RT-PCR检测结构分枝杆菌mRNA在利福平快速药敏试验中的初步应用
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摘要
结核病的卷土重来已引起全球性的关注。结核病与艾滋病的合并感染以及多药耐药结核杆菌的流行，使得结核病一跃成为感染性疾病中的头号杀乎。目前控制结核病的关键是及时合理地实施全程督导化疗方案，这就要求实验室能够尽快提供结核杆菌诊断和药敏试验结果。如美国疾病控制中心规定临床实验室在收到标本后应该在30天内报告结核杆菌诊断和药敏试验结果。传统的结核杆菌诊断和药敏试验方法普遍存在周期长的缺点。即使是液体放射性或非放射性快速培养法能显著缩短报告时间，平均耗时仍需要5-12天。近年来，随着分子生物学技术的发展，基因诊断技术尤其是PCR技术以其快速、敏感、特异的优点曾一度受到实验室人员和医生的普遍欢迎。但是，基于结核杆菌DNA的PCR检测技术要想在临床推广使用，仍有几个问题亟待解决。如PCR实验室中产物DNA污染临床标本而导致的假阳性；检测结核杆菌DNA难以鉴别死菌和活菌；基于DNA检测的PCR方法不能用于药物敏感试验等。有关假阳性的问题国内外学者研究很多，并提出了一些切实可行的解决办法。近年来，人们开始把目光集中于寻找可以鉴别死菌和活菌的分子标志物，研究较多的有ATP，16srRNA和mRNA，其中mRNA以其半衰期短、细菌死亡后即很快降解的优点而倍受瞩目。Hellyer等比较了mRNA、rRNA和DNA在结核病化疗过程中的变化规律，认为mRNA是结核杆菌活菌诊断的良好分子标志物，优于DNA和rRNA。本文即以此为理论依据，建立一种快速敏感的结核杆菌mRNA的检测方法，并探讨其在结核分枝杆菌快速药敏试验中的应用价值。
[目的]建立一种快速敏感的结核杆菌mRNA的检测方法；摸索检测结核杆菌mRNA用于利福平快速药敏试验时的各项参数，从而建立
南京医科大学硕士学位论文
一种新的利福平分子生物学药敏试验方法；比较新建方法与
BICThlERT 3D快速培养法对20株临床分离株的药敏检测结果，分
析检测结核杆菌m伽a在利福平临床快速药敏试验中的应用价值。
［材料与方法］实验第一部分，以结核杆菌标准菌株H37RV（ATCC
27”4）为实验菌株，经 M 7H9液体培养基培养，获得其对数生长期
的菌悬液；采用Tr i pure总RNA分离试剂辅以超声粉碎的方法提取
结核杆菌总 RNA；然后用 DNase消化总 RNA中污染的基困组DNA；针
对。抗原“B的特定编码序列设计引物，逆转录扩增结核杆菌lMA，
产物用琼脂糖凝胶电泳检测分析，建立结核杆菌 mRNA的盯干CR检测
方法。用 Vitek Special DR100 Colorimeter调节初始菌悬液浊度为
2．0 McFarland（约 16’CFU／ml），然后用 M7Hg 液体培养基 10倍系歹
稀释为每ml 含 10‘、10’、104、10’、10’和 10 CFU，各取 1．0ml按上法
检测mRNA，得出该检测方法的敏感度。实验第H部分，取0．1
McFarland浊度的结核杆菌H37Rv菌悬液，分q接种于…g／ml、
2卜m／ml、4Pm加 的利福平含药液体培养基中，于培养0、12、18、24、
48小时收集细菌，用上述方法检测结核杆菌。RNA，并用KOdak凝胶
成像分析系统对mRNA进行相对定量，得出结核杆菌mRNA检测用于利
福平快速药敏试验时的最佳给药剂量和检测时间点，初步确主药敏试
验的方案。然后将药敏试验的初始菌量改为2．0、1．0、0．l、0．of和
0．00 McFarland标准浊度，考核药敏试验时的菌量容许范围。实验第
三部分，收集本室20m～2003年的结核杆菌11＄床分离菌株共20株，
经 BacT八 3D全自动快速培养系统培养到对数生长期门叫 天）
后，分别用 BacT八 3D快速培养的方法和实验第一、二部分所
建立的方法进行利福平药物敏感试验，对两种方法的药敏检测结果按
2 X 2列联表资料进行 X’检验分析，评价 RTICR检测结核分枝杆菌
mRNA在利福平快速药敏试验中的应用价值。
［结果］1．超声粉碎加 Tripure总 RNA分离试剂相结合提取结核杆菌
总 RNA时，超声粉碎的最佳条件是冰浴下 120w工作 10秒钟、间隔
3 0秒共 3个循环。2．所提结核杆菌总 RNA中总会有少量的基因组
DNA污染，污染DNA可被PCR反应体系直接扩增出目的片段，逆
3
南京医科大学硕士学位论文
转录之前用 DNase处理结核杆菌总 RNA，V以完金去除污染的 DNA。
3．本研究所建立的结核杆菌 m删的 RTPCR检测方法的敏感度较
高，最低检测下限 为m0o 个细菌。4．结核杆菌在1、2、41令11牙才
平含药液体培养基中培养 24小时后，mRNA均检测不到。在对临床
分离敏感株的重复性检测时，以4ug／ml浓度的重复性最佳，即假耐
药率最低。在确定药敏试验的最佳检测时间点为川小时，利福平最
佳给药剂量为 4fig／ffil时，药敏初始菌量容许范围为 1．0－0．0＊
McFarland标准浊度（约 10‘－10勺之间。5．20株临床分离结核杆菌
药敏试验结果；BacT叭lERT 3D快速培养的方法敏感 12株耐药 8株，
RTPCR方法的结果为敏感 10株，耐药 10株。两者?
Research backgrounds:
The resurgence of tuberculosis has attracted worldwide attention to the need for more methods of rapid diagnosis and susceptibility test. Tuberculosis has become to be the leading killer among infectious diseases due to the outbreaks of multi-drug resistant tuberculosis (MDR-TB) and the epidemic together with human immunodeficiency virus (HIV). At present, to perform directly observed treatment (DOT) project immediately and reasonably is the key point in the control of tuberculosis. It requires the clinical microbiology