# Development and Evaluation of a Dual-Target One-Step Nested PCR for the Detection of Spotted Fever Group Rickettsia spp. in Ticks

## TL;DR

A new PCR method was developed to detect Spotted Fever Group Rickettsia in ticks more quickly and with less risk of contamination.

## Contribution

A dual-target one-step nested PCR assay was developed and evaluated for improved detection of SFG Rickettsia.

## Key findings

- The dual-target one-step nested PCR detected 10 gene copies for the 17 kDa gene and 1000 gene copies for ompA.
- The new assay reduced contamination risk and assay time compared to conventional nested PCR.
- One tick specimen out of 184 tested positive for the Rickettsia 17 kDa gene.

## Abstract

Spotted fever group (SFG) rickettsioses are tick-borne infectious diseases caused by more than 30 Rickettsia species. As ticks may harbor and transmit multiple pathogens during a single blood meal, sensitive and specific molecular detection methods are essential for early diagnosis. Conventional nested PCR is commonly used but is time-consuming and prone to cross-contamination due to multiple amplification steps. This study evaluated a dual-target one-step nested PCR assay developed as a rapid alternative to conventional nested PCR for SFG Rickettsia detection. Gene-specific primers targeting the Rickettsia outer membrane protein A (ompA) gene and the 17 kDa antigen gene were designed, with a Plasmodium falciparum thrombospondin-related anonymous protein (TRAP) gene included as an internal amplification control. Primer specificity was verified in silico, and assay performance was assessed using synthetic DNA templates. The dual-target one-step nested PCR achieved detection limits of 10 gene copies for the 17 kDa gene and 1000 gene copies for ompA, compared with 10 and 100,000 gene copies, respectively, using conventional nested PCR. Screening of 184 tick specimens identified one positive sample (0.54%) for the Rickettsia 17 kDa gene. Overall, the dual-target one-step nested PCR demonstrated comparable sensitivity to conventional nested PCR while reducing assay time and contamination risk, indicating its potential as a reliable tool for SFG Rickettsia detection.