結核菌分子檢驗技術之簡介與原理. The Techniques for Molecular Detection of Mycobacterium tuberculosis 全國結核病實驗室品質監測及人員認證計畫

Transcription

1 結核菌分子檢驗技術之簡介與原理 The Techniques for Molecular Detection of Mycobacterium tuberculosis 全國結核病實驗室品質監測及人員認證計畫

2 Topics Introduction of Mycobacterium tuberculosis Nucleic acids basic structure General methods for DNA extraction Comparison of different DNA extraction for M. tuberculosis Verification or Validation of FDA-approved tests and laboratory-developed tests for Infectious Diseases Physical and Chemical Methods for Nucleic Acid Detection Nucleic Acid Amplification Technologies 結核病分子生物檢驗技術最新發展現況 (CDC 提供 )

3 Mycobacterium tuberculosis Mycobacterium tuberculosis (MTB) is a pathogenic bacterial species in the family Mycobacteriaceae and the causative agent of most cases of tuberculosis (TB). M. tuberculosis has an unusual, waxy coating on its cell surface (primarily mycolic acid) and acidfast detection techniques are used. The genome of the H37Rv strain was published in Its size is 4 million base pairs, with 3959 genes. M. tuberculosis bacterial colonies M. tuberculosis visualization using the Ziehl Neelsen stain (acid-fast stain)

4 Cell wall of M. tuberculosis Schematic diagram of Mycobacterial cell wall. 1. outer lipids 2. mycolic acid 3. polysaccharides (arabinogalactan) 4. peptidoglycan 5. plasma membrane 6. lipoarabinomannan (LAM) 7. phosphatidylinositol mannoside 8. cell wall skeleton

5 Nucleic acids basic structure G Biochemistry, by Voet & Voet, 3rd edition - Chapter 05 (Nucleic Acids, Gene Expression, and Recombinant DNA Technology)

6 General methods for DNA extraction Essentials of Nucleic Acid Analysis: A Robust Approach; Chap. 4, DNA Extraction

7 General methods for DNA extraction Purification of the DNA is often the key step for success in molecular biology analysis. Cell or Membrane Lysis This step disrupts the cell wall/membrane and frees the DNA from cellular and organelle membranes. This can be accomplished by chemical (usually detergents), mechanical, enzymatic, microwave, sonication, heat or freeze/thaw treatment. Protection and Stabilization of Released DNA An extraction buffer contains a combination of chemical components which protect the released DNA in its new environment from degradation by cellular nucleases liberated during lysis. Separation of Nucleic Acids from Cell Debris or Sample Matrix The separation of released DNA from cellular debris has traditionally been achieved by phenol:chloroform and chloroform extractions. Concentration of DNA Both alcohol precipitation and commercial columns or bead-based chemistries can concentrate the DNA. Commercial kits are generally based on silica columns or magnetic beads over a wide range of chemistries.

8 Type and Amount of Detergent or Denaturant Used for cell or membrane lysis (CetylTrimethyl Ammonium Bromide)

9 Type and Amount of Detergent or Denaturant Used for cell or membrane lysis Essentials of Nucleic Acid Analysis: A Robust Approach; Chap. 4, DNA Extraction

10 Common enzymes used in cell and membrane lysis Essentials of Nucleic Acid Analysis: A Robust Approach; Chap. 4, DNA Extraction

11 Precipitation and Concentration of DNA Volume and Temperature of Alcohol Used and Precipitation Times Either a 2 volume of ethanol or a 0.6 volume of isopropanol is usually recommended. DNA precipitations are undertaken at room temperature or at -20 C. Precipitation can be improved by the addition of MgCl 2 or glycogen to a final concentration of 10mM and 10 mg/ml, respectively. Concentration and Type of Salt

12 Comparison of different DNA extraction for M. tuberculosis

13 (i) IDI extraction. The TE suspension was placed into an IDI lysis tube (Infectio Diagnostic, Inc), which contains a glass bead matrix. Tubes were vigorously mixed for 5 min on the highest setting of a Vortex Genie 2 and then boiling for 15 min. (ii) QIAGEN QIAamp DNA mini kit: 30 mg/ml lysozyme added to the tissue lysis buffer was followed by boiling for 15 min, and the proteinase K step was incubated at 56 C for 1 h. (iii) PrepMan extraction: A 200µl of PrepMan Ultra sample preparation reagent with boiling for 15 min. (iv) TE boil extraction: A 200µl TE was placed in a boiling water bath for 15 min Glass bead+boiling

14 The combination Z-HM (surface coated magnetic particles) was selected for its simplicity, high sensitivity, low cost relative to commercial spin columns, and potential for highthroughput automation. Zirconia 氧化鋯是一種堅硬 無色及光學上無瑕的結晶, 是鑽石的替代品

15 Verification or Validation of FDAapproved tests and laboratorydeveloped tests for Infectious Diseases Burd EM. Clin Microbiol Rev Jul;23(3):

