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Dr .

Bhim Rao Ambedkar University , Agra

DNA fingerprinting of
M.tuberculosis isolates from
Chakkipath region of Agra
by using IS6110 probe

Under supervision of :-
Submitted By:-
Dr. D.S Chauhan
Lata Kumari
Scientist D
INTRODUCTIO
N
 Tuberculosis is an ancient and
infectious disease caused by various
strains of Mycobacterium tuberculosis.
 In 1882 Robert koch isolate and
identified Mycobacterium tuberculosis
& hence known as koch bacillus.
 These are slim gram positive acid
fast bacilli (0.4µm x 2-10µm).
 They are nonmotile obligate
aerobes and non sporing .
One third of world population has
been infected with M.tuberculosis, and
new infections occur at a rate of one
per second on a global scale.
 According to WHO annual report 2012
there were an estimate of 8.7 million
incident cases of TB globally.
In India Uttar Pradesh has highest
incidence rate of about 175507 cases
per years.
 There are several diagnostic methods
are available for the epidemiological
studies of mycobacterium strains but
OBJECTIVES OF THE
STUDY
•The main objective of study “DNA
fingerprinting of Mycobacterium
tuberculosis isolates from
Chakkipath region of Agra by
using IS6110 probe”.
•To observe the transmission of the
disease by using this marker .
IS6110 – Restriction
Fragment Length
Polymorphism
The term RFLP refers to the differences in
homologous DNA sequences detected by
presence of fragments with varying lengths after
digestion of DNA .
In RFLP analysis, the DNA sample is broken into
pieces (digested) by restriction enzymes and the
resulting restriction fragments are separated
according to their lengths by agarose gel
electrophoresis.
It uses the insertion sequence IS6110 which is
present in most of the strains of tubercle bacilli
(copy no. 0-25) as a probe to enable the strain
IS6110 RFLP has been used in many
epidemiological studies to detect
outbreaks and to track TB transmission in
large population, to uncover laboratory
cross-contamination and to differentiate
relapse caused by endogenous
reactivation from re- infection by strain .

These studies are based on the


assumption that the DNA polymorphism of
IS6110 patterns among unrelated clinical
isolates is high, whereas epidemiologically
related M. tuberculosis strains show
identical or similar (one- or two-band
METHO Preparation of probe

D DNA Isolation

Restriction of DNA

Electrophoresis

Southern blotting

Prehybridization

Hybridization with IS6110 probe

Detection

Analysis of results
RESULT
1 2 3 4 5 6 7 8 9 10 11 M
S.No Lane No. Isolate No. Copy No.
1 Lane No. 1 PM 14 9
2 Lane No. 2 PM 16 10
3 Lane No. 3 PM 19 6
4 Lane No. 4 PM 30 9
5 Lane No. 5 PM 34 10
6 Lane No. 6 PM 37 11
7 Lane No. 7 PM 43 16
8 Lane No. 8 PM 57 12
9 Lane No. 9 PM 58 4
10 Lane No. 10 PM 59 14
11 Lane No. 11 PM 61 0
12 Lane No 12 Mol. Marker

Group Copy Number Number of isolates


Group A 0 to 5 copy number 2
Group B 6 to 15 copy number 8
Group C More than 15 copy number 1
In the DISCUSSION
present study IS6110
fingerprinting was done and we have taken 11
DNA

isolates from the Mycobacterial Repository


Center at NJIL & OMD Agra, belonged to
Chakkipath region, different hybridization
patterns were obtained suggesting differences in
copy number and genomic location of element.
Results show that maximum number of isolates
having multiple IS6110 copies where as few
were having less copies. Our observations are in
concordance with previous studies published
from JALMA (Chauhan et al 2004 and 2007).
Presence of low copy number of IS6110 or no
copies does not correlate with earlier studies
carried out in South India.
CONCLUSION
Though the number of isolates is less in our
study but the observations indicates that the
IS6110 DNA based fingerprinting could be applied
in such type of settings and the technique
appeals to be very useful for molecular
epidemiology of the tuberculosis in this area.
 Other complimentary techniques are also
required with other genotyping methods with
greater discriminatory power especially in low
copy or zero copy no. of IS6110 probe.
ACKNOWLEDGEME
NT :-
 Dr. Shri Kant
Tripathi
Dr. D.S Chauhan
Mrs. Parul Gupta
Dr. Ajay Vir Singh
All the staff of