A Loop-Mediated Isothermal Amplification (LAMP) Assay for the Detection of Treponema pallidum subsp. pertenue

Abstract

Abstract Objectives The eradication of yaws, a childhood disease caused by Treponema pallidum subsp. pertenue, is constrained by the lack of rapid, accurate diagnosis. We sought to develop a molecular point-of-care test for the diagnosis of yaws. Methods A loop-mediated isothermal amplification (LAMP) assay with primers targeting the conserved gene, tp0967, with visual detection by lateral flow test strip was developed. The assay was optimized by varying the concentrations of reagents as well as the temperature and time of the reaction. The limit of detection and selectivity of the assay were evaluated. Subsequently, 63 clinical samples from yaws-infected lesions were used to determine the sensitivity of the assay from both unextracted and DNA extracted swab samples as compared to the current molecular testing protocol (CDC PCR assay) as the gold standard. A further five clinical samples from lesions containing PCR-confirmed syphilis pathogen (Treponema pallidum subsp. pallidum) were tested to ensure specificity of the assay for yaws alone. Results The developed LAMP assay was found to be optimal when run at 65oC for 30 minutes. The limit of detection was 2.7*104 copies DNA per mL. Out of the 63 yaws samples tested, the CDC assay, extracted DNA LAMP assay, and unextracted DNA using the LAMP assay resulted in 12, 14, and 8 positive results, respectively. None of the syphilis samples tested positive in any of the assays. Conclusion We show the development of a fast and sensitive LAMP assay for Treponema pallidum subsp. pertenue detected by lateral flow test strip. Using extracted DNA, the assay sensitivity is on par with gold standard detection. Further, the assay can be adapted to minimal sample processing required for in-field detection without DNA extraction.