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MATERIAL AND METHODS

Sample
collection:

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The sputum samples were collected from 375 patients
having both male and female of different ages at Provincial TB Reference Laboratory,
Hayatabad Medical complex from Khyber pakhtunkhwa, Pakistan in the year of 2015.
The sputum samples were collected early in the morning in a sterile wide mouth
screw capped bottle. Collected samples were labeled with laboratory entry
number.

Sample
Processing and Culturing:

 I take sputum sample (at least 2 ml, not more than 5 ml) into a wide mouth screw
capped bottle. Add an equal volume of N-Acetyl-L-Cysteine-Sodium Hydroxide (NALC-NaOH) solution. Tighten the cap of the tube and shake or
vortex-mix for 20 seconds. Then keep at room temperature for 15 minutes with occasional hand shaking for decontamination. Fill the tube with sterile phosphate buffer to reached 50 ml volume. Vortex, mix and Centrifuge at 3000 rpm for 15 minutes. Then carefully pour off the
supernatant into a
discard can and resuspend the sediment in sterile buffer.

Inoculate 0.5 ml sample into Mycobacterium growth indicator tube
(MGIT). A BBL
MGIT tube (from Becton Dickinson) containing 7 mL modified middle brook 7H9
broth media. Lyophilized MGIT PANTA (containing polymyxin B, azlocillin,
nalidixic acid, trimethoprim, amphotericin B) was reconstituted with MGIT
growth supplement (containing oleic acid, albumin, dextrose, catalase, polyoxyethylene
stearate), and 0.8 ml of this was added prior to sample inoculation to the MGIT
tube. The inoculated media were incubated at 37 °C, MGIT tubes were incubated
inside the M960 instrument for 6 weeks.  M960 instrument detects the growth automatically;
flashing red light to indicate instrument positive tubes and green for negative
ones.
2-4 drops sample were also inoculated onto Lowenstein Jensen (LJ) media, and were incubated for 8 weeks and examined
every week,
also make a slid for microscopic examination through FM staining method.

Drugs
Susceptibility test (DST)

Prior to setting up the
DST, Drug vials of Streptomycin (STR) Isoniazid (INH) Rifampicin (RIF)
Ethambutol (EMBT) were reconstituted with 4 ml of sterile deionized (DI) water
and mixed well. Sets of five MGIT tubes were prepared per specimen for
performing the DST. One tube was labeled “growth control” (GC), one for each
STR, INH, RIF and EMBT. Then 0.8 ml of SIRE supplement was added into each of
the five labeled MGIT tubes. The next step was the addition of drugs, 0.1 ml of
reconstituted lyophilized STR, INH (0.1 µg/ml final concentrations), RIF (1.0 µg/ml)
and EMBT drug was added to particular labeled tube. After mixing the medium,
0.5 ml of the well mixed reconstituted sediment was inoculated in each of the
four drug containing tubes. For the control, the resuspended sediment was
diluted 1:10 by adding 0.2 ml of the well-mixed sediment into 1.8 ml of sterile
distilled water. After being thoroughly mixed, 0.5 ml was inoculated into GC
tubes. The tubes were mixed again by inverting several times. Growth control
and four drug containing tubes were placed in one set carrier. These set
carriers were entered in the MGIT M960
instrument for 4 to 13 days. The first tube in the set carrier was
always the control tube.

