Ethical Baltistan and Fedrally Administered Trible Area

Ethical
Consideration

The present study was approved by the
ASRB committee of Abdul Wali Khan University Mardan (AWKUM).

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Study
Areas

 The present study was designed keeping in
consideration the country of Pakistan. Pakistan with an area of 796,096 km²
divided into five provinces namely Punjab, Sindh, Balochistan, KPK, Gilgit Baltistan
and Fedrally Administered Trible Area (FATA). Our area of focus was the
province of KPK spread over 74, 521 km² area with a population of over 22
million.

 Blood sampling was done from different
districts of  Hazara division for this
research work. The current study was conducted from July 2016 to feb 2017 in
Molecular lab of Biochemistry department Abdul Wali Khan University Mardan
along with different hospital’s labs and private labs from where the samples
were collected.

Data
Collection for Prevalence of Malaria

Data was collected from different
districts of hazara division. Different labs and hospitals were visited
specially DHQs (District headquarter) hospitals. Three years data, from year
2015 to 2017 was collected with the help of different lab technicians and
hospital administrative. Especially the team manager of Malaria roll back
programme Hari pur helped alot in the provision and collection of data.

Blood Sample Collection

Blood samples were collected from malaria positive
patients using 5 ml syringes and were preserved
in sterile vacutainer
tubes containing Ethylenediamine tetra acetic acid (EDTA) as an anticoagulant. These
samples were collected from districts of Hazara with varying numbers of samples
on the basis of a division Haripur, Abbottabad and Mansehra. Blood samples collection
was done in session ( i.e. may 2016 
to  September 2016 ,  during which malaria prevails at a high
rate). These samples were stored at -20°C in the Molecular laboratory of biochemistry department AWKUM.

Purification and Extraction of
Genomic DNA from Blood.

For the purpose to study all the
samples and to characterize different genotypes of P. falciparum, the human genomic DNA was extracted and purified
from whole blood samples using Qiagen Mini kit (Cat. No.
51304).

Procedure

1. Twenty microliter  Protease (or proteinase K) was taken into the
bottom of a 1.5ml microcentrifuge tube.

2. Then 200?l of whole blood sample was
added to the microcentrifuge tube.

3. Two hundred microliter (400?l) volume
of  Lysis Buffer  was added to the sample and mixed by
pulse-vortexing for 15 seconds.

In order to ensure efficient lysis, it is essential that the
sample and Buffer AL are mixed thoroughly to yield a homogeneous solution.

4. The samples were then incubated at
56°C for 10 minutes.

5. Samples were centrifuged the 1.5 to
remove drops from the inside of the lid of microcentrifuge tubes.

6. About 200 ?l ethanol (96–100%) was
added to the samples and mixed again by pulse-vortexing for 15 seconds. After
mixing, sample tubes were centrifuged to remove drops from the inside of the
lid.

7. The mixture from step 6 was applied to
the  GeneJET Genomic DNA Purification
column (in a 2ml collection tube) without wetting the rim. Closed the cap and
centrifuged at 6000 x g

(8000 rpm) for 1 minute. Placed the
GeneJET Genomic DNA column in a clean 2 ml collection tube (provided) and
discarded the tube containing the filtrate.

8. The GeneJET Genomic DNA column was
carefully opened and 500?l Wash  Buffer I
was added without wetting the rim. The cap was closed and centrifuged at 8000 x
g (8000 rpm) for 1 minute. Discord the flow through and placed the column back
into the collection tube.

9. The 
spin column was opened and 500?l Wash Buffer II  was added without wetting the rim. The cap was
closed and centrifuged at full speed (>12,000 x g; 14,000 rpm) for 3 minutes.

10. 
The GeneJET Genomic DNA column was placed in a clean 1.5ml
microcentrifuge tube (not provided), and discarded the collection tube containing
the filtrate. Carefully opened the  spin
column and added 200?l Elution Buffer  or
distilled water.

Incubated at room temperature (15–25°C)
for 2 minute, and then centrifuged at 8000 x g (8000 rpm) for 1 minut

A second elusion step with further 200?l Elution  Buffer  increased the yield up to 15%.

Discord the purification column.

Gel
Electrophoresis

1% Agarose gel was prepared to check the extracted DNA from both
quantitative and qualitative point of view and was detected by using Ethidium
bromide. The extracted DNA was visualized under ultraviolet illuminator, at a
wavelength of 254 nm using Gel documentation system.

Nested
Polymerase Chain Reaction

Nested PCR is used to
increase the specificity and yield of amplification of target DNA, when ever
primer has a potential to bind sequence other than target area. Two sets of
primers were used in Nested PCR. Role of first primer set was to bind to the sequences
outside the target DNA, same as standard PCR but it may also get attached to
other areas of the template. The second primer set role was to bind with sequences
in the target DNA that is within the part amplified by the first set. Thus with
the products of the first reaction, the second set of primers bound and
amplified the target DNA.

Primer Used in
Nested PCR for Detection of Plasmodium
falciparum Gene

During present study, P. falciparum merozoits surface protein
specific primers pfmsp1 and Pfmsp2 and types specific primers of pfmsp1 and pfmsp2 such as K1, MAD20 and RO33 family of pfmsp1 and FC27 family and 3D7/IC1 of pfmsp2 were designed by eurofins mwg/operon. The sequence of these
primers is given in Table No 3.1

Table
1: Primers
sequences used for Nested PCR analysis

 

PCR
Analysis

Initial PCR

For initial PCR, 20.5µl of the PCR master mix was taken into a PCR tube and 3µl of gDNA was added to it. Then 0.5µl of both forward and reverse primer of pfmsp1 and pfmsp2 were added. PCR was run under the following conditions. To
each tube, 21.5µl of PCR master and 2µl of extracted DNA were added. Then 0.5µl of the primers of family pfmsp1 and pfmsp2 were added.  

The PCR was run using
the following program:

The initial
Denaturation at 95°C for 5 minutes and 35 cycles of, denaturation at 94°C for
30 seconds, annealing at 55°C for 30 seconds and extension at 72°C for 1
minute. Lastly, extension at 72°C for 5 minutes was performed. PCR was run and
initial PCR products were obtained.

2nd PCR

Five sets of each tube
of the initial PCR products were made and labeled according to the desire. To
each tube, 21.5µl of PCR master and 2µl of extracted
DNA were added. Then 0.5µl of the initial
primers of family pfmsp1 and pfmsp2 were added. The initial denaturation at 95°C for 5 minutes and 35
cycles of, denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds
and extension at 72°C for 1 minute. Final extension at 72°C for 5 minutes was
performed. PCR was run and final PCR products were obtained.

Visualization
of PCR Products by Gel Electrophoresis

Agarose gel Electrophoresis was conceded out to
study the amplified DNA samples in    2 % agarose
gel and were detected by Ethidium
bromide staining. The amplified products were visualized under
ultraviolet illuminator at a wave length of 254 nm using Gel documentation
system.