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Using Molecular Genotyping Methods to
Investigate of the Genetic Diversity of
Mycobacterium tuberculosis Strains in Taiwan
Peter Chin
NDSRA
Mycobacteria Reference Laboratory
Division of Laboratory Research and Development
Center for Disease Control, Kun-Yang Office
Nankang, Taipei, Taiwan
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• Non-motile, non-pigmented
• Acid-Fast stain, rod-like, 0.4x3.0 micron
• Obligate aerobic, slow grower, need special
media for culture
• Reduce nitrate, produce catalase
• Produce long chain fatty acids
• Genome of H37Rv sequenced in 1998
• Person to person transmission
through aerosol droplet
Mycobacterium tuberculosis
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Global TB Disease Burden – WHO Report 2002
Reprinted from www.who.int/entity/tb/publications/ global_report/2004/en/annex5.pdf
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Genome of Mycobacterium tuberculosis H37Rv
• more than 4.4 million base
pairs
• 3924 genes detected initially,
13 more genes uncovered
through protemics and
compatative genomics
• more than 25 genetic markers
identified for typing
Cole ST et al.
Nature 1998; 393: 537.
Reprinted from
http://www.cbs.dtu.dk/services/GenomeAtlas/atlasdata/Bacteria/Mtuberculosis_H37Rv_
Main/Mtuberculosis_H37Rv_Main.ZDNAatlas.nfp.png
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Purpose of genotyping of Mycobacterium tuberculosis
• To investigate biodiversity and population structure
of Mycobacterium tuberculosis
• To define predominant genotype
• To trace the origin of strains and their potential
spatial relatedness
• To built-up genomic database
• To initiate systematic TB molecular epidemiological
studies
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Genotyping Methodology
• Non-DNA Typing Methods
– Phage Typing
– Antibiotics Typing
– Serological Typing
– Biochemical Typing
• DNA-based Typing Methods
– Restriction Fragment Length Polymorphism w/o Hybridization
– Pulsed Field Gel Electrophoresis
– Transposable Element Typing (DR, IR, IS…etc. )
– 16s/32s rDNA(RNA) Typing
– Minisatellite-based Typing
• Polymorphic GC-rich Repetitive Sequence
• Major Polymorphic Tandem Repeat
• Variable Number Tandem Repeat
• Mycobacterial Interspersed Repeat Units
12. 疾病管制局 RFLP Flowchart
Heat
Inactivation
DNA Extraction
TB Culture
DNA Digestion
Gel Electrophoresis Overnight GelMembrane
Blotting
Hybridization with
Probes
Detection &
Analysis
Sample and
Internal marker
probe
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• a PCR based method
• DNA polymorphism at “Direct Repeat” (DR) region of
chromosomal locus
Spoligotyping Principle
A
B
C
D
E
DR:36bp DVRNon-repetitive
DNA spacer
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Apply Oligos in an array
using a miniblotter
Oligos are bound covalently
in line pattern to membrane
Rotate filter 90° apply biotin-labeled
PCR products and hybridize
Incubate with HRP-conjugate and
substrate and expose X-ray film
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MIRU Principle
• Application of MTB H37Rv Genome Project
• Mid-short tandem repeat around the genome, so
called minisatellite genetic marker
• Multi nucleotide repeat unit
– ex. (ATCG)n
R. Frothingham and W.A. Meeker-O’Connell, 1998
P. Supply et al. 2000
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Objective
• Evaluating the genotyping efficiency between four method we
performed
• Finding the proper balance between accuracy, throughput and
timing
• Choosing the selective genetic markers
• Cutting down the cost
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Approach For MIRU Typing
• PCR each 15 locus
– Simply
– Lowest throughput, difficult to analysis
– Heavy loading, time-cost and expensive (15 PCR reactions/sample)
• Multiplex PCR With Dye-labeling Primer by Gel EP
– More complex
– Mid throughput, but easy data handling
– Heavy loading
• Multiplex PCR With Dye-labeling Primer by Cap EP
– Most complex and most expensive
– High throughput and easy data handling
– Lowest Labor Loading
– Bottle neck :upstream DNA extraction
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Stage Progress Period
2003
六月 七月 八月 九月
1 88dConstructing The TB Ref. LAB. Facility
2 88dEstablishment of VNTR Typing Protocol
3 21dEstablishment of MIRU Typing Protocol
4 133dSetup Automation and HTP Protocol
2004
十月 一月 二月 三月 四月 五月 六月 七月 八月 九月
5 23dFirst-stage Data Analysis
2004年10月7日
頁面 1
Progress Chart for The High-Throughput TB Genotyping Methodology Establishment
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• Time interval: 2002~2004^
• Space: northern, central, southern and eastern area in Taiwan
• Criteria: AND (RFLP, Spoligotyping, VNTR, MIRU)
• Sample size: 479
Description of Sample Model
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Introduction of Linkage Disequilibrium
• Disobeying Principle of Segregation
• Example
– 2 alleles (A & a) at locus A: P(A)=p
– 2 alleles (B & b) at locus A: P(B)=r
– 2 alleles (C & c) at locus A: P(C)=q
– D=P(AB)-prq
– D’=prq
• Useful marker for genome-wide scan analysis
– Pedigree analysis
– Sib-pair study
a
b
c C
A
B
C
b
A
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• Monte Carlo simulation
– Developed in 1948 by Los Alamos National Lab.
– Von Neumann, Fermi, Ulam and Metropolis
– Simulation of the H-bond explosion by resampling formula
– Calculating Physics, Chemistry, Biology, Economics
– Resampling the loci without replacement and computing a VD
value for each resampled data set
– Resampling for about 10000 times
• Parametric Method
– L = Ve + 1.654 √ Var(VD)
• Null hypothesis for Ve=VD
LIAN Version 3.1: Detecting LD in Multilocus Data
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Conclusion
• Lower discrimination power compared with other publications
– Diverse genotype observed in Taiwan
• Different PT value can effect discrimination power obviously in
RFLP pattern
– Stability of IS6110, Electrophoresis Bias, Subjective Judgment of Data
• Linkage analysis showed highly clonal separation in Taiwan
– Need Further Surveillance
• Combination of VNTR & MIRU achieves compatible selective power
as RFLP
• Genotyping Strategy
– MIRU-VNTR as frontline, RFLP as confirmation and spoligotyping for
Beijing type surveillance
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Outlook
• Selection for appropriate LD markers
– Pairwise Association Analysis for Each Locus
• Redefinition the roles of RFLP, Spoligotyping, VNTR
and MIRU
– Flowchart or Pipeline for Genotyping Methodology
• Throughput Is Minority, Discrimination Power is
Majority
– More high-diversity markers (h>0.5)
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Acknowledgements
• Ruwen Jou- Principal Investigator
• Wei-Lun Huang- Assistant Research Fellow
• Strain Identification, General Affair
• Su-Ying Chang- Medical Technician
• Bacteria Culture, MDR surveillance
• Hsuan Liu- Research Assistant
• RFLP, DNA Extraction
• Huang-Yau Chen- Research Assitant
• Strain Identification, TB Virulence Research
• Meng-Hsun Chen- NDSRA
• Spoligotyping, MDR Surveillance
• Ting-Yung Kou- Research Assistant
• Proteomic, MDR Surveillance
• Pei-Ju Chin
• MIRU-VNTR, TB susceptibility Research, Bioinformatics
Reprinted from http://profiles.nlm.nih.gov/VC/B/B/B/T/_/vcbbbt.jpg
