Ptc 150

Mice were bled from the orbital venous sinus, and 200 μL of blood was collected at the 1^st and 6^th month after treatment. PCR was performed only in samples from negative animals as determined by fresh blood examination. DNA extraction and PCR were performed according to Gomes et al. [20 (link)] with some modifications. The primers used for the parasite minicircle amplification were the following: S35 5’-AAATAATGTACGGG(T/G)GAGATGCATGA-3’ and S36 5’-GGGTTCGATTGGGGTTGGTGT-3’ [21 (link)]. Thirty-five amplification cycles were carried out in a Research Programmable Thermal Controller (MJ Research, model PTC-150). The cycles consisted of an initial denaturation of 5 minutes at 95°C, followed by 35 cycles of 1 minute each at 95°C for denaturation, 1 min at 65°C for primer annealing and 1 minute at 72°C for primer extension. Five microliters of the PCR product was analyzed by electrophoresis on a 6% polyacrylamide gel and visualized by silver staining. Positive and negative blood samples and reagent controls were processed in parallel in each assay, and all experiments were conducted under controlled conditions. To avoid contamination, DNA extraction, mixing, and electrophoresis were performed in separate, delineated areas. To confirm the absence of inhibition factors, an internal control corresponding to a segment of the murine TNF-α gene was amplified [22 (link)].

Detection of aminoglycoside resistance genes rrs (rrs1 and rrs2), aacC2 (aacC1-1, aacC1-2, aacC2-1, aacC2-2, aacC3-1, aacC3-2, aacC4-1, aacC4-2, aadC-1, and aadC-2), aacA-aphD (aacA-aphD-1 and aacA-aphD-2), and aphA3 (aphA3-1 and aphA3-2) from E. coli was done by PCR technique using Thermal Cycler (PTC 150, MJ Research). Blue mix DNA polymerase master mix, deoxynucleotide triphosphates (dNTPs), taq DNA polymerase, and the reaction buffer containing magnesium ions and the other required components were obtained from RBC Bioscience, India. Primers for the genes were purchased from Invitrogen (USA) through JOYVEL Biotech, India. Template DNA was isolated from E. coli. The working concentration of the primer was taken as 200 mM. The amount of PCR reaction mixture was taken as 50 μL which included 25 μL of blue mix DNA master mix, 5 μL of forward primer, 5 μL of reverse primer, 5 μL of template DNA, and 10 μL of TAE buffer. The PCR was performed with initial denaturation at 95°C for 3 min followed by 32 cycles each of denaturation at 94°C for 30 sec, annealing at 60°C for 45 sec, and extension at 72°C for two min for each gene.
The amplification products were analyzed by 0.8% agarose gel electrophoresis and the product size was compared with DNA markers. After treatment with ethidium bromide (0.5 μg/mL), the gels were visualized using gel-doc system (Bio-Rad Laboratories, USA).