How are ticks tested for borreliosis in a laboratory? - briefly
In the laboratory, tick specimens are homogenized, DNA is extracted, and a PCR assay amplifies Borrelia‑specific gene fragments such as flaB or ospA. Positive amplifications are confirmed by sequencing or by immunofluorescence staining of the spirochete in tick tissues.
How are ticks tested for borreliosis in a laboratory? - in detail
Ticks collected from the environment are first placed in sterile vials containing ethanol (70 %) or RNAlater to preserve nucleic acids. Each specimen is identified to species and developmental stage, then logged in a database with collection site, date, and host information. After labeling, the tick is surface‑sterilized with a brief bleach wash followed by sterile water rinses to eliminate external contaminants.
The next step isolates genetic material. The tick is macerated with a sterile pestle or bead‑beater in lysis buffer, then subjected to proteinase K digestion at 56 °C for 1–2 hours. DNA extraction follows either a silica‑column kit or magnetic‑bead protocol, with an elution volume of 50–100 µL. Extraction controls (blank and positive) are processed in parallel to monitor contamination and assay performance.
Detection of Borrelia DNA relies on polymerase chain reaction (PCR). Real‑time quantitative PCR (qPCR) targeting the 16S rRNA gene or the flagellin (flaB) gene provides high sensitivity. Reaction mixes contain primers, a hydrolysis probe, dNTPs, MgCl₂, and a thermostable polymerase. Thermal cycling typically includes: initial denaturation at 95 °C for 3 min; 40 cycles of 95 °C for 15 s and 60 °C for 60 s. Amplification curves are evaluated against a standard curve derived from known copy numbers to estimate pathogen load.
When qPCR yields positive results, confirmatory sequencing is performed. Amplified fragments are purified and sequenced using Sanger technology. Resulting reads are aligned with reference Borrelia genomes in databases such as GenBank to verify species identity (e.g., B. burgdorferi sensu stricto, B. garinii, B. afzelii). Phylogenetic analysis may be applied to assess strain variation.
Alternative or complementary methods include:
- Culture: Inoculation of tick homogenate into Barbour‑Stoenner‑Kelly (BSK) medium under microaerophilic conditions; incubation at 33 °C for up to 6 weeks; observation of spirochete growth by dark‑field microscopy.
- Immunofluorescence assay (IFA): Fixed tick sections incubated with Borrelia‑specific monoclonal antibodies, followed by fluorescent secondary antibodies; visualization under a fluorescence microscope.
- Loop‑mediated isothermal amplification (LAMP): Amplification at a constant temperature (65 °C) using a set of six primers; detection by turbidity or fluorescence, suitable for field laboratories.
Quality assurance incorporates internal controls (e.g., tick actin gene amplification) to confirm DNA integrity, and external proficiency testing with reference laboratories. Results are reported with cycle threshold (Ct) values, estimated copy numbers, species identification, and any culture or serological confirmation. This comprehensive workflow ensures reliable detection of Lyme‑disease agents in tick vectors.