Detection of Chlamydia trachomatis ompA DNA in urine by loop-mediated isothermal amplification (LAMP) assay.

OBJECTIVES

Efficient real-time PCR kits are commercially available for the detection of Chlamydia trachomatis (CT) genetic material, but at a price. As a result, cost-effective, sensitive and specific CT diagnostic tests are essential for resource limited countries. This study aims to describe the optimization of a loop mediated isothermal amplification (LAMP) assay for the detection of CT ompA DNA in urine.

METHODS

Cost-saving modifications included using Bsm polymerase and a nucleic acid gel stain. Crude DNA extraction (method-1) involved centrifuging urine at 14,000 g for 30 min, heating the deposit at 95 °C for 5 min, and centrifuging again at 17,000 g for 1 min. To boost sensitivity, urinary inhibitors were diluted with phosphate-buffered saline washes and a larger urine volume was used (method-2). The LAMP-SYBR GOLD assay was incubated at 56 °C for 60 min, with nucleic acid gel stain color changes observed under UV light. Urine from 326 sexually transmitted diseases clinic attendees was tested with both LAMP-SYBR GOLD and real-time PCR, comparing sensitivity and specificity.

RESULTS

Analytical sensitivity of the LAMP-SYBR GOLD assay was 0.8 copies per reaction volume. Compared to real-time PCR, LAMP-SYBR GOLD assay had sensitivity, specificity, positive predictive and negative predictive values of 71.4 , 99.7, 96.2, 96.7 % respectively. Five of the seven false negative results obtained with method-1 were re-tested using method-2, providing in all the cases the expected positive results.

CONCLUSIONS

The LAMP-SYBR GOLD assay showed a sensitivity of 71 % and a high specificity for detecting CT in urine with extraction method-1. The use of method-2 could increase this sensitivity, likely due to the removal of urine inhibitors.