MATERIALS AND METHODSEthicalConsiderationThe design studywas approved by ASRB, the ethical committee of department of biochemistry AbdulWali Khan University, Mardan (AWKUM).Sampling Area The current research was arranged keeping in attentionto the country of Pakistan, a malaria epidemic country located within Southasia cover an area of 796,096 km² having five provinces and Federally AdministratedTribal Area (FATA). According to 2017 sensus Pakistan population of was 207,774,520 (Khilji et al., 2017). Congested regions were focused of the southof Khyber Pakhtunkhwa (KP) such as Kohatregion 2,545 km² with population 993,874,and Bannu region 1,227 km² with population 1.168 million.
The above regionshaving warm and temperate climate creates fertile and productive environmentfor mosquitoes. Bloodsampling Blood was collected from different falciparumpositive patients in the selected regions of south of KP for conductingresearch work. The present study was organized from July, 2017 to feb, 2018 in Molecularlaboratory of Biochemistry of the department of Abdul Wali Khan University,Mardan. A number of P.falciparum positivesamples done based on assistance and accessibility of the donor’s personnelin private and hospital’s lab during thestudy. MalariaPrevalence and Data collectionData was collectedfrom different regional hospitals of south of KP provides full assistance indata collection, and malaria prevalenc during visit of District HeadquarterHospitals (DHQs) and other clinical laboratories nearby. Different laboratorytechnicians and hospital personells helped us in collection of three years ofdata from 2016 to 2018. Collectionof Blood SamplesFrom malaria positive patients blood samples were collectedusing 5 ml syringes and saved in sterile vacutainer tubesenclose an anticoagulant agent Ethylenediaminetetra acetic acid (EDTA).
These samples were attained from south of KP including Hangu, Kohat, Bannu and Dera Ismail Khan .Collectionof blood samples was design in period ( i.e. march 2017 to September 2017 , during whichmalaria show epidemicity with high rate). These samples were preserved at -20°C in theMolecular lab of biochemistry department AWKUM.DNA Extraction from Human BloodDNAwas extracted from human blood using a phenol chloroform method to study anddifferentiate various genotypes of P.falcipaum (Loparev etal., 1991) Table 2.
2 Compositions of solutions used in DNA extraction S.No. SOLUTIONS Composition of chemical 1.
Solution A 0.3 M sucrose 10 mM Tris pH 7.5 5 mM MgCl2 1% (v/v) Triton X-100 2. Solution B 10 mM Tris pH 7.5 400mM NaCl 2 mM EDTA pH 8.0 3. Solution C Saturated Phenol 4. Solution D Chloroform 24% (v/v) Isoamyl alcohol 1% (v/v) Procedure1.
700 µl of of positive blood sample and 700 µl of sol Awere taken in 2ml of Eppendorf tube forRBC lysis.2. Blood sample was mixed with sol A inthe tubes by inverting 4-6 times and incubate at room temperature for 5-10mins.3.
The tubewas Centrifuge at 13000 rpm for 1 min.4.The supernatantwas discarded again and 400 µl of Sol.A was added in pellet and dissolved it byvortexing.
5. Centrifugationwas followed again for 1 min at 13000 rpm. The supernatant was discarded andre-suspend the nuclear pellet in 400 µl of Sol.
B, 17 µl of 20% SDS and 8 µl ofProteinase K.6. Overnight incubationwas done at 37C or °65?for 3 hours.7.
500 µl of freshSol C and Sol D mixture of equal volume were added to the sample afterincubation which separate layer. Invert the mixture 2-3 times and centrifuge13000 rpm for 10 min.8. The aqueous phase(Upper layer) was transfer to a new eppendorf tube.
55 µl of Sodium Acetate(3M, PH 6) and equal volume of 500 µl of Isopropanol were added and severaltime mix the tubes for DNA precipitation.9. The tubes werethen centrifuge at 13000 rpm for 10 mins, the supernatant was discarded. 200 µlof 70% ethanol to DNA pellet was added for washing, and Centrifugation wasfollowed at 13000 rpm for 7 min and the ethanol should be discarded.
10. For half anhour DNA was dried by keeping the tubes for for 30 mins at 37?.11. The precipitatedDNA was dissolved in 200 µl of TE and stored at -20?. GelElectrophoresisExtracted DNA was confirmedby prepared 1% agarose gel from both qualitative and quantitative point of viewand was seen by using Ethidium bromide.
Visualization of extracted DNA was doneusing ultraviolet illuminator, at a wavelength of 254 nm using Geldocumentation system.Nested Polymerase Chain ReactionNested PCR is carried to amplify the target DNA and inhance thespecificity of target region of DNA. Some time the primer bind other than thetarget sequence and creates problem in the desired results.