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20 Determine probits by looking up those corresponding to the % responded in Finney s table (Finney 1952):

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22 Physical and Chemical Methods for Nucleic Acid Detection

23 Physical and Chemical Methods for Nucleic Acid Detection Spectral Absorption The component bases of nucleic acids maximally absorb light within the ultraviolet (UV) spectrum at 260 nm. the purity of nucleic acids taken from biological sources with ratios (260/280) greater than 1.8 and 2.0, indicating high purity of DNA and RNA. Intercalating Dyes Ethidium bromide Ethidium bromide binds with DNA and slips in between its hydrophobic base pairs and stretches the DNA fragment, removing water molecules from the ethidium cation. The result of this dehydrogenation is an increase in fluorescence of the ethidium. SYBR Green I and II Minor groove of double-stranded nucleic acids. Probes Non-fluorescent Detection Probes by optical or electrical chemiluminescence, antigenic groups, and enzymes. Fluorescent Detection Probes 5 Nuclease Probes FRET Hybridization Probes Molecular Beacons

24 5 Nuclease Probes Fluorogenic 5 nuclease probes are oligonucleotides containing a 5 reporter dye and a 3 quencher dye. Excitation FRET Emission Amplicon Reporter ANNEALING Quencher EXTENSION Amplicon 5-3 exonuclease

25 FRET Hybridization Probes Fluorescent resonance energy transfer (FRET) is achieved with hybridization probes when a probe containing a fluorescent donor dye and a second probe containing a fluorescent reporter dye hybridize to adjacent sequences in a target nucleic acid. When donor and reporter dyes are separated by only one to five nucleotides, light excitation driven FRET leads to the emission of a characteristic wavelength of light from the reporter dye. FRET hybridization probes are typically used as fluorescent reporters for real-time PCR with probe colocalization occurring during the annealing phase of PCR.

26 FRET Hybridization Probes FITC Red 640 Excitation FRET Emission P Phosphate P Amplicon

27 Molecular Beacons Molecular beacons are oligonucleotides containing terminal reporter and quencher molecules flanked by inverted terminal repeat sequences permitting the formation of a stem-loop structure.

28 Nucleic Acid Amplification Technologies

29 Nucleic Acid Amplification Technologies Target Amplification Polymerase Chain Reaction (PCR) Transcription-Based Amplification System (TAS, TMA) Strand Displacement Amplification (SDA) Cross-Priming Amplification Loop-mediated isothermal amplification Probe Amplification Ligase Amplification Reaction (LAR, LCR) FEN-1 DNA Polymerase-Based Amplification Cycling Probe Signal Amplification branched DNA (bdna) testing Hybrid capture technology

30 Polymerase Chain Reaction (PCR)

31 Polymerase Chain Reaction (PCR)

32 Polymerase Chain Reaction (PCR) The actual amount of product that accumulates is as follows: N (total) = N (initial)(1+y) X, where Y is the efficiency per cycle.

33 Real time PCR Five major detection chemistries DNA-binding fluorophores a pair of adjacent, fluorogenic hybridization oligoprobes 5' Nuclease oligoprobes Molecular beacons (hairpin oligoprobes) Self-fluorescing amplicon Sunrise primers Scorpion primers Advantages rapidity, sensitivity, reproducibility and the reduced risk of carry-over contamination 33

34 Real-Time PCR

35 Real time PCR Hairpin oligoprobes 5 Nuclease oligoprobes. Adjacent oligoprobes. 35

36 Real time PCR Sunrise primers Scorpion primers. 36

37 Transcription-Based Amplification System (TAS) or TMA

38 Transcription-Based Amplification System (TAS)

39 Strand Displacement Amplification (SDA)

40 Strand Displacement Amplification (SDA)

41 Cross-Priming Amplification J Clin Microbiol Mar;47(3):845-7.

42 Loopmediated isothermal amplification Notomi T et al. Nucl. Acids Res. 2000;28:e63-e63

43 Ligase Amplification Reaction (LAR)

44 Ligase Amplification Reaction (LAR)

45 FEN- DNA polymerasebased amplification. 1

46 Cycling Probe DNA--RNA--DNA Tm(short probe) Incubation Temp: Tm(long probe)-5 C

47 Branched DNA (bdna) signal amplification.

48 Hybrid capture.

49 Hybrid capture.

50 結核病分子生物檢驗技術 最新發展現況 黃偉倫 疾病管制局研究檢驗中心 分枝桿菌實驗室

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52 FDA-approved NAA tests Roche Amplicor MTB system - amplifies 16S rrna sequence that and detect PCR product by hybridization with a MTBC-specific probe - discontinued in early 2010 Gen-probe amplified Mycobacterium tuberculosis Direct (MTD) test - September 30, 1999

