Highly Sensitive Molecular Assay for the Detection of Treponema pallidum infection

Abstract

Introduction: According to the World Health Organization (WHO) more than 7 million new Treponema pallidum (T. pallidum) infections were reported among people aged 15–49 years in 2020 globally, the majority of them in developing countries. Syphilis, which is caused by T. pallidum is transmitted through contact with active lesions of a sexual partner or from an infected pregnant woman to her foetus. Gold standard T. pallidum laboratory diagnosis methods include dark-field microscopy, silver staining, direct fluorescence immunoassays and the rabbit infectivity test. However, these tests are associated with false positive or negative results. The gold standard 16S ribosomal (rRNA) gene polymerase chain reaction (PCR) is used for routine amplification of T. pallidum conserved genes. Here we report on an ultrasensitive syphilis diagnostic method, based on de novo genome mining of the T. pallidum DNA to identify identical multi repeat sequences (IMRS) as amplification primers. Methods: We used genome-mining approaches to find IMRS distributed throughout the T. pallidum genome to design a primer pair that target four repeat sequences. Genomic T. pallidum DNA was diluted from 8.14×104 to 8.14×10− 2 genome copies/𝜇l and used as template in the IMRS-based amplification assay. For performance comparison, 16S rRNA PCR assay was employed. Probit analysis was used to calculate the lower limit of detection of the T. pallidum-IMRS PCR and the conventional 16S rRNA PCR assays. Results: Probit analysis confirmed that the T. pallidum-IMRS primers offered higher test sensitivity of 0.03 fg/𝜇L compared to the 16S rRNA PCR (0.714 pg/𝜇L). Using the T. pallidum-IMRS primers, we were able to observe considerable isothermal amplification of genomic DNA at a starting concentration of 0.01 pg/µL. Conclusion: De novo genome mining of T. pallidum IMRS as amplification primers can serve as a platform for developing ultrasensitive diagnostics for Syphilis and potentially a wide range of infectious pathogens.