laboratories to provide the diagnosis and drug susceptibility data of Mycobacterium tuberculosis as soon as possible. In America, the Centers for Disease Control and Prevention recommend that all isolates of Mycobacterium tuberculosis be tested for their susceptibility to antibiotics, and that susceptibility data for first-line drugs be available within 30 days after receipt of a specimen. But conventional culture-based techniques for diagnosis and susceptibility all have the shortcoming of a
long turnround time. Although both radiometric and non-radiometric liquid culture systems have significantly shortened the turnround period, the results are still not available for 5-12 days after receipt of an isolate. In recent years, with the development of molecular biological techniques, gene diagnosis techniques, especially PCR, have become popular for their rapidness, sensitivity and specificity virtues. However, several
problems must be resolved before PCR can be widely accepted in clinical application to patients with tuberculosis, such as the risk of obtaining false-positive results due to contamination of clinical specimens with M tuberculosis DNA product from the PCR laboratory, the inability of the PCR method to detect a difference between viable and ncnviable organisms, and the incapacity of the PCR method to determine drug susceptibility. Considerable work has already gone into resolving the problem of contamination. So many people began to focus their attention on finding good molecule markers for assessing microbial viability, such as ATP, 16s RNA and mRNA. The mRNA molecule is considered to be the promising one for its short half-life and ability to distinguish viable and dead bacterium. Hellyer et al. has studied the changes of mRNA, rRNA and DNA during anti-tuberculosis therapy and drawn a conclusion that mRNA should be a good molecule marker for viable Mycobacterium tuberculosis diagnosis compared with DNA and rRNA. Based on this theory, we have developed a rapid and sensitive method to detect the mRNA of the M. tuberculosis and investigated its application in rifampin rapid susceptibility. Objectives:
To develop a rapid and sensitive detection method of Mycobacterium tuberculosis mRNA; study all the parameters of rifampin rapid susceptibility test through detection of Mycobacterium tuberculosis mRNA and draw a protocol of rifampin new molecular biological susceptibility test method. Compare the results of 20 clinical isolates of rifampin susceptibility by the method we set up and Bact/ALERT 3D auto culture system. Evaluate the value of detection Mycobacterium tuberculosis mRNA in rifampin rapid susceptibility. Materials and methods: 1. The first part
1.1 To develop a method to detect M. tuberculosis a antigen 85B
messenger RNA with the RT-PCR technique.
