International Journal of Advancements in Research & Technology, Volume 2, Issue4, April-2013
ISSN 2278-7763
38
Identification of Leishmania tropica by PCR and
RFLP Techniques in Kohat Region of Khyber
Pakhtunkhwa,Pakistan.
Sultan Ayaz*1, Marukh1, Shoaib Nawaz3, Shakir Ullah Khan2, Mehboob Nawaz2,Farzana
Raza1 and Abdul Malik tareen4
1
Department of zoology, kohat university of Science and technology Kohat.26000
Department of Microbiology, Kohat University of Science and Technology Kohat 26000
Pakistan
3
Department of Biotechnology and Genetic Engeneering, Kohat University of Science
and Technology Kohat 26000 Pakistan.
4
Department of Biotechnology, University of Science and Technology Quetta . Pakistan.
Corresponding author;
Sultan_Ayaz@yahoo.com.
Chairman, Department of Zoology
Kohat University of Science and Technology Kohat 26000, Pakistan
Cell; +92-3348684196.
2
ABSTRACT
Cutaneous Leishmaniasis is a worldwide public health and a social problem in many
developing countries. This disease is also found in Pakistan in general and Khyber
Pakhtunkhwa in particular. A total of 100 samples were examined from clinically
suspected Cutaneous Leishmaniasis patients under sterilized conditions in village Sordag,
District Karak, Khyber Pakhtunkhwa. The DNA was extracted and amplified through
PCR and reconfirmed by RFLP technique. The PCR showed the result 53% (53/100)
were positive, in which 67.30% (35/100) were female while 37.50% (18/100) male
positive. These results were reconfirmed through RFLP which also showed 186bp
amplified fragment. It is concluded that PCR-RFLP appears to be most sensitive and
appropriate techniques for the detection of Leishmania tropica.
Keywords: Cutaneous Leishmaniasis, DNA, PCR and RFLP
Copyright © 2013 SciResPub.
International Journal of Advancements in Research & Technology, Volume 2, Issue4, April-2013
ISSN 2278-7763
1
INTRODUCTION
the
Himalayan
39
sub-mountain
range
(Azad Kashmir, Mansehra, Rawalpindi,
Globally Leishmaniais is one of the most
Abbotabad) [9].
ignored tropical disease with high
prevalence rate[1] caused by infectivity
Extra-cellular promastigotes and intra-
of protozoa of the genus Leishmania [2]
cellular
in
morphological forms of Leishmania. The
their
different
forms
[3,4].
amastigotes
Leishmaniasis is transmitted by Female
elongated
Phlebotomine
promastigotes
Sand
flies
(Family:
and
are
motile
are
the
two
flagellated
initiated
in
the
Psycodidae, Order: Diptera) [5, 6]. It has
alimentary canal of the sandfly while
been reported that about 350 million
nonmotile and ovoid amastigotes exists
people are at the risk of Leishmaniasis
and reproduce in the phagocytosomes of
[7] in 88 countries among which 22 are
host macrophages [10]. Taxonomy of
in the new world and 66 in the old world
Leishmania is as; Kingdom-Protozoa;
[8] and annually 2,357,000 new cases
Subkingdom-Protista;
are reported [7].
Sarcomastigophora;
PhylumClass-
Zoomastigophora; Order-Kinetoplastida;
Approximately 90% of the Cutaneous
Suborder-Trypanosomatina;
Genus-
Leishmaniasis cases came about in
Leishmania;
Species:
L.
tropica
complex,
donovani
complex,
Pakistan, Saudi Arabia, Iran, Brazil,
Syria,
Afghanistan
and
L.
L.
