Development of a Real Time Polymerase Chain Reaction Assay to Detect the Causative Agent of Lyme's Disease

AbstractThe tick borne pathogen Borrelia burgdorferi, causative agent of Lyme disease, causes significant morbidity in the surrounding region. Current diagnosis of this disease is reliant on either physical identification of patient symptoms, or the detection of antibodies specific to Borrelia. The purpose of this study is to design and optimize a diagnostic assay to detect presence of B. burgdorferi in ticks utilizing real-time polymerase chain reaction (RT-PRC) amplification of the bacterial recA chromosomal gene. Knowledge of the infectivity status of the suspect tick would aid in diagnosis of patients suspected to have been infected, but may not yet be showing outward symptoms. A detection limit of 125 copies of purified B. burgdorferi DNA was achieved under optimized conditions. Currently, isolation of B. burgdorferi DNA from tick vectors is being optimized through a variety of methods from ammonium hydroxide extraction to zirconium bead beating and will be further discussed.

Ben Bonis and Lloyd TurtinenUniversity of Wisconsin-Eau Claire, Department of Biology

IntroductionThe polymerase chain reaction (PCR) is a technique used for the amplification of template DNA to detectable levels. Utilizing heat sensitive DNA polymerase enzymes, amplification involves the periodic fluctuation of temperature to activate and deactivate the enzymes as well as annealing and melting the DNA. In a process known as Real-Time PCR (RT-PCR), one can visually determine the progress of the reaction as well as the quantity of original DNA by monitoring the fluorescence. While DNA itself does not emit light during the reaction, coupling it to a detector molecule such as SYBR Green, which emits at a characteristic wavelength when bound to double stranded DNA, allows the researcher utilize this method (Figure 1).

Figure 1. Binding of SYBR Green to double stranded DNA.

Funding was provided by Center of Excellence for Faculty/Student Research Collaboration

Methods

DNA ExtractionThe following extraction methods were tested on ticks collected from the surrounding region to determine any inhibition of the PCR reaction. DNA extracts were PCR amplified with B. burgdorferi specific primers and analyzed against B. burgdorferi DNA spiked samples (Figure 4).

•Boiling in NH4OH2

•Zirconium bead beating homogenization3

•Sonciation in InstaGene DNA extraction matrix

Real-Time PCRDue to reagent depletion during a reaction, and therefore changing reaction kinetics, endpoint fluorescence is an unreliable method for initial copy number determination1. However, the point at which the amplification of the DNA becomes noticeably exponential is a consistent measure during amplification for similar DNA concentrations. Known as the crossing time or Ct, this feature allows for a reliable measurement in situ (Figure 2).

Condition OptimizationRT-PCR amplification of Borrelia burgdorferi DNA was conducted varying controllable conditions to achieve optimal amplification. Primer concentration, elongation time, annealing temperature, and Mg2+ concentration were altered as single variables using a range of DNA concentrations to determine the conditions yielding the lowest Ct (Figure 3). These optimized conditions were then utilized to determine the detection limit of the reaction, as well as analyzing various extraction techniques.

References

Figure 2. Representative Crossing Time of a RT-PCR Reaction.

Figure 3. RT-PCR Condition Results. Ct was the primary indicator of reaction efficiency. Primer concentrations yielded similar results. Altering elongation time had no apparent effect on Ct. Annealing temperature was most linear when compared to DNA copy number at 59⁰C. Mg2+

concentration optimization was conducted using an Epicentre™ FailSafe premixed enzyme blend, and are therefore designated by premix identification rather than concentration.

ConclusionsOptimum conditions were determined by lowest Ct as well as purity of product as determined by a melting assay (Figure 5). • Differing primer concentrations yielded similar results, so

2µM was chosen to conserve materials. • Extending elongation time had no apparent effect on Ct, and

was kept to 10 seconds in the interest of time. • An annealing temperature of 59⁰C yielded a standard curve

with the highest linearity and product purity. • Mg2+ concentration optimization was conducted using an

Epicentre™ FailSafe premixed enzyme blend. Mixes K and L were chosen due to product purity and fluorescence of the products on a gel.• Zirconium bead beat has yielded the best results in preliminary

studies

Results

1. Real time PCR guide: Understanding real time PCR protocol and real time quantitation. (2009) Premier Biosoft International. http://www.premierbiosoft.com

2. Kirstein F, Rijpkema S, Molkenboer M, Gray JS. (1997) Local variations in the distribution and prevalence of Borrelia burgdorferi senu lato genomospecies in Ixodes ricinus ticks. Applied and Environmental Microbiology 63(3): 1102-06

3. Rijpkema SG, Molkenboer MJ, Schouls LM, Jongejan F, Schellenkens JF. (1995) Simultaneous detection and genotyping of three genomic groups of Borrelia burgdorferi sensu lato in Dutch Ixodes ricinus ticks by characterization of the amplified intergenic spacer region between 5S and 23S rRNA genes. Journal of Clinical Microbiology 33(12): 3091-95

Figure 4. DNA Extraction Using Zirconium Bead Beating. The tick was transferred to a microfuge tube containing zirconium beads and InstaGene DNA extraction matrix. B. burgdorferi DNA was extracted from the tick by zirconium bead beating in a MixerMill 8000D for 6min at full speed and stored at -20°C until analysis. 125fg B. burgdorferi DNA was added to both the spiked sample and the control. These results suggests this tick was positive for B. burgdorferi , as the addition of purified DNA to the extracted DNA shifted the curve downward below that of either sample alone. The melting curve obtained from this assay supports this observation (results not shown).

12.5ng 1.25ng 125pg 12.5pg 1.25pg 125fg 12.5fg 1.25fg0

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Sensitivity Limit

DNA Content

Ct

Figure 5. Sensitivity Limit Assay. A standard curve was created by the serial dilution of B. burgdorferi DNA to determine the minimum DNA content detectable under the optimized conditions. A sensitivity limit of 125 copies was determined and supported by melting curve analysis (results not shown).

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