M tuberculosis H37Rv (ATCC 27294) was selected as standard strain. Bacterium suspension was obtained when the strains of M. tuberculosis grew to the mid-logarithmic phase in Middlebrook 7H9 broth for 7 to 14 days; Total RNA of M tuberculosis was isolated with Tripure total RNA isolation reagents associated with ultrasonic disruption; The genomic DNA contamination in total RNA was digested with RNA free DNase; A pair of primers was designed according to the specific sequence encoding alpha antigen 85B. 130 bp product was obtained through amplification of a antigen 85B messenger RNA using reverse transcription PCR and gel electrophoresis. 1.2 To assess the sensitivi
[目的]建立一种快速敏感的结核杆菌mRNA的检测方法；摸索检测结核杆菌mRNA用于利福平快速药敏试验时的各项参数，从而建立
南京医科大学硕士学位论文
一种新的利福平分子生物学药敏试验方法；比较新建方法与
BICThlERT 3D快速培养法对20株临床分离株的药敏检测结果，分
析检测结核杆菌m伽a在利福平临床快速药敏试验中的应用价值。
［材料与方法］实验第一部分，以结核杆菌标准菌株H37RV（ATCC
27”4）为实验菌株，经 M 7H9液体培养基培养，获得其对数生长期
的菌悬液；采用Tr i pure总RNA分离试剂辅以超声粉碎的方法提取
结核杆菌总 RNA；然后用 DNase消化总 RNA中污染的基困组DNA；针
对。抗原“B的特定编码序列设计引物，逆转录扩增结核杆菌lMA，
产物用琼脂糖凝胶电泳检测分析，建立结核杆菌 mRNA的盯干CR检测
方法。用 Vitek Special DR100 Colorimeter调节初始菌悬液浊度为
2．0 McFarland（约 16’CFU／ml），然后用 M7Hg 液体培养基 10倍系歹
稀释为每ml 含 10‘、10’、104、10’、10’和 10 CFU，各取 1．0ml按上法
检测mRNA，得出该检测方法的敏感度。实验第H部分，取0．1
McFarland浊度的结核杆菌H37Rv菌悬液，分q接种于…g／ml、
2卜m／ml、4Pm加 的利福平含药液体培养基中，于培养0、12、18、24、
48小时收集细菌，用上述方法检测结核杆菌。RNA，并用KOdak凝胶
成像分析系统对mRNA进行相对定量，得出结核杆菌mRNA检测用于利
福平快速药敏试验时的最佳给药剂量和检测时间点，初步确主药敏试
验的方案。然后将药敏试验的初始菌量改为2．0、1．0、0．l、0．of和
0．00 McFarland标准浊度，考核药敏试验时的菌量容许范围。实验第
三部分，收集本室20m～2003年的结核杆菌11＄床分离菌株共20株，
经 BacT八 3D全自动快速培养系统培养到对数生长期门叫 天）
后，分别用 BacT八 3D快速培养的方法和实验第一、二部分所
建立的方法进行利福平药物敏感试验，对两种方法的药敏检测结果按
2 X 2列联表资料进行 X’检验分析，评价 RTICR检测结核分枝杆菌
mRNA在利福平快速药敏试验中的应用价值。
［结果］1．超声粉碎加 Tripure总 RNA分离试剂相结合提取结核杆菌
总 RNA时，超声粉碎的最佳条件是冰浴下 120w工作 10秒钟、间隔
3 0秒共 3个循环。2．所提结核杆菌总 RNA中总会有少量的基因组
DNA污染，污染DNA可被PCR反应体系直接扩增出目的片段，逆
3
南京医科大学硕士学位论文
转录之前用 DNase处理结核杆菌总 RNA，V以完金去除污染的 DNA。
3．本研究所建立的结核杆菌 m删的 RTPCR检测方法的敏感度较
高，最低检测下限 为m0o 个细菌。4．结核杆菌在1、2、41令11牙才
平含药液体培养基中培养 24小时后，mRNA均检测不到。在对临床
分离敏感株的重复性检测时，以4ug／ml浓度的重复性最佳，即假耐
药率最低。在确定药敏试验的最佳检测时间点为川小时，利福平最
佳给药剂量为 4fig／ffil时，药敏初始菌量容许范围为 1．0－0．0＊
McFarland标准浊度（约 10‘－10勺之间。5．20株临床分离结核杆菌
药敏试验结果；BacT叭lERT 3D快速培养的方法敏感 12株耐药 8株，
RTPCR方法的结果为敏感 10株，耐药 10株。两者?