In Nested PCR twosets of primers are apply. First role of primer set was possible to getattached to the outside sequences of the target region of DNA, standard PCR isthe same, but it can also get bound to other region of the DNA template. Thetarget region of DNA amplified by firstset of primer was subjected to the second set of primer, to amplify the regionwithin first amplification. Thus to the products of the first PCR reaction, theprimers of second set gets attached and amplified the DNA region.Primer set Used in NestedPCR During the currentstudy, P.
falciparum merozoites surface protein specific primers pfmsp1, Pfmsp2 and Pf GLURP typesspecific primers such as K1, RO33 and MAD20 allelic family of pfmsp1, 3D7/IC1 and FC27 allelic familyof pfmsp2 and polymorphic alleleGLURP R2 repeat region were designed by eurofinsm wg/operon. The sequence ofthese primers is given in Table No 0.0 Table1: Primers sequences used for Nested PCRanalysis Primer Sequence Pf mspl (N1) F: 5′-CTAGAAGCTTTAGAAGATGCAGTATTG-3′ R: 5′-CTTAAATAGTATTCTAATTCAAGTGGATCA-3′ Pfmsp-2 (N1) F: 5 ‘-ATGAAGGTAATTAAAACATTGTCTATTATA-3, R: 5′-CTTTGTTACCATCGGTACATTCTT-3′ Pf GLURP (N1) F: 5?-TGAATTTGAAGATGTTCACACTGAAC-3?; R:5’GTGGAATTGCTTTTTCTTCAACACTAA-3? K1 family (N2) F: 5′-AAATGAAGAAGAAATTACTACAAAAGGTGC-3′ R: 5′-GCTTGCATCAGCTGGAGGGCTTGCACCAGA-3’ R033 family(N2) F: 5 ‘-TAAAGGATGGAGCAAATACTCAAGTTGTTG-3 ‘ R: 5 ‘-CATCTGAAGGATTTGCAGCACCTGGAGATC-3 ‘ MAD20family (N2) F: 5’AAATGAAGGAACAAGTGGAACAGCTGTTAC-3′ R:5’ATCTGAAGGATTTGTACGTCTTGAATTACC-3′ FC27family (N2) F: 5′-AATACTAAGAGTGTAGGTGCARATGCTCCA-3’ R: 5 ‘-TTITATTTGGTGCATTGCCAGAACTTGAAC-3 3D7/I C1family (N2) F: 5 ‘-AGAAGTATGGCAGAAAGTAAKCCTYCTACT-3 ‘ R: 5 ‘-GATTGTAATTCGGGGGATTCAGTTTGTTCG-3 ‘ Pf GLURP (N2) F: 5?-TGTTCACACTGAACAATTAGATTTAGATCA-3’ R:5’GTGGAATTGCTTTTTCTTCAACACTAA-3 PCR AnalysisInitial PCRFor initial PCR, a 25µl of total volumeprepared in PCR tube .
One PCR tube contain 16.2µl PCR water, 1.8µl Magneciumchloride, 2.5 µl Taq buffer,0.5 µl dNTPs, 0.5µl of forward and reversed primerof pfmsp1, pfmsp2or glurp, 0.
5µl Taq polymerase and1.5µl of gDNA was added, each tube has the same components and equal volune.Master mix prepapation took place on ice because Taq pol is temperaturesensitive. The PCR run was optimized using the following program:The initial Denaturation at 95°C for 5 minutes and 30 cycles of,denaturation at 95°C for 50 seconds, annealing at 58°C for 50 seconds andextension at 72°C for 1 minute and 45 seconds. Final, extension at 72°C for 5minutes was performed.
PCR was run and initial PCR products were obtained.2nd PCRInitial PCR products were taken for which five sets of PCRtubes were labelled for each product of pfmsp1 and pfmsp2 and a separate PCR tubefor GLURP R2 nested according to the desire. A total of 25µl volumeof product prepared for each, to which 16.2µl PCR water, 1.8 µl Magnecium chloride, 2.5µl Taq buffer, 0.5 µl dNTPs, 0.5 µl forward and reversed primers of family of pfmsp1 and pfmsp2 and Pf Glurp R2repeat nested, 0.
5 µl Taq pol and 2.5 µl initial PCR product added to each tube. The PCR run was again optimized using the following program: The initialdenaturation at 95°C for 5 minutes and 30 cycles of, denaturation at 95°C for 45seconds, annealing at 58°C for 50 seconds and extension at 72°C for 1 minuteand 45 seconds. Final extension at 72°C for 5 minutes was performed. PCR wasrun and final PCR products were preserved at -20Co.
Visualization of PCRProducts by Gel ElectrophoresisA2 % agarose gel was prepared to studythe amplified DNA samples using agarose gel electrophoresis technique, and wereseen by staining with Ethidium bromide. Visualizationof the amplified products were done under ultraviolet illuminator at a wavelength of 254 nm using Gel documentation system.