DNA
Extraction

The INH resistant samples were further processed for
DNA isolation. Pipet 1 ml of bacterial
culture into a 1.5 ml microcentrifuge tube, and centrifuge for 5 min at 5000 x g
(7500 rpm). Calculate the volume of the pellet and add Buffer ATL (supplied
in the QIAamp DNA Mini Kit) to a total volume of 180 ?l. Add 20 ?l Proteinase
K, mix by vortexing, and incubate at 56°C until the cell is completely lysed.
Vortex occasionally during incubation to disperse the sample. Briefly
centrifuge the 1.5 ml microcentrifuge tube to remove drops from the inside of the
lid. Add 200 ?l Buffer AL to the sample, mix by pulse-vortexing for 15 s, and
incubate at 70°C for 10 min. Briefly centrifuge the 1.5 ml microcentrifuge tube
to remove drops from inside the lid. Add 200 ?l ethanol (96–100%) to the sample
and mix by pulse-vortexing for 15 s. After mixing, briefly centrifuge the 1.5
ml microcentrifuge tube to remove drops from inside the lid. Carefully apply
the mixture to the QIAamp Spin Column (in a 2 ml collection tube) without
wetting the rim. Close the cap, and centrifuge at 6000 x g (8000 rpm)
for 1 min. Place the QIAamp Spin Column in a clean 2 ml collection tube
(provided), and discard the tube containing the filtrate. Carefully open the
QIAamp Spin Column and add 500 ?l Buffer AW1 without wetting the rim. Close the
cap, and centrifuge at 6000 x g (8000 rpm) for 1 min. Place the QIAamp
Spin Column in a clean 2 ml collection tube (provided), and discard the
collection tube containing the filtrate. Carefully open the QIAamp Spin Column
and add 500 ?l Buffer AW2 without wetting the rim. Close the cap and centrifuge
at full speed (20,000 x g; 14,000 rpm) for 3 min. Place the QIAamp Spin
Column in a clean 1.5 ml microcentrifuge tube (not provided), and discard the
collection tube containing the filtrate. Carefully open the QIAamp Spin Column
and add 200 ?l Buffer AE or distilled water. Incubate at room temperature for 1
min, and then centrifuge at 6000 x g (8000 rpm) for 1 min. the DNA were
store in -20°C.

Polymerase
Chain Reaction (PCR):

KatG gene specific set of primers
i.e. TB86 and TB87 were used for amplification in GTQ-Cycler 96 (Hain Life
Science, Germany). The 5x FIREPol
Master Mix were used which contained 5X Reaction buffer B (0.4 M Tris-HCl, 0.1 M (NH4)2SO4, 0.1% w/v
Tween-20), 7.5 mM MgCl2 (1x PCR
solution – 1.5 mM MgCl2), 1 mM
of each dNTPs (1x PCR solution
– 200 ?M dATP, 200 ?M dCTP, 200 ?M dGTP and 200 ?M dTTP), 2-5 U FIREPol DNA Polymerase (0.05-0.5 U/
?l). For each individual sample, the PCR amplification mix (25 ?l) was prepared
in a DNA-free room which include 5 ?l 5x
FIREPol Master Mix, 0.5 ?l Forward primer (10 `pmol/?l), 0.5 ?l Reverse
primer (10 pmol/?l) , 5 ?l DNA template and PCR grade water up to 25 ?l (Master
Mix is know 1x concentration of each reagent). All the samples were loaded with
one negative control containing 5 ?l of sterile distilled H2O and PCR program
were set to an initial denaturation at 95°C for 10 minutes was followed by 35
cycles of denaturation at 95°C for 45 seconds, annealing at 62°C for 1 minute
and extension at 72°C for 40 seconds. A final extension step at 72°C for 8
minutes concluded the reaction program. The amplified regions have a length of
230 bp (KatG).

Gel
Electrophoresis:

PCR product will checked on 2% Agarose gel
Electrophoresis for confirmation of products. For each agarose gel, the
appropriate weight of Agarose-1000 was weighed, added to the appropriate volume
of 1X tris borate EDTA (TBE), and left to hydrate for 15 minutes. The
agarose-TBE mixture was then heated in a microwave until boiling, removed and
mixed. The agarose was then left to cool for 5 minutes at room temperature and
add 5 ?l ethidium bromide. The agarose was poured into the gel tray, and gel
comb was inserted, left to set for 30 minutes at room temperature. The gel was
then inserted into the gel tank, TBE was added such that there was at least 0.5
cm TBE buffer above the gel, and gel combs were removed. 100 bp ladder was
added to first wells and 5 ?l of each amplicon was mix with loading dye and
added to the corresponding well. The gel tank lid was placed and connected to a
power supply unit. Voltage and time was set to the desired values. Image was
taken using an Image Master Gel Documentation System.

Sequencing

PCR
products were submitted for PCR clean-up and sequencing at the Central Analytic
Facility of Stellenbosch University. Sequences were then compared with the
whole genome sequence of M. tuberculosis H37Rv reference
(http://genolist.pasteur.fr/TubercuList) to determine if mutations are present
in the candidate genes from different M. tuberculosis strain families.

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