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

• Kamani Mangalika Gunasekera ^[1] ; Hemali Attanayake ^[2] ; Jayanthi Elvitigala ^[2] ; Charitha Goonasekara ^[3] ; Nalaka Abeygunasekera ^[4]
  1. [1] Department of Microbiology, Faculty of Medical Sciences, University of Sri Jayewardenepura, Nugegoda, Sri Lanka, E-mail: kamani@sjp.ac.lk. https:// orcid.org/0000-0002-1132-5121
  2. [2] National STD AIDS Control Program, Colombo, Sri Lanka, E-mail: hemaliattanayake@gmail.com (H. Attanayake), jayanthi_lk@yahoo.com (J. Elvitigala)
  3. [3] Department of Pre Clinical Sciences, Faculty of Medicine, General Sir John Kotelawala Defence University, Ratmalana, Sri Lanka, E-mail: charithalg@hotmail.comb
  4. [4] STD Clinic, Colombo South Teaching Hospital, Kalubowila, Sri Lanka, E-mail: m13094@pgim.cmb.ac.lk

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• Localización: Advances in Laboratory Medicine / Avances en Medicina de Laboratorio, ISSN-e 2628-491X, Vol. 6, Nº. 3, 2025, págs. 314-319
• Idioma: inglés
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  • 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 realtime PCR, comparing sensitivity and specificity.

    Results: Analytical sensitivity of the LAMP-SYBR GOLD assay was 0.8 copies per reaction volume. Compared to realtime 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.

    Keywords: Chlamydia trachomatis; crude extract; loop mediated isothermal assay (LAMP); urine