The application of loop mediated isothermal amplification for the detection of the sexually transmitted pathogens Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, and Trichomonas vaginalis, at the point of care.

Abstract

The purpose of this multi-partnered project was the production of a fully integrated POC system, combining automated nucleic acid extraction in a centrifugally operated microfluidic disk (the LabDisk), with loop mediated isothermal amplification (LAMP) and optical detection, capable of detecting the sexually transmitted pathogens Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium and Trichomonas vaginalis in clinical urine and swab samples. LAMP is a novel nucleic acid amplification method, designed to amplify target nucleic acid in a highly specific and rapid manner, under isothermal conditions. The work detailed in this thesis presents the development of a rapid total nucleic acid extraction process, based on the capture of target nucleic acid by magnetic silica beads, optimised for use on the LabDisk platform. The extraction process was capable of the purification of target nucleic acid from a clinical sample within 5 minutes, and was robust when challenged with a range of inhibitory compounds potentially encountered in samples for STI testing. The system was capable of tolerating N. gonorrhoeae (1 x 105 CFU/ml) urine suspensions containing samples containing 50% total blood volume, 1x108 E. coli cells per ml, and 10mg/ml of BSA, without any effects on the downstream amplification time of the N. gonorrhoeae specific LAMP assay. A freeze dried lysis buffer pellet was developed, that was able to increase the sample volume, thereby decreasing the time to detection, whilst minimising the stored fluid volume on the LabDisk. LAMP assays were designed for the detection N. gonorrhoeae and M. genitalium, and the limits of detection and specificity of the assays were evaluated. The N. gonorrhoeae ORF1 assay was able to detect a minimum of 20 copies of the N. gonorrhoeae genome per reaction, whilst the M. genitalium pdhD assay was capable of detecting 16 genome copies. The tolerance of the ORF1 LAMP assay to urea, and blood, was found to be 1.8M, and 20% reaction volume, respectively. The increased tolerance of the LAMP assay to these inhibitors in comparison to PCR demonstrates the suitability of LAMP when processing urine samples for STI’s. To our knowledge this is the first application of LAMP technology for the detection of these organisms, and the first attempt at commercialising a fully integrated molecular diagnostics system based on LAMP.