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Detecting the Lyme Disease Spirochete, Borrelia Burgdorferi, in Ticks Using Nested PCR
Published on: February 4, 2018
Development and Evaluation of a Dual-Target One-Step Nested PCR for the Detection of Spotted Fever Group Rickettsia
Phiaw Chong Foo1, Canedy Jacob1, Christina Injan Mawang1
1Acarology Unit, Infectious Disease Research Centre, Institute for Medical Research, Ministry of Health Malaysia, National Institutes of Health Complex, Bandar Setia Alam, Shah Alam 40170, Selangor, Malaysia.
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.
Insights
A new dual-target one-step PCR assay offers a faster, safer method for detecting Spotted Fever Group (SFG) rickettsioses. This molecular diagnostic tool shows promise for early identification of these tick-borne diseases.
Area of Science:
- Microbiology
- Infectious Diseases
- Molecular Diagnostics
Background:
- Spotted fever group (SFG) rickettsioses are tick-borne diseases caused by over 30 *Rickettsia* species.
- Accurate and rapid detection is crucial due to pathogen co-transmission by ticks.
- Conventional nested PCR methods are effective but time-consuming and prone to contamination.
Purpose of the Study:
- To evaluate a novel dual-target one-step nested PCR assay as a rapid alternative for SFG *Rickettsia* detection.
- To compare the sensitivity, speed, and contamination risk of the new assay against conventional nested PCR.
- To assess the utility of the assay for screening tick specimens.
Main Methods:
- Designed gene-specific primers for *Rickettsia* outer membrane protein A (*ompA*) and the 17 kDa antigen gene.
- Incorporated a *Plasmodium falciparum* thrombospondin-related anonymous protein (*TRAP*) gene as an internal control.
- Validated primer specificity in silico and assay performance using synthetic DNA and 184 tick specimens.
Main Results:
- The dual-target one-step PCR achieved detection limits of 10 copies for the 17 kDa gene and 1000 copies for *ompA*.
- Conventional nested PCR had detection limits of 10 copies for the 17 kDa gene and 100,000 copies for *ompA*.
- One of 184 tick specimens (0.54%) tested positive for the *Rickettsia* 17 kDa gene.
Conclusions:
- The dual-target one-step nested PCR is a sensitive and specific molecular tool for SFG *Rickettsia* detection.
- This assay reduces assay time and contamination risk compared to conventional nested PCR.
- The developed method is a reliable alternative for early diagnosis and surveillance of SFG rickettsioses.