Research backgrounds:
The resurgence of tuberculosis has attracted worldwide attention to the need for more methods of rapid diagnosis and susceptibility test. Tuberculosis has become to be the leading killer among infectious diseases due to the outbreaks of multi-drug resistant tuberculosis (MDR-TB) and the epidemic together with human immunodeficiency virus (HIV). At present, to perform directly observed treatment (DOT) project immediately and reasonably is the key point in the control of tuberculosis. It requires the clinical microbiology laboratories to provide the diagnosis and drug susceptibility data of Mycobacterium tuberculosis as soon as possible. In America, the Centers for Disease Control and Prevention recommend that all isolates of Mycobacterium tuberculosis be tested for their susceptibility to antibiotics, and that susceptibility data for first-line drugs be available within 30 days after receipt of a specimen. But conventional culture-based techniques for diagnosis and susceptibility all have the shortcoming of a
long turnround time. Although both radiometric and non-radiometric liquid culture systems have significantly shortened the turnround period, the results are still not available for 5-12 days after receipt of an isolate. In recent years, with the development of molecular biological techniques, gene diagnosis techniques, especially PCR, have become popular for their rapidness, sensitivity and specificity virtues. However, several
problems must be resolved before PCR can be widely accepted in clinical application to patients with tuberculosis, such as the risk of obtaining false-positive results due to contamination of clinical specimens with M tuberculosis DNA product from the PCR laboratory, the inability of the PCR method to detect a difference between viable and ncnviable organisms, and the incapacity of the PCR method to determine drug susceptibility. Considerable work has already gone into resolving the problem of contamination. So many people began to focus their attention on finding good molecule markers for assessing microbial viability, such as ATP, 16s RNA and mRNA. The mRNA molecule is considered to be the promising one for its short half-life and ability to distinguish viable and dead bacterium. Hellyer et al. has studied the changes of mRNA, rRNA and DNA during anti-tuberculosis therapy and drawn a conclusion that mRNA should be a good molecule marker for viable Mycobacterium tuberculosis diagnosis compared with DNA and rRNA. Based on this theory, we have developed a rapid and sensitive method to detect the mRNA of the M. tuberculosis and investigated its application in rifampin rapid susceptibility. Objectives:
To develop a rapid and sensitive detection method of Mycobacterium tuberculosis mRNA; study all the parameters of rifampin rapid susceptibility test through detection of Mycobacterium tuberculosis mRNA and draw a protocol of rifampin new molecular biological susceptibility test method. Compare the results of 20 clinical isolates of rifampin susceptibility by the method we set up and Bact/ALERT 3D auto culture system. Evaluate the value of detection Mycobacterium tuberculosis mRNA in rifampin rapid susceptibility. Materials and methods: 1. The first part
1.1 To develop a method to detect M. tuberculosis a antigen 85B
messenger RNA with the RT-PCR technique.
M tuberculosis H37Rv (ATCC 27294) was selected as standard strain. Bacterium suspension was obtained when the strains of M. tuberculosis grew to the mid-logarithmic phase in Middlebrook 7H9 broth for 7 to 14 days; Total RNA of M tuberculosis was isolated with Tripure total RNA isolation reagents associated with ultrasonic disruption; The genomic DNA contamination in total RNA was digested with RNA free DNase; A pair of primers was designed according to the specific sequence encoding alpha antigen 85B. 130 bp product was obtained through amplification of a antigen 85B messenger RNA using reverse transcription PCR and gel electrophoresis. 1.2 To assess the sensitivi
引文
1. World Health Organization. Global tuberculosis control. WHO report 2000.Geneva: WHO, 2000
2. Dye C, Scheele S, Dolin P, et al. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. WHO global surveillance a monitorine project, JAMA, 1999,282:677-686
3．全国结核病流行病学抽样调查技术指导组．2000年全国结核病流行病学抽样调查报告．中国防痨杂志，2002，25(1)：3-7
4. Tenover FC,Crawford JC,Koga H,et al. The Resurgence of Tuberculosis:Is Your Laboratory Ready?J Clin Microbiol[J], 1993,31(4):767-770.
