Non tuberculosis Mycobacteria and Mycobacteria tuberculosis complex differentiation in clinical

isolates and its relevance

Project Report

Submitted to

Hemwati Nandan Bahuguna Central University, Srinagar, Uttarakhand

For Partial Fulfillment of Award of Degree in Master of Science in Microbiology

Under the Guidance of

Supervisor Co-Supervisor
Dr. NAROTAM SHARMA Dr. Nita Garg
Department Scientist, Central Professor and Head, Biochemistry
Molecular Research Laboratory

Biochemistry Department,

Central Molecular Research Laboratory ,Shri Guru Ram Rai Institute of Medical & Health
Science, Patel Nagar, Dehradun (Uttarakhand), 248001

Submitted by

PREETIKA RANA

Department of Microbiology, Hemwati Nandan Bahuguna Central Garhwal University Srinagar,

Uttarakhand
 Declaration
I, the undersigned, hereby declare that the Project entitled, Non tuberculosis Mycobacteria
and Mycobacteria tuberculosis complex diffrentiation clinical isolates specimens and its
relevence , is a study conducted at Central Molecular Research Laboratory, Department of
Biochemistry of Shri Guru Ram Rai Institute of Medical & Health Sciences, Patel Nagar,
Dehradun, Uttarakhand, India, under the supervision of Dr. R.K Singh, Professor and Head,
Biochemistry Department, and co-supervised by Dr. Narotam Sharma, Scientist, Central
Molecular Research Laboratory, Shri Guru Ram Rai Institute of Medical & Health Sciences,
Patel Nagar, Dehradun, Uttarakhand, India. The pragmatic finding in this project is based on the
data and information collected by myself.

PREETIKA RANA

M.Sc. Microbiology [4th sem]

HNB Garhwal University, Srinagar, U.K.

 Acknowledgements
It is my privilege to express our sincerest regards to our Head of the Department Dr. A.B. Bhatt
HNB Garhwal University Dr. Narotam Sharma scientist at central molecular research
laboratory of Shri guru ram rai institute of health and medical science Dehradun.

I am also thankful to my project coordinator Dr. Nita Garg for their valuable inputs and able
guidance, encouragement, whole hearted cooperation and constructive criticism throughout the
duration of my project.

I deeply express my sincere thanks to Dr. Narotam Sharma for encouraging and allowing us to
present the project on the topic Non tuberculosis Mycobacteria and mycobacteria tuberculosis
complex differentiation in clinical isolates specimens and its relevance and I am also
thankful to Mr. Satish Chandra Nautiyal who continuously helping me regarding the project
and give me the chance to work on the diagnostic samples that helpful for me in the future.

I take this opportunity to thank all my lectures Dr. Seema Rawat have directly or indirectly held
my project.

I am also thankful to all the staff members of Biochemistry Department of Shri Guru Ram Rai

Institute of Medical & Health Sciences for helping me whenever I approached them.I cannot

express in words the sacrifices of my parents. Their motivation, inspiration and care are

incredible. I will always remain indebted to them

.
 Contents

Particulars Page

Sr.no.

1 Introduction 8-16

2 Review of literature 16-50

3 Aims and objectives 51

4 Materials and methods 52-

5 Results

6 Conclusion

7 References

List of Abbreviations

BT Buffer tissue

EB Elution buffer

EtBr Ethidium bromide

EDTA Ethylenediamine tetra acetic acid

HEPA High efficiency particulate air

IS Insertion sequence

MCT Micro centrifugal tube

NFW Nuclease free water

PCR Polymerase Chain Reaction

TAE Tri-acetate EDTA

Table No. Description Page No.

1 Master Mix Preperation For tb
2
3
ULPA Ultra low penetrate air filter

BR Binding Rack

WR Washing Rack

ER Elution Rack

BSC Bio-Safety cabinet

BSL Bio Safety Levels

PK proteinase K

IC Internal Control

4-MATERIALS AND METHODOLOGY

Site of Implementation of the work:-
All the experiments for current work, were carried out at lab Central
Molecular Research Laboratory, Department of Biochemistry, Shri Guru Ram
Rai Institute of Medical and Health Sciences (SGRRIM&HS), Patel Nagar,
Dehradun (U.K.).

