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

Spotted fever group (SFG) rickettsioses are tick-borne infectious diseases caused by more than 30 <i>Rickettsia</i> 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 <i>Rickettsia</i> detection. Gene-specific primers targeting the <i>Rickettsia</i> outer membrane protein A (<i>ompA</i>) gene and the 17 kDa antigen gene were designed, with a <i>Plasmodium falciparum</i> thrombospondin-related anonymous protein (<i>TRAP</i>) 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 <i>ompA</i>, 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 <i>Rickettsia</i> 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 <i>Rickettsia</i> detection.