5．张敦熔．现代结核病学[M]．北京：人民军医出版社，2000：36-66．
6．刘根焰，童明庆．结核分枝杆菌快速药敏方法研究进展[J]．临床检验杂志，2002，20(3)：179-181
7. Brisson-Noel A, Gicquel B, Lecossier D, et al. Rapid diagnosis of tuberculosis by amplification of mycobacterial DNA in clinical samples[J].Lancet, 1989,4:1069-71
8. Cimino GDK,Metachette JW, Tessman JE, et al. Post-PCR sterilization:a method to control carryover contamination for the pholymerase chain reaction.Nucleic Acids Res, 1991,19:99-107
9. Kitchin PA,Szotyori Z,Fromhole C,et al. Avoidance of false positive.Nature, 1990,344:201-206
10. Gabain Von,Bilasco JG,Schottel JL,et al. Decay of mRNA in Escherichia coli:investigation of the fate of specific segments of transcrips.Proc Natl Acad Sci USA, 1983,80:653-657
11. Hellyer TJ, Fletcher TW, Bates JH, et al. Strand Displacement Amplification and the Polymerase Chain Reaction for Monitoring Response to Treatment in Patients with Pulmonary Tuber culosis. J Infect Dis[J], 1996,173:934-94
12. Hellyer TJ,Desjardin LE,Hehman GL,et al. Quantitative Analysis of
mRNA as a Mark for Viability of Mycobacterium tuberculosis[J]. J Clin Microbiol, 1999, 37(12):290-295.
13. Lewine B.The messenger RNA template.In Genes IV ed. Lewine B.pp. 171-184, 1990, Oxford: Oxford University Press.
14. Jonas V, Alden MJ,Curry JI, et al. Detection and identification of Mycobacterium tuberculosis directly from sputum sediments by amplification of rRNA [J]. J Clin Microbiol, 1993, 31(9):2410-2416.
15. Rajagopalan M, Boggaram V and Madiraju MVVS. A rapid protocol for isolation of RNA from Mycobacteria [J]. Letters in Applied Microbiology, 1995,21(1): 14-17
16．Ausubel F, Brent R,.Kingston RE, et al，精编分子生物学实验指南[M]．第三版，颜子颖、王海林译，北京：科学出版社，1998，128-129
17. Brennan PJ and Nikaido H.The envelope of mycobacteria [J]. Annu Rev Biochem, 1995; 64:29-63
18. Mangan JA, Sole KM, Mitchison DA, et al. An effective method of RNA extraction from bacteria refractory to disruption including mycobacteria [J]. Nucleic Acids Research, 1997;25(3):675-676
19. Jou NT, Yoshimori RB,Mason GR,et al. Single-tube,nested,reverse transcriptase PCR for detection of viable Mycobacterium tuberculosis[J].J Clin Microbiol,1997;35:1161-1165
20．王民，刘枫春，赵兵．结核分支杆菌H_(37)Rv与H_(37)Ra株mRNA基因差异显示研究[J]，中华结核和呼吸杂志，2000；23(11)：669～671
21. Kikuta-Oshima LC, Quinn FD, Butler WR, et al. Isolation of RNA from Mycobacterium tuberculosis using a nitrogen decompression chamber. Biotechniques. 1995;18(6):987-90
22. Belisle JT, Vissa VD,Sievert T, et al. Role of the major antigen of Mycobacterium tuberculosis in cell wall biogenesis. Science, 1997;276:1420-1422
23. Wiker HG and Harboe.The antigen 85 complex: a major secretion production of Mycobacterium tuberculosis. Microbiol
Rev. 1992,56:648-661
24.Lee BY and Horwitz MA. Identification of macrophage and stress-induced protein of Mycobacterium tuberculosis.J Clin Invest. 1995;96:245-249
25.Wilker HG, Harboe M and Lea T. Purification and characterization of two protein antigens from heterogeneous BCG 85B complex in Mycobacterium bovis BCG. Int Arch Allergy Appl Immunol, 1986;81:298-306
26.Patel BK, Banerjee DK, Butcher PD,et al. Determination of Mycobacterium leprae viability by polymerase chain reaction amplification of 71-kDa heat-shock protein mRNA. J Infect Dis. 1993; 168(3):799-800.
