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Bronchial Aspirates in the Diagnosis of Pulmonary Tuberculosis: Outward PCR Analysis

In one case with a positive CA result in which contamination was suspected through the washing procedure, outward PCR genetic relatedness was tested by molecular fingerprinting employing the IS6110-based outward PCR method. In brief, outward PCR employed the single primer based on the invert-repeat fragment located at the ends of IS6110 whose sequence was 5′-GACIIICCGGGGCGGTTCA-3′, where “I” was inosine.
The 3′ end of the primer was directed outwardly from both sides of the inverted repeats present in IS6110, amplifying the flanking sequences between two copies of IS6110. http://buy-asthma-inhalers-online.com there PCR was carried out with approximately 10 ng of genomic DNA in a final volume of 50 |j,L in a reaction buffer containing 0.4 pmol primers, 2 mM MgCl2, 200 |j,L of deoxynucleotide triphosphates, and 2.5 U of Taq polymerase. DNA samples were denatured by incubation for 3 min at 95°C before amplification for 35 cycles at 94°C for 1 min, 58°C for 1 min, and 72°C for 2 min using a thermocycler (GeneAmp PCR System 9600; Perkin Elmer; Foster City, CA).After the last amplification cycle, the samples were incubated for 7 min at 72°C to complete the elongation of the PCR intermediate products. Positive and negative controls were used for each reaction. The positive control was the reference strain M tuberculosis H37Rv, and the negative control was the PCR mix without DNA. The PCR products were analyzed by electrophoresis through a 1.2% agarose gel and stained with ethidium bromide. Each sample was tested at least three times in order to verify the reproducibility of the results.
Statistical Analysis
Statistical analysis was performed using software (SPSS for Windows version 7.5; SPSS; Chicago, IL). The McNemar test was used to compare of the sensitivity of direct AFB smear and CA testing. Data are expressed as mean ± SD, and statistical significance was defined as p < 0.05.