According to (154) with some modification in the method of collecting the aspiration fluid, the lesion sites were cleaned with 70% alcohol. there my a syringe (1ml,30G*1/2 )when intradermal pentostam injection at the periphery was done , we were aspirated the fluids and the blood that oozing from the sites by capillary tubes with anti-coagulants and prepared slide to examine directly by microscopy after staining and the other was saved in screw cap container with the Sterile NaCl solution as dilution fluid and incubated in refrigerator (4 c) until DNA extraction.
This examination conducted on each sample of aspiration prepared the smear by transferring a small portion of the sample onto the clean slide. Staining with Giemsa or Leishman’s stain solution and the examined under the light microscope with a 100 objective lens. according to:
Preparation showing the amastigotes is consider to be positive (+ ve ?) for the Lishmania spp. and preparation with no amastigotes is considered to be negative (-ve) for the Leishmania spp. All the results were recorded (125,154).
The isolation of Leshmania spp. DNA was extracted from the aspiration fluid by using the (DNA extraction for the intracellular organism) Genomic DNA Mini Kit ( Geneaid , Taiwan ) according to the manufacturer’s protocol and stored at _20oC (Appendix No.
The components of the kit are as follows:-
2 ml collection Tube 200 pcs
(( Added absolute ethanol (see the bottle label for volume) to wash buffer prior to initial use ))
“DNA samples that prepared from the aspiration blood were quantified by the Ultraviolet spectrophotometer (Unico, USA) reading first at 260 and 280 nm (155). All the samples were stored at the ( -20 oC) until use.”
The DNA for assessment was removed from cold storage for 30 minutes, flicking the bottom of tubes ensured that the entire DNA was suitably resuspended.
Samples were then centrifuged at 8000 xg for 5 minutes.
A blank was prepared by taking 1000 ?l of TE buffer and used to zero the spectrophotometer = first Quartz cuvette.
In the second Quartz cuvette, 10 ?l of the DNA sample was mixed with 990 ?l TE buffer (sample cuvette). After zeroing the spectrophotometer (Unico, USA) on the first cuvette was brought into place to obtain an absorbance reading.
“Reading was taken of wavelengths of 260 nm (OD260) for the DNA of the sample and the 280 nm (OD280) for detected the protein concentration of the sample; the spectrophotometer were re zeroed between each of the wavelength reading.”
The ratio between the readings of the (OD260/OD280) it is provided to determination of the sample purity, which is should be become between (1-2). Values of (OD260/OD280) of the less than 1 indicate the contamination of the DNA by protein (Sambrook et al., 1989).
The concentration of the DNA (?g/?l ) were determined by using of the following equation:
“DNA Concentration (?g/?l ) = (reading in OD260) X (total/sample volume) X constant (50 ?g/1000 ?l ). & ?((OD in 260 nm x( dilution factor )x constant 50µg)/(1000µ²))”
The yield was estimated by (concentration X total volume).
Electrophoresis of DNA extracted (Pre_PCR) on agarose gel was checked by illumination after staining with ethidium bromide as a method that showed by (156) with some slight modification. After staining, ultraviolet transillumination was used for illumination of the DNA bands in the gel.
agarose gel electrophoresis was adopted to confirm the presence and integrity of the extracted DNA (155).
Bromophenol blue in 1% glycerol (loading buffer).
The casting of the horizontal agarose gel :
After sealing both edges of the gel tray with cellophane tapes and fixing the comb in 1 cm way from one edge, the agarose solution was poured into the gel tray, the agarose was allowed to solidify at room temperature for 30 min. The fixed comb was carefully removed and the gel tray was placed in the gel tank. The tank was filled with 1X TBE buffer until it reached 1-2 mm over the surface of the gel (155).
“All suspension samples examined for the DNA extraction which was the assayed by the PCR amplification process. The specific all primers were synthesized from IDT (IDT, Inc ( USA ), and they were designed on the basis of the sequence information of the genes repeated the unit that amplifies a highly repeated the sequence of Cutaneous Leishmania DNA according to the following” :
“The forward primer (LITSR) 5-CTGGATCATTTTCCGATG-3 and reverse primer (L5.8S) 5-TGATACCACTTATCGCACTT-3, specific to the ribosomal ITS1 region that occurs between the genes encoding the small subunit (18S) ribosomal RNA and 5.8S rRNA” according to (157,158), (appendix No.8).
“Amplification of the mini-exone genes performing as a single PCR with the forward (5′-TATTGGTATGCGAAACTTCCG-3′) and the reverse (5′-ACAGAAACTGATACTTAT-AT AGCG-3′) primers as the described in (120)”, (Appendix No. 7).
The kDNA PCR using the primers 13A (5′-GTG GGG GAG GGG CGT TCT-3′) and 13B (5′-ATT TTC CAC CAA CCC CCA GTT-3′) according to (159, 160), (Appendix No. 6).
All primers were supplied by IDT Company, these Primers (lyophilized product of different picomols concentrations) were resolved according to the instructions of the manufacturer, where resuspension using deionized water to Primer’s tube, then placed in microcentrifuge with speed 8,000rpm for 1mintues for obtaining on stock solution with concentration 100 picomols, then was taken 10 µ² from stock solution in Eppendorf tube( 1.5 ml ) and 90µ² of deionized water was added for obtaining a working solution with a concentration of 10 picomols /?l of suspension.
Table(3.1): The preparation of the final concentration of the study PCR primers of cutaneous leishmania genes.
The final concentration of the stock solution (picomole/µ²) Concentration (picomole) Sequences Primer name
PCR reaction kit (PCR PreMix) was purchased from Bioneer company. The PCR reaction was carried out in 20 ?l solution containing (Taq DNA polymerase), 1 U, each dNTP (dATP, dGTP, dCTP, dTTP) 250 ?M, 1.5 mM MgCl2, Tris-HCl (pH 9.0), 10 mM, KCl, 30 mM, Template DNA, 5 – 50 ng, Primer, 5 – 10 pmole
The Reaction mixture was prepared in a pre-mix tube as shown in the following table(Table 3.2). according to BIONEER company instructions (Appendix No. 5).
After the addition was completed all the components of a mixed reaction in pre-mix tube mixed well by vortex, all tubes were centrifuged for 30 seconds at 10,000 rpm according to manufacture company, then all tubes were transferred into Thermal cycler apparatus for starting Thermal cycler program.
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