27.Desiardin LE,Perkins MD,Wolski K,et al. Measurement of sputum Mycobacterium tuberculosis messenger RNA as a surrogate for response to chemotherapy[J]. Am J Respir Crit Care Med, 1999; 160:203-210
28.Gabrielle ME, Schepers P, Schukking RAF, et al. Assessment of Mycobacterial Viability by RNA Amplification. Anti Agents Chemother [J], 1994, 38(9):1959-1965.
29.Watterson SA, Wilson SM,. Yates MD, et al. Comparison of Three Molecular Assays for Rapid Detection of Rifampin Resistance in Mycobacterium tuberculosis [J].J Clin Microbiol, 1998;36(7): 1969-1973
30.Microbiology Manual of Clinical Microbiology [M]. Editor in chief, Patrick R M. editors, Ellen J, Baron C et al. Pressed by ASM .7th. ed. 1999,1602-1603
31.Watterson S A, Drobniewski F A. Modern laboratory diagnosis of mycobacterial infections [J]. J Clin Pathol,2000;53:727-732
32.Kim B J, Lee K H, Park B N, et al. Detection of Rifampin-Resistant Mycobacterium tuberculosis in Sputa by Nested PCR-Linked
Single-Strand Conformation Polymorphism and DNA Sequencing[J].J Clin Microbiol, 2001; 39: 2610-2617.
33.Felmlee T A, Liu Q, Whelen A C,et al. Genotypic detection of Mycobacterium tuberculosis rifampin resistance: comparison of single-strand conformation polymorphism and dideoxy fingerprinting[J].J Clin Microbiol, 1995; 33: 1617-1623.
34.Saribas Z, Kocagoz T, Alp A, and Ayfer G(?)nalp. Rapid Detection of Rifampin Resistance in Mycobacterium tuberculosis Isolates by Heteroduplex Analysis and Determination of Rifamycin Cross-Resistance in Rifampin-Resistant Isolates [Jl. J Clin Microbiol, 2003; 41: 816-818.
35.Bartfai Z, Somoskovi A, Kodmon C, et al. Molecular Characterization of Rifampin-Resistant Isolates of Mycobacterium tuberculosis from Hungary by DNA Sequencing and the Line Probe Assay[J].J Clin Microbiol, 2001; 39:3736-3739.
36.Eltringham I J,Dribniewski F A,Mangan J A, et al. Evaluation of reverse transcription-PCR and a bacteriophage-based assay for rapid phenotypic detection of rifampin resistance in clinical isolates of Mycobacterium tuberculosis. J Clin Microbiol, 1999;37(11):3524-3527
37.Telenti A, Imboden P, Marchesi F, et al. Detection of rifampicin-resistance mutations in Mycobacteriurn tuberculosis. Lancet, 1993 ;341:647-650
38.Roggenkamp A, Hornef MW, Masch A, et al. Comparison of MB/BacT and BACTEC 460 TB systems for recovery ofinycobacteria in a routine diagnostic laboratory[J].J Clin Microbiol. 1999;37(11):3711-2
39.Mirovic V, Lepsanovic Z. Evaluation of the MB/BacT system for recovery of mycobacteria from clinical specimens in comparison to Lowenstein-Jensen medium [J].Clin Microbiol Infect.
