Magna pure machine

DNA from genital swab samples was extracted using the MagNA Pure 96 DNA and Viral NA small volume kit (Roche) on the MagNA pure machine (Roche) according to the manufacturer’s instructions.
The oligonucleotide primer set was designed for detection of all species of Chlamydiae. The sense primer, 16S DIR 5′-CGCCTGAGGAGTACACTCGC-3′, and anti-sense primer, 16S Rev 5′-CCAACACCTCACGGCACGAG-3′, were designed to amplify a 208-bp fragment of the Chlamydial 16S ribosomal subunit gene, conserved across Chlamydia strains and serovars. Primers were obtained from Sigma Genosys (The Woodlands, TX), and the probe, 16S Fam-5′-CACAAGCAGTGGAGCATGTGGTTTAA-3′ Tamra, was synthesized by Applied Biosystems, (Foster City, CA).
The 50-μL reaction mixtures consisted of 1× QuantiTect Multiplex PCR master mix without ROX (Qiagen), 100 nmol/L 16S probe, 200 nmol/L primer 16S DIR, 400 nmol/L primer 16S Rev, 30 nmol/L ROX reference dye, and 5 μL of sample DNA. Nontemplate controls consisting of the reaction master mix, primers, and probe, but no DNA, were included in each assay run. Reaction conditions were set as follows: 1 cycle at 95 °C for 15 min, followed by 40 cycles at 94 °C for 1 min and at 60 °C for 1 min. Thermal cycling, fluorescent data collection, and data analysis were performed using the Stratagene Mx3005P system (Stratagene) according to the manufacturer’s instructions.