hydrodynamic injection time on peak heights for the GABA-NDA derivative and side-products in the in-column derivatization mode. 108 Table 2 2 Comparison of the in-column and off-column CE approaches to the determination of GABA in CSF samples. 109 Table 4 1 Resolution and theoretical plate number of BCAAs with change of poly(ethylene oxide) concentration. 165 Table 4 2 LOD, LOQ and linearity. 166 Table 4 3 Recovery of the CE-LEDIF method for ascites BCAAs. 167 Table 4 4 Precision of the CE-LEDIF method for BCAAs quantitative. 168 Table 5 1 Mycobacterium strains studied for the RFLP pattern of hsp65 gene by capillary electrophoresis. 193 Table 5 2 Fragment sizes (bp) of hsp65 genes from 12 Mycobacterium species after HaeIII digestion and capillary electrophoresis in comparison with those deduced from sequence analysis. 194 Table 5 3 Mean and standard deviation of fragment length (bp) of mycobacterial hsp65 gene detected by capillary electrophoresis in comparison with those deduced from sequence analysis. 195 Table 6 1 Mycobacterium and Nocardia strains studied for the hsp65 gene patterns by the PCR-RFLP analysis with capillary electrophoresis. 225 Table 6 2 Fragment sizes (bp) of hsp65 genes from 12 Mycobacterium and 7 Nocardia species after HaeIII digestion and CE in comparison with those deduced from sequence analysis. 226 Table 6 3 Mean and standard deviation of hsp65 gene fragment sizes (bp) from the strains of three Mycobacterium species detected by the capillary electrophoresis in comparison with those deduced from the sequence analysis. 227 Table 7 1 Primers and thermocycles designed for traditional and nested PCR. 256 Table 7 2 Comparison of results between AFB smear, culture, PRACE, and sequence analysis for mycobacteria in sputum. 2575462456 bytesapplication/pdfen-US毛細管電泳雷射誘發螢光發光二極體誘發螢光電化學發光胺基酸生物鹼結核分枝桿菌Capillary electrophoresisLaser-induced fluorescenceLight-emitting-diode induced fluorescenceelectrochemiluminescenceamino acidalkaloidMycobacterium tuberculosis[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/51975/1/ntu-96-D92223001-1.pdf