Peru.
mexicana complex, braziliensis complex
Widespread areas of disease in Pakistan
[11]. The lesions are generally found on
include the Karakoram and Hindukush
the uncovered areas of the skin [12,13].
sub-mountainous range (Gilgat, Dir and
Chatral); the Toba Kakar sub-mountain
The lesions or ulcers leave a scratch
mark on infected area [14]. Increase
range (Qila Abdullah, Quetta, Qila
tissue demolition and disfiguring of the
Saifullah, Pishin); the Suleman and
skin are caused by secondary fungal or
Kirthar sub-mountain range (Jacobabad,
D.G.Khan,
Rajanpur,
Derabughti,
Khuzdar, Lasbela, Dadu and Larkana);
Copyright © 2013 SciResPub.
bacterial infection of the ulcers [13].
Keeping in view the importance of the
disease; research is design to carry out
International Journal of Advancements in Research & Technology, Volume 2, Issue4, April-2013
ISSN 2278-7763
40
the identification of leishmania parasite
added in it and mixed well through
through PCR and RFLP techniques.
vortex and Centrifuged at 12000rpm for
5 minutes. The upper layer (aqueous)
above
2
MATERIALS AND METHODS
the
middle
removed.500μl
of
layers
100%
was
absolute
ethanol was added in it and mixed well
2.1 Sample Collection
Specimens
were
leishmania
infected
Cutaneous
through vortex and again Centrifuged at
collected
from
patients
Leishmaniasis.
The
of
skin
12000rpm
for
5
minutes.
The
supernatant was discarded.
2.4 Step.2 Washing of DNA
scrapings were made with the help of
surgical blades in one direction till the
500μl of trisodium citrate was added and
blood oozes out of the lesion and an
centrifuged at 12000rpm for 5 minutes,
incision were given mostly in the
and supernatant was discarded. Then
inflamed border of the lesion. Distilled
incubated for 20 minutes in orbital
water was sprinkled with the help of
shaker at roomtemperature.500μl of 70%
sterilized syringe on the lesion and after
ethanol was added and centrifuged at
that samples were collected in sterilized
12000rpm for 5 minutes and the
eppendorf tubes.
supernatant was discarded. Incubated at
room temperature for 10 minutes in
2.2 DNA Extraction
orbital shaker. 500μl of 8mM of NaOH
By using the DNAzole kit (Trizole
was added and centrifuged at 14000rpm
USA), DNA was extracted from biopsies
for 5 minutes .From the supernatant 30μ
/skin scrapings through a standard
l of DNA was taken in a new sterilized
protocol of the
eppendorf tube and was kept at -40 C
manufacturer.
The
following steps were involved,
2.5 DNA Amplification
2.3 Step.1 Cell lyses / Denaturation
The target DNA was amplified in 20μl
300µl of sample was taken in a sterilized
reaction mixture containing 10x PCR
eppendorf tube and 500µl of trizole was
buffer 2μl (ammonium sulphate), 1μl
added in it and mixed well through
dNTPs (10mM), 2.4μ l MgCl2 (25mM),
vortex. Then 160μl of chloroform was
Copyright © 2013 SciResPub.
International Journal of Advancements in Research & Technology, Volume 2, Issue4, April-2013
ISSN 2278-7763
1μl
of
forward
5/-
primer
TTT́ CTTTGGATGGGTTTCTGG-3/
41
hours and gently vortex occasionally
during the incubation procedure
(10pM), 1μl of reverse primer 3/-
2.7 Gel Electrophoreses
CAACACCAACGTAAGCGTAAC5/(10pM), deionized water 8.3μl, target
10μl of PCR product was mixed with 5μl
DNA 4ul and 0.3μl of Taq DNA
of
polymerase (5μl). The designed program
Similarly 5μl of ladder was mixed with
for
DNA
5μl of loading dye. Then gel tray was
amplification was initial denaturation
placed in gel tank containing 1000ml
cycle1 at 92oc for 3 minutes,25 cycle at
0.5X TBE buffer. The ladder was loaded
initial cycle 1 is on 92 oc for40 second,
in the 1st well and 10μl of each sample
cycle 2 for 50oc for 40 second, cycle 3
was loaded in the remaining wells. The
for
leishmania
72oc
for
for
1minute
the
and
finally
extension at 72oc for 7 minutes.,
loading
dye
through
pippting.
gel was run for 20-25 minutes at a
voltage of 120 volts and 500ampere
current. Gel was then examined by UV
2.6
Restriction
Fragment
Length
Polymorphism
Tran illuminator.
