Transcript Project Update

Pathways of technology adoption and adaptation
Saheer Gharbia
Applied and Functional Genomics
Centre for Infection
Health Protection Agency
Methods and approaches to
elucidate the spread of infectious
diseases are as old as human
civilisation and stretch in their
logic from magical, philosophical to
scientific.
Pheno-, Sero-, phage and biotypes
extremely valuable, able to identify cellular
components associated with virulence.
Limited discriminatory power, resolving
pathogens into only a few types.
Challenged by Diversity, mutations and
acquisition of new traits
Technology in diagnostics is a
composite of:
•Refining what is established
•Exploring what is new (concept or tool)
•Adapting the new to the function
Examples of Streamlining:
VIDAS®: automated system for Rapid Pathogen Monitoring
VITEK® 2 Compact: automated system for microbial identification
API® / ID 32/ APIWEB®: microbial identification with internet-base tool
TEMPO®: enumeration of quality indicators in food
air IDEAL®: aerobiocontamination control
Count-Tact™ range: monitoring of surface and air biocontamination
BacT/ALERT® 3D microbial detection system
Multiplex Fluorescent Immunoassay
RaPET Rapid Particle Enhanced Technology
Salvador Luria and Max Delbruck
(1943)
Provided a statistical
demonstration that
inheritance in bacteria
follows Darwinian
principles. Mutations occur
randomly in bacterial
populations. They occur in
small numbers in some
populations and in large
numbers in other cultures.
awarded the Nobel Prize in
Medicine or Physiology in 1969.
Classical Molecular Genetics for the
analysis of infectious agents was a
natural follow up especially with the
publication of the Watson-Crick DNA
model.
Gross DNA structure: Restriction Diget,
southern hybridisation, RFLP,AFLP,
plasmid profiling, PFGE, fAFLP and
ribotyping
PCR lead to the next phase by accelerating the
process, adding higher resolution, selective
amplification and quantitative measurements
The power of PCR-based methods is the ease
with which they can be applied to many
bacterial pathogens and their multilocus
discrimination.
Such methods have proven valuable for genetic
dissection of pathogens for which previous
methods have failed.
Dendrogram of MroI FAFLP
However, a limitation of many PCRbased approaches is the biallelic
(binary) nature of their data resulting
from the presence or absence of a
marker fragment.
Draft Microbial
Genomes
The first pathogens to be
sequenced under the
current program are
members of the Bacillus,
Brucella, Clostridium,
Francisella, Shigella, and
Yersinia groups. In many of
these groups, several
strains or related species
will be sequenced, for
example, two strains of
Bacillus anthracis (anthrax)
and one of the similar
species Bacillus
thuringiensis.
Sequence-derived DNA typing is more
rapid and has an even greater
capacity for genetic dissection of
bacterial pathogens.
It is limited only by the genome size
and the technology.
Because most microbial genomes
consist of millions of nucleotides,
technology is invariably limiting.
The flagellum is composed of 20,000
flagellin subunits
• Flagellin subunit is the antigenic determinant
H antigen typing of Salmonella
Kauffmann-White scheme
• Expression of antigens is determined by agglutination with
specific antisera
• In accordance with this scheme, routine clinical laboratories
classify Salmonella by their particular combination of flagellar (H)
and somatic (O) antigens.
• O antigens (60 have been distinguished)
• H1 antigens (63 have been distinguished)
• H2 antigens, not always present (37 have been
distinguished)
• H-antigens are designated by letters of the alphabet
z1, z2 etc.) and by Arabic numerals.
(a, to z,
Identification of unique amino acid
motifs
-_gallinarum_M84979
-_pullorum_B51
g,m_enteritidis_B16
g,m_enteritidis_B18
g,m,s_emek_B20
-_gallinarum_M84975
-_gallinarum_M84976
g,m_enteritidis_B17
g,m,[t]_othmarschen_U06455
f,g,m,p_enteritidis
g,m_essen_U05299
g,q_moscow_Z15086
g,p_dublin2_M84972
g,p_dublin3_M84973
g,p_dublin1_z15067
g,p_dublin_B12
g,p,u_rostock
g,p,s_naestved
g,m_enteritidis_B19
g,m,[p],s_montevideo
g,m,{p},s_montevideo_B31
[g,s,t]_simsbury
g,s,t_senftenberg
g,[s],t_B59
g,t_budapest
f,g,s_agona_B01
f,g_derby
f,g_derby_B09
f,g_adelaide
f,g,t_berta
m,t_banana
m,t_oranienburg
g,z51_C15
g,z51_C09
g,z51_newmexico
Sequence distances of fliC
of different serotypes
g-complex sequences
r_A37_heidleberg
r_A30_heidleberg
r_A40_heidleberg
r_A32_heidleberg
r_A31_heidleberg
i_typhimurium_C01
i_typhimurium_A01
i_typhimurium
e,h_anatum_B02
e,h_saintpaul_A22
e,h_newport_B36
c_choleraesuis_B04
c_choleraesuis_AF159459
z41_bongori_C11
z_indiana_B25
z10_hadar
z10_haifa_B22
z35_arizonae_C08
k_thompson_B62
l,v_panama_B39
l,v_B03
a_miami_B28
d_muenchen_A63
d_muenchen_X03395
d_duisberg_B15
b_paratyphiB_A41
z4,z24_seminole_C16
z4,z23_stanleyville_B61
z4,z23_arizonae_C05
68.6
60
50
40
30
Nucleotide Substitutions (x100)
20
10
0
“non-g” sequences
Analysis of several SNPs
5’-C/TGGCCGGGTCACGAT/GGCCC-3’
Identification of H1:g,p vs. H1:g,m
•A test was designed specifically for differentiation
between dublin and enteritidis serotypes.
