Transcript 2. FT-IR

A MOLECULAR APPROACH TO INVESTIGATE TUBERCULOSIS CASES IN A GOTHIC POPULATION
FROM GHERĂSENI NECROPOLIS, BUZĂU COUNTY
CHIRIAC
1Molecular
1,3
Cecilia ,
LUPAN
1,2
Iulia ,
RADU
1
Claudia ,
KELEMEN
1,2
Beatrice
Biology Center, Interdisciplinary Research Institute on Bio-Nano-Sciences, “Babeș-Bolyai” University, Cluj-Napoca, Romania
2Faculty of Biology and Geology, Babes-Bolyai University, Cluj-Napoca, Romania
3NIRDBS, Institute of Biological Research, Cluj-Napoca, Romania
INTRODUCTION:
MATERIALS AND METHODS:
World Health Organization (WHO) ranks tuberculosis as the second most dominant infectious
disease, exceeded just by HIV/AIDS. Given the incidence of the disease today and the appearance
of multi-drug-resistant strains, it is important to obtain informations about the conserved and
variable genomic loci and about the mutation rate of the pathogen. For this reason, ancient cases
of tuberculosis have to be investigated and the co-evolution of the pathogens with modern
humans should be tracked. Species pathogenic to humans are included in the M. tuberculosis
complex (MTBC).
1. Anthropological and anthropometrical analysis for estimation of gender, age and
possible diseases of individuals;
2. FT-IR spectroscopy analysis - JASCO FT-IR 6000 Spectrometer
tuberculosis - mycolic acids markers
3. ancient-DNA (aDNA) extraction - phenol - chloroform;
4. PCR, Cloning and Sequencing
The aim of this study is to confirm through
molecular methods the presence of MTBC
in human remains from 4TH-5TH century
necropolis.
Figure 1. Geographic location of
Gherăseni necropolis
6. Bioinformatic analysis
RESULTS AND DISSCUSION:
1. Anthropological and
anthropometrical analysis
2. FT-IR
4. PCR, Cloning and Sequencing
A
C
Figure 3. Differences between FT-IR spectra of
tuberculosis infected and not infected skeletal remains.
Figure 2. A. The inferior view of a healthy V lombar
vertebrae comparing with (B) a tuberculosis affected
one. C. Lateral view of a normal toracic vertebrae
versus (D) two fused vertebrae from an individual
suffering of Pott's disease.
B
D
Figure 5. A. A serial dilution was made to overcome aDNA contamination with soil substances that
inhibit PCR; B. A fragment of pyrazinamidase gene (117 bp) was obtaind from two bones with
tuberculosis injuries; C. The regulator of hydrogen peroxide-inducible genes (oxyR pseudogene in
MTBC) was amplified (110 bp) in tuberculosis infected bone samples. D. TbD1 (deletion 1) fragment
(112 bp) was obtained from three bone samples and the amplicons are to be sequenced.
5. Bioinformatic analysis
A
3. aDNA extraction
Figure 4. aDNA extraction
1. rib, 2. vertebrae, 3.
negative control. As a
consequence of post –
mortem degradation, the
majority of genetic material
has below 50 bp in lenght.
B
Figure 6. 3D structure of pncA
fragment determined with The
mfold Web Server, State
University of New York.
A
C
B
C
Figure 7. Sequences obtained for pncA were subjected to multiple alignment using ClustalW algorithm in
Mega 5. As an outgroup we used homologous sequences from Corynebacterium glutamicum.
Figure 8. The evolutionary history was inferred by using the Maximum Likelihood method based on the
Tamura 3-parameter. The percentage of trees in which the associated taxa clustered together is shown
next to the branches. A discrete Gamma distribution was used to model evolutionary rate differences
among sites (5 categories (+G, parameter = 3.0035)). The tree is drawn to scale, with branch lengths
measured in the number of substitutions per site. The analysis involved 45 nucleotide sequences. All
positions containing gaps and missing data were eliminated. There were a total of 73 positions in the final
dataset. Evolutionary analyses were conducted in MEGA5.
CONCLUSIONS
Figure 9. Sequencing oxyR F3-R1 PCR products clearly reveal
their identity with modern M. tuberculosis strains.
Osteologic signs of Pott’s disease (spinal tuberculosis) was confirmed using physical and molecular
techniques:
A. the presence of mycolic acids in bone samples was determined by FT-IR spectroscopy;
B. the pncA fragments obtained throught sequencing formed a cluster with MTBC in the ML
phylogenetic tree;
C. oxyR sequences were identical with modern ones with only one sequence having a SNP
ACKNOWLEDGMENT: This study was supported by funding from the project Genetic Evolution: New Evidences for the Study of Interconnected Structures (GENESIS). A Biomolecular Journey around the Carpathians from Ancient to Medieval Times
(CNCSIS-UEFISCDI _PNII_PCCA_1153/2011).