The PCR products (amplified ITS1
3
region) from ITS1 PCR were digested
A total of 100 samples of skin scarping
using restriction endonuclease enzyme
/biopsies were collected from the clinical
Hae
as
patients in village Sordag, District Karak
recommended by manufacture (BioLabs
under sterile conditions and examined
Inc, New England). Briefly, for clinical
through poly merease chain reaction
diagnostic samples that were amplified
(PCR) and 53%(53/100) were found
by ITS PCR, quantity of 15-20μl of
positive. Among these(37.50%)
DNA was incubated and restricted
male and (67.30%) were female and 186
byaddition of 1μl (5 units) of Hae III
bp DNA was amplified (Fig 1and
enzyme and 5μl of corresponding 10 X
table.1)The
N.E buffer and 5-10μl of deionized
reconfirmed through RFLP and the
III
(Haemophilis
III)
o
water and incubated at 37 C for 1.5-2
RESULTS
result
result showed
of
PCR
were
was
the same result as
determined by PCR. ( Fig.2 and Table.2)
Copyright © 2013 SciResPub.
International Journal of Advancements in Research & Technology, Volume 2, Issue4, April-2013
ISSN 2278-7763
42
TABLE NO 1: PCR diagnostic ratio of Cutaneous leishmanisis in district Karak
Khyber Pakhtunkhwa.
Sex
Percentage of Samples
Samples
Female
Male
Female
Male
Positive
35
18
67.30%
37.50%
Negative
17
30
32.70%
62.50%
Total
52
48
100%
100%
TABLE: 2. Comparison between PCR and RFLP diagnostic techniques.
Test
Positive
Negative
Total
PCR
53
47
100
RFLP
53
47
100
186bp
Fig.2; RFLP product showing the band of 186bp
Copyright © 2013 SciResPub.
International Journal of Advancements in Research & Technology, Volume 2, Issue4, April-2013
ISSN 2278-7763
4
DISCUSSION
facilities are available (Laboratory and
Cutaneous Leishmaniasis is widespread
in many tropical and sub-tropical areas
of
Equator
43
[15].
Cutaneous
Leishmaniasis are reported in Iran,
Afghanistan, India and Pakistan where
Skilled persons). Direct microscopic
detection of Leishmaniasis is cheap and
easy but its sensitivity is very low, even
if it is carried out by skilled persons [17,
18].
and
PCR offer certain compensation over
Leishmania tropica were found that
conventional methods for the diagnosis
initiate Leishmaniasis in challenging
and
areas [16].
Leishmaniasis.
both
Leishmania
donovani
characterization
of
When
Cutaneous
approximately
applied, PCR can be more specific,
It is a main society health problem
largely disturbing populations living in
distant areas where essential services of
life were not simply accessible and
where
the
detection
of
versatile
conventional
and
methods;
rapid
in
than
addition
genetic information can be obtained in
the process [19].
Cutaneous
Leishmaniasis was base on clinical
characteristics. Affirmative laboratory
tests are basically used and very limited
Copyright © 2013 SciResPub.
sensitive,
Cutaneous Leishmaniasis is prevalent in
Pakistan and has been reported from all
the Provinces [20]. From the finding of
International Journal of Advancements in Research & Technology, Volume 2, Issue4, April-2013
ISSN 2278-7763
44
the present study it has been revealed
to the Kohat University of Science and
that Cutaneous Leishmaniasis is caused
Technology Kohat, Pakistan.
by a parasite, Leishmania tropica, in
6
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