Validation of a nested PCR OmpB-RFLP for identification of Rickettsia species found in Colombia

Introduction Human rickettsiosis is an emerging and reemerging disease caused by bacteria of the genus Rickettsia, transmitted through bites from infected ticks and other arthropods. This pathogen induces a potentially lethal acute febrile syndrome in human beings. While cases have been documented in Colombia, the absence of accessible diagnostic services highlights an urgent need to develop rapid, economical, and reliable strategies to enable timely detection and identification of the most pathogenic species. The objective of this study was to standardize and analytically validate a nested PCR followed by RFLP as a preliminary assay for the genotyping and differentiation of Rickettsia species reported in Colombia. Methods For preliminary validation, key analytical parameters were assessed, including the limit of detection, repeatability, reproducibility, robustness, and digestion profiles used for the identification of each Rickettsia species. Results The limit of detection was established at a single copy of the ompB gene, and the assay demonstrated a reproducibility of 96.7%. The technique proved robust against variations in reagent concentrations, except for the amount of Taq DNA polymerase, which significantly affected the results. Digestion profiles were determined for most circulating Rickettsia species, and these profiles were consistent with in silico analyses. Conclusions The analytically validated nested PCR-RFLP assay is a reliable technique for the diagnosis of Rickettsia. Species identification using a single restriction enzyme represents a cost-effective strategy for screening patient and tick samples, which could strengthen the diagnosis and surveillance of rickettsioses in Colombia.