Lyme disease is a debilitating illness caused by the spirochetal pathogen Borrelia burgdorferi sensu lato. The bacterium is transmitted to humans through the bite of an infected tick, and it causes multi-system symptoms that can affect the skin, joints, heart, and nervous system. Everyday activities like dog walking and hiking can put people at increased risk of contracting Lyme disease.
Strategies to combat this growing threat therefore include robust surveillance measures that can indicate the pathogen prevalence in ticks and identify geographic regions of concern. This video demonstrates the utility of nested polymerous chain reaction as a molecular screening tool for Borrelia. To increase specifically, both outer-surface protein A and flagellin B genes are target for amplification.
The workflow begins with tick collection, DNA extraction, and then an initial round of PCR to detect Borrelia-specific loci. Second-round PCR uses the product of the first reaction as a new template to generate smaller internal amplification fragments. A tick is considered positive for the pathogen when inner amplicons from both Borrelia genes can be detected by agarose gel electrophoresis.
Passive surveillance enlists the help of citizens and veterinarians to collect and submit ticks for testing. As part of this process, it is important to capture information such as the tick host, date, and location of the encounter. In the laboratory, ticks should first be photographed and identified based on morphological comparison to standard keys.
The sensitivity of nested PCR makes this technique vulnerable to contamination, so each stage of sample processing should occur in a separate, clean location, and multiple controls should be included to ensure that the reagents and environment are free of contaminants. Once the workspace has been suitably prepared, bisect the tick in a sterilized biological safety cabinet and place half into a microcentrifuge tube. Any DNA isolation procedure that yields PCR-compatible template can be used.
This video demonstrates a simple chelation-based DNA extraction. To begin, add an appropriate volume of the lytic chelation buffer depending on the size of the tick fragment and homogenize using a microtube pestle. Incubate samples in a water bath at 60 degrees Celsius for 45 minutes, vortex, and then centrifuge the contents.
While waiting, prepare fresh microcentrifuge tubes for each specimen by aliquoting 50 micrometers of isopropanol. Transfer the supernatant into the new microcentrifuge tube, mix by inversion, and re-centrifuge as before. This time, discard the supernatant and rinse the DNA pellet with 70%ethanol.
Remove residual liquid using a pipette and air-dry the pellet for 15 minutes at room temperature. Finally, add 50 micrometers of 1-millimolar Tris to each pellet, and incubate for one hour in a 60-degree water bath to resuspend the DNA. Samples can now be stored at negative 20 degrees Celsius for future molecular analyses.
Begin by sterilizing a PCR cabinet using UV light and 70%ethanol. The reaction cocktail consists of master mix containing the polymerase, water, forward and reverse outer primers, and the DNA extracted in the previous procedure. Separate reactions are set up to detect OspA and FlaB independently in each tick specimen.
Pulse-centrifuge the samples, place them in a thermal cycler, and program the machine. The initial PCR experiment uses gene-specific outer primers that produce long amplicons. PCR products from the first reaction then become the template for subsequent amplification.
This nested PCR uses gene-specific primers that anneal within the first amplicon to produce new, shorter fragments of DNA. Product from the second reaction can now be loaded next to a molecular mass reference ladder on a 1.2%agarose gel and electrophoresed for one hour. Finally, view the gel using a transilluminator.
In this example, each lane represents a different tick specimen, analyzed for both OspA and FlaB. Amplicons are present in only some of the lanes. Although these two samples both generated PCR products for OspA, only one of them captured FlaB.
A sample must produce bands for both genes to be considered positive. Visual inspection of PCR products thus allows the Borrelia status of each tick to be determined. Overall, nested PCR is a sensitive, specific, and relatively straightforward technique that can be applied to Borrelia surveillance.
Data generated from such initiatives can help to identify regions of geographic concern for Lyme disease and estimate the prevalence of this pathogen in nature.
