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Updated: Jan 13, 2026
Detecting the Lyme Disease Spirochete, Borrelia Burgdorferi, in Ticks Using Nested PCR
Published on: February 4, 2018
CRISPR/Cas Tools for the Detection of Borrelia sensu lato in Human Samples
Ermanno Nardon1, Eros Azzalini1, Dino Paladin2
1Department of Medical Sciences (DSM), University of Trieste, 34149 Trieste, Italy.
Background/Objectives:
Lyme disease diagnosis remains challenging due to the limitations of current methods. While PCR-based assays are widely used, their sensitivity can be affected by sample type and the inhibition of host DNA. This study aimed to evaluate the feasibility and sensitivity of a CRISPR/Cas12-based detection system for Borrelia burgdorferi sensu lato, comparing its performance with real-time PCR.
Methods:
DNA from three Borrelia genospecies (B. burgdorferi, B. garinii, and B. afzelii) was amplified targeting the OspA gene. Detection was performed using a Cas12/crRNA system with a fluorescent ssDNA reporter. Sensitivity assays were conducted on serial dilutions of Borrelia DNA, with and without human genomic DNA, and results were compared with qPCR.
Results:
Direct detection of Borrelia DNA without amplification was not feasible. However, when combined with PCR, the Cas12/crRNA system reliably detected as few as 5 genome copies per reaction. End-point PCR extended to 60 cycles improved detection robustness for B. garinii and B. afzelii, although sensitivity decreased in the presence of human genomic DNA.
Conclusions:
The Cas12/crRNA-based system offers a sensitive and accessible alternative to qPCR, especially in settings lacking real-time PCR instrumentation. Future developments may include integration with isothermal amplification and microfluidic platforms to enhance direct detection capabilities.
Insights
A new CRISPR/Cas12 system shows promise for detecting Lyme disease bacteria, offering a sensitive alternative to traditional PCR methods. This approach enhances Lyme disease diagnostics, particularly where advanced equipment is unavailable.
Area of Science:
- Molecular Biology
- Infectious Disease Diagnostics
- Genetics
Background:
- Lyme disease diagnosis is hindered by current method limitations.
- PCR assays for Lyme disease can be affected by sample type and host DNA inhibition.
- Novel diagnostic tools are needed for improved Borrelia burgdorferi detection.
Purpose of the Study:
- To assess the sensitivity and feasibility of a CRISPR/Cas12 detection system for Borrelia burgdorferi.
- To compare the performance of the CRISPR/Cas12 system against real-time PCR.
- To explore new diagnostic strategies for Lyme disease.
Main Methods:
- Targeted amplification of the OspA gene from Borrelia genospecies DNA.
- Detection using a Cas12/crRNA system with a fluorescent reporter.
- Sensitivity testing with serial dilutions of Borrelia DNA, with and without human DNA, compared to qPCR.
Main Results:
- Direct detection of Borrelia DNA without amplification was not successful.
- The Cas12/crRNA system, when coupled with PCR, detected as few as 5 genome copies.
- Extended PCR cycles improved detection for some Borrelia species, but human DNA reduced sensitivity.
Conclusions:
- The Cas12/crRNA system provides a sensitive and accessible diagnostic alternative to qPCR.
- This system is particularly valuable in resource-limited settings lacking real-time PCR.
- Future research may integrate Cas12 with isothermal amplification and microfluidics for enhanced direct detection.
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