The clinical specimens work form , Shri Guru Ram Rai Institute of Medical and
Health Sciences (SGRRIM&HS),

Fig.20.Shri Guru Ram Rai Institute of Medical and Health Sciences

Biosafety and Precaution Taken:-

Biosafety A biosafety level is a level of the biocontainment precautions required to isolate

dangerous biological agents in an enclosed laboratory facility. The levels of containments range
from the lowest biosafety level 1 (BSL-1) to the higest at level 4 (BSL- 4). An agent of
biological origin that has the capacity to produce denterious effects on human.

Biosafety levels Levels of contentment having the more the facilities grow the dangerous
pathogen (BSL-2, BSL-3) example H1N1 virus
 BSL-1- this level is suitable for work involving well characterized agents not known to
consistently cause disease in healthy adult humans, and of minimal potential hazard to
laboratory personnel and the environment. It includes several kinds of bacteria and
viruses including canine hepatitis, non pathogenic Escherichia coli, as well as some cell
cultures and non infectious bacteria.

BSL-2- this level is similar to biosafety level 1 and is suitable for work involving agents
of moderate potential hazard to personnel and the bacteria and the viruses that causes
only mild disease to humans, or are difficult to contract aerosol in lab setting, such as C,
human immunodeficiency virus (HIV), orthopoxviruses ,infiuenza A, Lyme
disease,salmonella,mumps, measles.
 BSL-3- Indigenous/ exotic agents associated with human disease with the potential for
aerosol transmission. This level is applicable to clinical,diagnostic, teaching,research,or
production facilities in which work is done.it includes various bacteria,parasites and
viruses that can cause severe to fatal disease in human .
 BSL-4- This level is required for work with dangerous and exotic agents that pose a high
individual risk of aerosol-transmitted laboratory infections, agents which causes server to
fatal disease in humans for such as bolivian and argentine hemorrhagic fevers, Marburg
virus ,Ebola virus,and various other hemorrhagic diseases. This level is also use for work
with agents such as smallpox that are considered contagious enough to require the
additional safety measures, regardless of vaccination availability.
 Precaution taken

We have take the precaution in laboratory. We have wear the personal protective equipments
(PPE), those undergoes to upper, lower, shocks, cap, gloves & mask. They equipments protected
our body highly infectious pathogens.

Instrumentation Facilities at BSL-2, BSL-3 and General Laboratory Areas at

Central Molecular Research Laboratory.

Instrumentation Facilities at BSL-2, BSL-3 and General Laboratory Areas at

Central Molecular Research Laboratory.
Sr. Name of Equipment Molecular Biology reagent Accessories
No
1 Real Time PCR Machine (CTM Buffer(10x)
Roche Taq Man 48) Latex gloves and powder free
gloves

Mask
2 Real Time PCR Machine MgCl2(25mm)
(Qiagen Rotor Gne-Q ) Cotton

3 UV Tran- illuminomester dNTPs(2mm)

Tissue paper

4 Gradient Thermal Cycler Taq DNA polymerase

5 Gradient Thermal Cycler Nuclease free water

Parafilm

6 Biosafety cabinet class II Protienase K

Measuring cylinder

7 Biosafety cabinet class III Carrier RNA

Gel Casting tray

8 Laminar Air flow cabinet

Aerosol barrier tips( 0.1-10l ,
10-20 l ,

9 Gel Electrophoretic Unit with Agarose Powder

Power Supply Pipettes (10l-100l,2s0l-
200l & 100l-1000l)

10 E-Gel Imager System with Tris- Acetate (1x)

UV Light Base Microcentrifuge tube(MCT)
11 Spectrophotometer Tris Acetate (50x) Flask
12 Cooling centrifuge Ethanol PCR tube
13 Cooling centrifuge Bromo phenol blue(tracking dye) MCT stand
14 Micropipette DNA molecular marker(DNA Pipette stand
ladder)
15 Vortexer Carbon fusion Spatula
16 Vortexer Decolourise Transportation box
17 PCR Cabinet Methylene blue Slides
18 Dry Bath Lysis Buffer Silica Column
19 Water Bath Washing Buffer Collection Tube
20 Deep freezer (-20) Elution Buffer
21 Refrigerator
22 Cooling centrifuge
23 Microfuge
24 Air Curtain
25 Split AC 1.5 ton for Thermal
cyclers
26 UPS 5kw for Thermal cycler
power backup
27 Biosafety cabinet class II
28 Non cooling centrifuge
29 Incubator
30 Distillation Unit
31 Veriti Gradient Thermal Cycler,
Veriti 96-Well Thermal Cycler
32 E-Gel Imager System with
UV Light Base
33 Biosafety cabinet vertical for
BSL III lab.
34 -70 Centigrade Refrigerator
35 Double door Horizontal
Autoclave
36 Cooling Centrifuge
37 Heating Block

Required Materials
 Fig.5. PCR workstation Fig. 6.Horizontal laminar air hood

Fig.10. Real Time PCR Fig.11.