53 Commercial Amplification Method for Direct Detection of Mycobacterium tuberculosis Complex in Clinical Samples International(2) Tests Method Target Sample Detection AMPLICOR PCR 16SrDNA 100 L Colorimetric AMTD2 TMA 16SrRNA 450 Chemiluminescence LCx LCR PAB 500 Fluorimetric DTB SDA IS Fluorimetric LiPA Nested PCR rpob 500 Colorimetric TMA: transcription-mediated amplification; LCR: ligase chain reaction; SDA: strand displacement amplification; PAB: protein antigen b (38 kd) Modified from Piersimoni C & Scarparo C J Clin Microbiol 2003;41:

54 Commercial Amplification Method for Direct Detection of Mycobacterium tuberculosis Complex in Clinical Samples International(3) Tests Assay time (h) Automation Internal control FDA approval AMPLICOR 6 Yes Yes Yes AMTD2 2.5 No Yes Yes LCx 6 Yes No No DTB 3 Yes No No LiPA 12 Yes No No Modified from Piersimoni & Scarparo J Clin Microbiol 2003;41:

55 Commercial Amplification Method for Direct Detection of Mycobacterium tuberculosis Complex in Clinical Samples International(4) Tests Sensitivity % Specificity % Sensitivity(%) for smear (+) smear (-) AMPLICOR 27.3~ ~ ~ ~71.1 AMTD2 77.3~ ~ ~ ~100 LCx 69.7~ ~ ~ ~100 DTB 96.5~ ~100 90~ ~90.4 LiPA 58.8~98.3 Modified from Piersimoni & Scarparo J Clin Microbiol 2003;41:

56 Update on WHO review of newer NAAT technologies The Eiken NAAT - the Loopamp MTB complex (MTBC) Detection Kit - manual assay using the loop-mediated amplification (LAMP) platform to detect TB DNA in sputum specimens - insufficient evidence to proceed with the development of policy guidance The Hain Lifescience NAAT - Genotype MTBDRsl - specificity for detecting resistance to fluoroquinolones and second-line injectables was high, its sensitivity was suboptimal - it cannot be used as a replacement test for conventional phenotypic drug susceptibility testing (DST)

57 Update on molecular TB Tests - Cepheid Xpert MTB/RIF assay - Eiken Loopamp MTBC detection kit - Epistem Genedrive Mycobacterium id test-kit - Molbio Diagnostics Truelab TB assay - Ustar biotechnologies easynat Tb Isothermal Amplification diagnostic Kit

58 Cepheid Xpert MTB/RIF assay

59 Xpert MTB/RIF simple and rapid diagnostic tools - a fully automated molecular test for tuberculosis case detection and drug resistance testing - a disposable plastic cartridge containing all reagents required for bacterial lysis, nucleic acid extraction, amplification, and amplicon detection

60 rpob Molecular Beacon Assay real-time PCR assay to amplify an MTB specific sequence of the rpob gene within 2 hours 3 specific primers and 5 unique molecular probes

61 Eiken Loopamp MTBC detection kit

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63 Epistem Genedrive Mycobacterium id

64 那項技術的發明者說, 他們的自動化磁帶機還可以用於識別抗結核病藥物治療的細菌

65 Genedrive Mycobacterium id test-kit Epistem Ltd. UK has developed the Genedrive real-time PCR instrument portable real-time PCR instrument capable of single-test processing with sample preparation, assay incubation, and test result in under 45 minutes cassette contains three reaction tubes made of high-quality optical material in which the test reactions are performed. These are used for MTB detection, rifampicin resistance screening, and an internal process control (IPC) limit of detection of 30 colony-forming units/ml

66 Molbio Diagnostics Truelab TB assay

67 Molbio Diagnostics Truelab TB assay PCR amplification with fluorescence detection in real time using an embedded Android cell phone controls the necessary operating software to perform and determine the test result The MTB assay target is IS6110 and the time to final result is 35 minutes The use of an Android-based device in the platform gives added utility in terms of wireless operation and communication with the ability to locate via global positioning system (GPS)

68 Molbio Diagnostics Truelab TB assay

69 Ustar biotechnologies easynat Tb Isothermal Amplification diagnostic Kit

70 Ustar EasyNAT TB Isothermal Amplification Diagnostic kit The sensitivity of CPA from smearand liquid culture-positive specimens was 96.9%, and that from smear-negative and liquid culturepositive specimens was 87.5%. The specificity of CPA in culturenegative specimens was 98.8%.

71 Ustar EasyNAT TB Isothermal Amplification Diagnostic kit limit of detection of 10 colony-forming units/ml developed and patented an enclosed LFS-based detection tool

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74 Conclusion ASSURED (WHO) for diagnostic tools suitable for use in developing world and low-resource regions - Affordable, Sensitive, Specific, User-friendly, Rapid/Robust, Equipment-free and Deliverable Standardized EQA (External Quality Assessment; Proficiency Testing) to ensure adequate performance of equipment and users Molecular diagnostic limitations - false positive, false negative, price