2002;8(11):709-14
40．吴龙章，蔡杏珊，潘美玉，等．BacT／A1ERT 3D全自动培养仪在细菌鉴定和药敏试验的临床应用[J]．中华检验医学杂志，2002；25(4)：243-244
41. Martel F, Grundemann D, and Schomig E.A simple method for elimination of false positive results in RT-PCR [J]. J Biochem Mol Biol, 2002; 35(2): 248-50
2. Dye C, Scheele S, Dolin P, et al. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. WHO global surveillance a monitorine project, JAMA, 1999,282:677-686
3．全国结核病流行病学抽样调查技术指导组．2000年全国结核病流行病学抽样调查报告．中国防痨杂志，2002，25(1)：3-7
4. Tenover FC,Crawford JC,Koga H,et al. The Resurgence of Tuberculosis:Is Your Laboratory Ready?J Clin Microbiol[J], 1993,31(4):767-770.
5．张敦熔．现代结核病学[M]．北京：人民军医出版社，2000：36-66．
6．刘根焰，童明庆．结核分枝杆菌快速药敏方法研究进展[J]．临床检验杂志，2002，20(3)：179-181
7. Brisson-Noel A, Gicquel B, Lecossier D, et al. Rapid diagnosis of tuberculosis by amplification of mycobacterial DNA in clinical samples[J].Lancet, 1989,4:1069-71
8. Cimino GDK,Metachette JW, Tessman JE, et al. Post-PCR sterilization:a method to control carryover contamination for the pholymerase chain reaction.Nucleic Acids Res, 1991,19:99-107
9. Kitchin PA,Szotyori Z,Fromhole C,et al. Avoidance of false positive.Nature, 1990,344:201-206
10. Gabain Von,Bilasco JG,Schottel JL,et al. Decay of mRNA in Escherichia coli:investigation of the fate of specific segments of transcrips.Proc Natl Acad Sci USA, 1983,80:653-657
11. Hellyer TJ, Fletcher TW, Bates JH, et al. Strand Displacement Amplification and the Polymerase Chain Reaction for Monitoring Response to Treatment in Patients with Pulmonary Tuber culosis. J Infect Dis[J], 1996,173:934-94
12. Hellyer TJ,Desjardin LE,Hehman GL,et al. Quantitative Analysis of
mRNA as a Mark for Viability of Mycobacterium tuberculosis[J]. J Clin Microbiol, 1999, 37(12):290-295.
13. Lewine B.The messenger RNA template.In Genes IV ed. Lewine B.pp. 171-184, 1990, Oxford: Oxford University Press.
14. Jonas V, Alden MJ,Curry JI, et al. Detection and identification of Mycobacterium tuberculosis directly from sputum sediments by amplification of rRNA [J]. J Clin Microbiol, 1993, 31(9):2410-2416.
15. Rajagopalan M, Boggaram V and Madiraju MVVS. A rapid protocol for isolation of RNA from Mycobacteria [J]. Letters in Applied Microbiology, 1995,21(1): 14-17
16．Ausubel F, Brent R,.Kingston RE, et al，精编分子生物学实验指南[M]．第三版，颜子颖、王海林译，北京：科学出版社，1998，128-129
17. Brennan PJ and Nikaido H.The envelope of mycobacteria [J]. Annu Rev Biochem, 1995; 64:29-63
18. Mangan JA, Sole KM, Mitchison DA, et al. An effective method of RNA extraction from bacteria refractory to disruption including mycobacteria [J]. Nucleic Acids Research, 1997;25(3):675-676
19. Jou NT, Yoshimori RB,Mason GR,et al. Single-tube,nested,reverse transcriptase PCR for detection of viable Mycobacterium tuberculosis[J].J Clin Microbiol,1997;35:1161-1165
20．王民，刘枫春，赵兵．结核分支杆菌H_(37)Rv与H_(37)Ra株mRNA基因差异显示研究[J]，中华结核和呼吸杂志，2000；23(11)：669～671
21. Kikuta-Oshima LC, Quinn FD, Butler WR, et al. Isolation of RNA from Mycobacterium tuberculosis using a nitrogen decompression chamber. Biotechniques. 1995;18(6):987-90
22. Belisle JT, Vissa VD,Sievert T, et al. Role of the major antigen of Mycobacterium tuberculosis in cell wall biogenesis. Science, 1997;276:1420-1422
23. Wiker HG and Harboe.The antigen 85 complex: a major secretion production of Mycobacterium tuberculosis. Microbiol
Rev. 1992,56:648-661
24.Lee BY and Horwitz MA. Identification of macrophage and stress-induced protein of Mycobacterium tuberculosis.J Clin Invest. 1995;96:245-249
25.Wilker HG, Harboe M and Lea T. Purification and characterization of two protein antigens from heterogeneous BCG 85B complex in Mycobacterium bovis BCG. Int Arch Allergy Appl Immunol, 1986;81:298-306
26.Patel BK, Banerjee DK, Butcher PD,et al. Determination of Mycobacterium leprae viability by polymerase chain reaction amplification of 71-kDa heat-shock protein mRNA. J Infect Dis. 1993; 168(3):799-800.