Vortexer

Fig.12. Bench top PCR Fig.13. ABI veriti PCR

Methodology

Sample Collection Pulmonary & Extra pulmonary

Clinical specimens or samples were collected from various Departments of
Shri Mahant Indiresh Hospital after the clearance of Ethical committee of the
Hospital.

Extra pulmonary specimens Pulmonary

1. Ascetic fluid 1. BAL

2. Blood 2. Plural fluid
3. CSF (Cerebrospinal fluid)
4. Drain fluid
 5. Endometrial biopsy
6. Endometrial tissue
7. FNAC(Fine Needle Aspirate Cytology)
8. Menstrual blood
9. Pus
10. Pendromial biopsy
11. Pigment breast
12. Tissue
13. Urine

(1)-Preprocessing of all clinical specimens (Urine, Tissue, BAL, Pus,

Endometrial tissue, Endometrial biopsy, Pendromial biopsy)

Transfer sample in 50ml MCT

Add sample washing buffer in sample

50ml MCT in Non cooling centrifuge for 15 minute

Discard the supernatant

 Add Pellet in MCT

(2)- DNA isolation specimen of pulmonary & extra pulmonary

Add 200l sample in MCT

Add 200l Lysis buffer

Add 25l Proteinase K

Incubate at 56 for 30 minute (Dry Bath)

Add 200l Lysis buffer 2

Add 250l chilled ethanol

Vortexing

Transfer the sample in labeled silica column

 Centrifuge at 13000rpm for 1 minute

Discard the collection tube

Transfer the silica new collection tube

Add 500l washing buffer 1

Centrifuge at 13000rpm for 1 minute

Decant the collection tube

Add 600l washing buffer 2

Centrifuge at 13000rpm for 1 minute

Decant the collection tube

Centrifuge at 13000rpm for 1 minute of the empty column (dry washing)

Discard the collection tube & transfer the silica column new labeled MCT
Preheated elution buffer add 60l (incubate the elution buffer at 70 for

15 minute)

Hold room temperature at 2 minute

Centrifuge at 13000rpm for 2 minute

Recover the DNA

(3)-Preparation of Master Mix for IS6110:-

1st Round amplification

1st amplification premix (purple cap) - 8.2l

Hot start Taq polymerase 0.33l

Uracil DNA glycasylase 0.5l

2nd Round amplification

2nd amplification premix (red cap) - 14.7l

Hot start Taq polymerase 0.33l

Pipette 9.03 l of the master mix into each PCR tube. Then add 3l of the
eluted sample DNA, and mix well by pipetting repeatedly up and down for
first round. Pipette 15.03l of the master mix take the each PCR tube

(4)- Preparation of Master Mix for Non tuberculosis Mycobacterium

(rpoB gene):-

Sr.no. Number of samples For 1 reaction For 10 reaction

1 Buffer (10x) 2.5l 2.5*10= 25l

2 dNTPs(2mm) 2l 2*10= 20l

3 MgCl 2(25mm) 3l 3*10= 30l

4 Primer 1(25mm) 0.30l 0.30*10= 3.0l

5 Primer 2(25mm) 0.30l 0.30*10= 3.0l

6 Taq (30/l) 0.12l 0.12*10= 1.2l

7 Nuclease free water 4.28l 4.28*10= 42.8l

Pipette 12.5l of the master mix into each PCR tube. Then add 12.5 l of the
eluted sample DNA, and mix well by pipetting repeatedly up and down.

Amplification
For the amplification of rpoB gene, cycling conditions as well as concentration and volume of
the reagents were initially standardized and optimized. For standardizing cycling parameters i.e.
denaturation, annealing, extension and final extension in terms of temperature (in degree
centigrade) and duration (in seconds) a PCR program was run.

