Detection of Chlamydia trachomatis ompA DNA in urine by loop-mediated isothermal amplification assay

Abstract

Abstract A cost-effective, sensitive and specific Chlamydia trachomatis (CT) diagnostic test is essential for resource limited countries. A loop mediated isothermal amplification (LAMP) assay was optimized for detection of CT ompA DNA in urine. Crude DNA extraction (method-1) from urine was by centrifugation at 14,000g for 30 minutes, heating at 95°C for five minutes with buffer and centrifugation at 17,000g for one minute. A 25µl LAMP reaction comprising, 7µl crude extract in 20mM Tris-HCl, 10mM KCl, 10mM (NH4)2SO4, 2mM MgSO4, 0.1% Tween 20, six LAMP primers, 1.4mM deoxynucleoside triphosphate, 0.8M Betaine, 6mM MgSO4 and 8U Bsm polymerase, were incubated at 56°C for 60 minutes and the colour change, when a nucleic acid gel stain was added, was noted by ultraviolet illumination. Urine from 326 sexually transmitted disease clinic attendees were tested, both by LAMP and real time PCR (rPCR). Analytical sensitivity was 0.8 copies per reaction volume. Compared to rPCR, LAMP assay had sensitivity, specificity, positive predictive and negative predictive values of 71.4%, 99.7%, 96.2%, 96.7% respectively. LAMP assay was highly specific for CT ompA DNA in urine. The sensitivity improved further when urinary inhibitors were diluted and volume of urine for extraction was increased (method-2).