27.Desiardin LE,Perkins MD,Wolski K,et al. Measurement of sputum Mycobacterium tuberculosis messenger RNA as a surrogate for response to chemotherapy[J]. Am J Respir Crit Care Med, 1999; 160:203-210
28.Gabrielle ME, Schepers P, Schukking RAF, et al. Assessment of Mycobacterial Viability by RNA Amplification. Anti Agents Chemother [J], 1994, 38(9):1959-1965.
29.Watterson SA, Wilson SM,. Yates MD, et al. Comparison of Three Molecular Assays for Rapid Detection of Rifampin Resistance in Mycobacterium tuberculosis [J].J Clin Microbiol, 1998;36(7): 1969-1973
30.Microbiology Manual of Clinical Microbiology [M]. Editor in chief, Patrick R M. editors, Ellen J, Baron C et al. Pressed by ASM .7th. ed. 1999,1602-1603
31.Watterson S A, Drobniewski F A. Modern laboratory diagnosis of mycobacterial infections [J]. J Clin Pathol,2000;53:727-732
32.Kim B J, Lee K H, Park B N, et al. Detection of Rifampin-Resistant Mycobacterium tuberculosis in Sputa by Nested PCR-Linked
Single-Strand Conformation Polymorphism and DNA Sequencing[J].J Clin Microbiol, 2001; 39: 2610-2617.
33.Felmlee T A, Liu Q, Whelen A C,et al. Genotypic detection of Mycobacterium tuberculosis rifampin resistance: comparison of single-strand conformation polymorphism and dideoxy fingerprinting[J].J Clin Microbiol, 1995; 33: 1617-1623.
34.Saribas Z, Kocagoz T, Alp A, and Ayfer G(?)nalp. Rapid Detection of Rifampin Resistance in Mycobacterium tuberculosis Isolates by Heteroduplex Analysis and Determination of Rifamycin Cross-Resistance in Rifampin-Resistant Isolates [Jl. J Clin Microbiol, 2003; 41: 816-818.
35.Bartfai Z, Somoskovi A, Kodmon C, et al. Molecular Characterization of Rifampin-Resistant Isolates of Mycobacterium tuberculosis from Hungary by DNA Sequencing and the Line Probe Assay[J].J Clin Microbiol, 2001; 39:3736-3739.
36.Eltringham I J,Dribniewski F A,Mangan J A, et al. Evaluation of reverse transcription-PCR and a bacteriophage-based assay for rapid phenotypic detection of rifampin resistance in clinical isolates of Mycobacterium tuberculosis. J Clin Microbiol, 1999;37(11):3524-3527
37.Telenti A, Imboden P, Marchesi F, et al. Detection of rifampicin-resistance mutations in Mycobacteriurn tuberculosis. Lancet, 1993 ;341:647-650
38.Roggenkamp A, Hornef MW, Masch A, et al. Comparison of MB/BacT and BACTEC 460 TB systems for recovery ofinycobacteria in a routine diagnostic laboratory[J].J Clin Microbiol. 1999;37(11):3711-2
39.Mirovic V, Lepsanovic Z. Evaluation of the MB/BacT system for recovery of mycobacteria from clinical specimens in comparison to Lowenstein-Jensen medium [J].Clin Microbiol Infect.
2002;8(11):709-14
40．吴龙章，蔡杏珊，潘美玉，等．BacT／A1ERT 3D全自动培养仪在细菌鉴定和药敏试验的临床应用[J]．中华检验医学杂志，2002；25(4)：243-244
41. Martel F, Grundemann D, and Schomig E.A simple method for elimination of false positive results in RT-PCR [J]. J Biochem Mol Biol, 2002; 35(2): 248-50