Nested PCR is a sensitive, specific, and straightforward technique that can be applied to tick DNA extracts to probe for Borrelia burgdorferi, the causative agent of Lyme disease. The initial PCR experiment uses gene-specific primers to generate long amplicons, which then become templates for a subsequent reaction using internal primers.
Total time
~3–4 hours per tick sample
Steps
1
Extract DNA from tick specimens
Isolate genomic DNA from tick samples using appropriate extraction protocols. This extracted DNA serves as the template for downstream PCR amplification.
▶ 01:42
2
Perform nested PCR for Borrelia detection
Conduct two sequential PCR reactions using gene-specific primers targeting OspA and FlaB genes. The first reaction generates long amplicons; the second uses internal primers on the first product for increased sensitivity and specificity.
▶ 04:06
3
Analyze and interpret nested PCR results
Examine amplicon bands on agarose gel electrophoresis to determine presence or absence of Borrelia burgdorferi. Compare results against positive and negative controls.
▶ 05:25