•Based on a SNP in the central variable region.
S. dublin genotype:
S. enteritidis genotype:
•One of the most recent developments in molecular analysis involves
the analysis of VNTR sequences.
•Short nucleotide sequences that are repeated multiple times often vary
in copy number, creating length polymorphisms that can be detected by
PCR using flanking primers.
•Satellites: Spanning megabases
•Minisatellites: Repeat units 6-100bp (spans 100’s bps
•Microsatellites: Repeat units 1-5 bp (spans 10’s bps)
The multi-locus
VNTR banding
patterns, as originally
described by Alec
Jeffreys enable us to
determine
relationships and
degrees of
relationship between
individuals. Bands on
the blot may be
classified as 'M' for
maternal, 'P' for
paternal, 'I' for
invariant, or 'X' for
non-parental.
VNTR Background
Polymorphism at a VNTR locus can occur either as a result of
nucleotide sequence changes between individual repeat units or as a
result of variation in the number of repeat units, hence creating allelic
variants.
TAGACTAGATAGC
TAGACTAGATAGC
TAGACTAGATAGC
GATCATCGGT
A
GATCATCGGTCATAGACTAT
T
A
G
GATCATCGGTCATAGACTATGATC
Electrophoretic analysis of VNTR fragments from
different B. anthracis isolates
Capillary Electrophoresis
VNTR
Cluster Analysis
Categorical
100
50
VNTR largest x-y
K@lisademo@00000006
18-NCTC_10. Nairobi.
.
1963 PH 80/63
.. 10329 and 10330 from same. Antiqua
K@lisademo@00000016
E168704B
.
K@lisademo@00000003
16-NCTC_10. Nairobi.
.
1958 13925/58 .. 10029 and 10030 from same. Antiqua
K@lisademo@00000004
17-NCTC_10. Nairobi.
.
1958 13927/58 .. 10029 and 10030 from same. Antiqua
K@lisademo@00000010
19-NCTC_10. Nairobi.
.
1963 PH 90/63
.. 10329 and 10330 from same. Medievalis
K@lisademo@00000013
Kenya
.
K@lisademo@00000001
13-NCTC_59. .Java
Yp2
.Kenya?
.1939 Tjiwidej
.
Yp1
..
Orientalis
K@lisademo@00000017
E16895B
K@lisademo@00000008
11-NCTC_20. .Java; . 1925 Java
K@lisademo@00000009
10-NCTC_570. India;
.
. 1920 Bombay 2. .. patient from Maratha plague . Orientalis
K@lisademo@00000005
09-NCTC_144. India;
.
. 1920 Parel
K@lisademo@00000007
12-NCTC_28. Bomb.
.
1928 Bombay 1 .. patient from Maratha plague . Orientalis
K@lisademo@00000002
08-NCTC_59. .Type s. 1939 Soemeda. ..
K@lisademo@00000014
A1122
K@lisademo@00000011
14-NCTC_87. .Probab.1953 139 L
.. sample received from India
.Orientalis
K@lisademo@00000012
15-NCTC_87. .Probab.1953 TS
.. sample received from India
.Orientalis
K@lisademo@00000015
FV-1
.
.Califor. 1939
.
.
.. received via Pasteur Institute .Orientalis
..
Orientalis
Orientalis
. Spermophilus beecheyi
Microarrays
Array Technology
High density oligonucleotide arrays
High density ‘spotted’ arrays
Low density ‘line-probe’ arrays
Low density addressable arrays
Pathogenesis
The CDC, NAID: SARS, smallpox
Disease Susceptibility
“susceptibility genes” in HIV
Vaccine Development
to examine transcriptional activity of all genes of pathogenic
microorganisms under in vivo conditions
Pathogen Identification
Identify pathogens using ribosomal DNA sequences
Drug Response
identify the presence of drug resistance genes
catalog individual genetic variations in drug resistance
Imagine a world where microscopic medical implants patrol our
arteries, diagnosing ailments and fighting disease; where military
battle-suits deflect explosions; where computer chips are no bigger
than specks of dust; and where clouds of miniature space probes
transmit data from the atmospheres of Mars or Titan.
Many incredible claims have been made about the future's
nanotechnological applications, but what exactly does nano mean,
and why has controversy plagued this emerging technology?
Nanotechnology is science and engineering at the scale of
atoms and molecules. It is the manipulation and use of
materials and devices so tiny that nothing can be built any
smaller.
Green Emission Quantum Dot
Fluorene Oligomer for Electronic Applications
Water Soluble & Functional NIR Dyes
Molecular diagnostics is becoming a driving force
in drug development. Applications have spread
from identifying infections to include screening for
cancer, hepatitis and genetic disorders
Personalised Medicine
The scientific community is progressing quite
rapidly in developing molecular diagnostics, and
industry is developing assay prototypes and
conducting larger validation studies to advance
this research to full clinical utility.
The risk associated with the newer
technologies is that the accuracy and
precision of the data generated will be
compromised. Highly specific assay
formats are required to detect DNA
sequence variations
To achieve such specificity, Careful
assay design, Validation, Quality Control
Standards are required