PCR

The polymerase chain reaction (PCR) is a biochemical technology in molecular

biology to amplify a single or a few copies of a piece of DNA across several orders of
magnitude, generating thousands to millions of copies of a particular DNA sequence. Developed
in 1983 by Kary Mullis [54,]. PCR is now a common and often indispensable technique used in
medical and biological research labs for a variety of applications. These include DNA
cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis
of hereditary diseases; the identification of genetic fingerprints (used in forensic
sciences and paternity testing); and the detection and diagnosis of infectious diseases. In 1993,
Mullis was awarded the Nobel Prize in Chemistry along with Michael Smith for his work on
PCR [55]. PCR is used to amplify a specific region of a DNA strand (the DNA target). Most PCR
methods typically amplify DNA fragments of between 0.1 and 10 kilo base pairs (kb), although
some techniques allow for amplification of fragments up to 40 kb in size[56]. The amount of
amplified product is determined by the available substrates in the reaction, which become
limiting as the reaction progresses [57].
Principle of PCR

PCR is used to amplify a specific region of a DNA strand (the DNA target). Most PCR methods
typically amplify DNA fragments of between 0.1 and 10 kilo base pairs (kb), although some
techniques allow for amplification of fragments up to 40 kb in size. The amount of amplified
product is determined by the available substrates in the reaction, which become limiting as the
reaction progresses. Typically, PCR consists of a series of 20-40 repeated temperature changes,
called cycles, with each cycle commonly consisting of 2-3 discrete temperature steps, usually
three. The cycling is often preceded by a single temperature step (called hold) at a high
temperature (>90C), and followed by one hold at the end for final product extension or brief
storage.

Step 1. Initialization: - This step consists of heating the reaction to a temperature of 9496
C (or 98 C if extremely thermo stable polymerases are used), which is held for 19 minutes. It
is only required for DNA polymerases that require heat activation by hot-start PCR.[59]

Step 2. Denaturation: - This step is the first regular cycling event and consists of heatingthe
reaction to 9498 C for 2030 seconds. It causes DNA melting of the DNA template
bydisrupting the hydrogen bonds between complementary bases, yielding single-stranded DNA
molecules.

Step3. Annealing: - The reaction temperature is lowered to 5065 C for 2040 seconds
allowing annealing of the primers to the single-stranded DNA template. Typically the annealing
temperature is about 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-DNA
hydrogen bonds are only formed when the primer sequence very closely matches the template
sequence. The polymerase binds to the primer-template hybrid and begins DNA formation.

Step4.Extension:-The temperature at this step depends on the DNA polymerase used; Taq
polymerase has its optimum activity temperature at 7580 C[60,61] and commonly a
temperature of 72 C is used with this enzyme. At this step the DNA polymerase synthesizes a
new DNA strand complementary to the DNA template strand by adding dNTPs that are
complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the
dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. The
extension time depends both on the DNA polymerase used and on the length of the DNA
fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase
will polymerize a thousand bases per minute. Under optimum conditions, i.e., if there are no
limitations due to limiting substrates or reagents, at each extension step, the amount of DNA
target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment.
Step 5.Final Extension:- This single step is occasionally performed at a temperature of 70
74 C for 515 minutes after the last PCR cycle to ensure that any remaining single-stranded
DNA is fully extended.

Step 6.Storage:- It is done at 415 C for an indefinite time may be employed for short-term
storage of the reaction.
Fig.20. Schematic drawing of the PCR cycle. (1) Denaturing at 9496 C. (2) Annealing at
~65 C (3) Elongation at 72 C. Four cycles are shown here. The blue lines represent the DNA
template to which primers (red arrows) anneal that are extended by the DNA polymerase (light
green circles), to give shorter DNA products (green lines), which themselves are used as
templates as PCR progresses.
Table no.3. Standardization of PCR cycling conditions for the amplification of rpoB gene:

Steps Initial Denaturatio Annealing Elongation Final

Denaturatio n extension
n
Temperature 94 C 94C 63.5 C 72 C 72 C
Duration 5 minutes 30 seconds 1 minute 1 minute 5 minutes
Number of 1 40 1
Cycles
Hold / 4 C
Storage of
Amplicons

Nested PCR
Introduction :- Nested polymerasechain reaction (Nested PCR) is a modification of polymerase
chain