1. Development & Optimization of a Sensitive & Specific Quantitative Real-Time PCR Assay for Borrelia lonestari HaoQi (Esther) Li Mentors: Allen Richards, Ph.D., Ju Jiang Rickettsial Diseases Department, Infectious Diseases Directorate U.S. NAVAL MEDICAL RESEARCH CENTER Silver Spring, MD ABSTRACT Borrelia lonestari is a spiral-shaped bacterium recently discovered in the lone star tick, Amblyomma americanum, located throughout the southeastern United States. This spirochete is suspected of inducing signs and symptoms in humans commonly associated with Lyme disease such as rash, fever, and fatigue. Due to these common symptoms the diagnosis of the B. lonestari infection is very challenging. Previous methods to detect B. lonestari included polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses. However advances in biotechnology have introduced quantitative real-time PCR (qPCR) as a more accurate and efficient detection procedure. Therefore we report the development of a qPCR assay, which is highly sensitive and specific for detecting B. lonestari. Using the programs ClustalW and GeneDoc, a unique region between 594–719bp in the B. lonestari flagellin gene was identified and primers and a molecular Beacon probe were designed. A plasmid containing the target B. lonestari flagellin gene sequence was constructed with the TOPO TA Cloning Kit. After calculating the copies of the cloned plasmid, a serial dilution (10 10 -10 0 copies/uL) was made for a standard curve to quantitatively demonstrate the sensitivity of the assay. By using various concentrations of the primers, the probe, MgCl 2, and changing the annealing temperature, an optimal condition was established. The limit of detection of the assay was determined to be 1 copies/uL. Seven related Borrelia spp. and twenty-three non-related bacterial genomic DNA samples were used to verify the specificity. The assay only responded positively to B. lonestari thus demonstrating that the assay is specific. These results show that the newly developed qPCR assay to be a sensitive and specific tool for detecting B. lonestari. BACKGROUND Bacteria Borrelia lonestari belongs to the same genus as Borrelia burgdorferi, the causative agent of Lyme disease. B. lonestari itself is vectored by the Lone Star tick Amblyomma americanum and is proposed to cause the infectious southern tick-associated rash illness (STARI), which exhibits symptoms similar to Lyme disease and similar to those of many common illnesses (1). These symptoms include rash, fever, and fatigue, and their commonplace nature, along with the absence of a current method of diagnosis, often makes diagnosing the STARI disease extremely difficult. Since this “Lyme disease-like infection” is found throughout the southern United States (2), the development of an efficient method of diagnosis for this disease will be very practical. Fortunately, in recent years qPCR has proved to be both an efficient and accurate method of detecting bacterial DNA. Therefore developing a qPCR assay that is sensitive and specific for B. lonestari will be extremely helpful in diagnosing and treating STARI. MATERIALS AND METHODS Assay primers and probe. The B. lonestari flagellin gene sequence (3) was obtained using NCBI GenBank (NCBI; Bethesda, MD). NCBI’s Basic Local Alignment Search Tool (BLAST) was used to identify 34 highly related sequences. The sequences were aligned using ClustalW (EBI; Cambridge, UK) and their base pair differences were colored-coded using GeneDoc (PSC; Pittsburgh, PA). Regions of uniqueness were identified and it was found that an 18bp region between 667-668bp was deleted only in the B. lonestari flagellin sequence. Targeting around this area, the software program Beacon Designer 4.0 (Premier Biosoft; Palo Alto, CA) was used to determine the best combination of primers and probe for qPCR analysis. The Beacon probe has a FAM Reporter on the 5’ end and a Black Hole Quencher 1 (DBH1) with a quench range of 480- 580nm on the 3’ end. Oligonucleotides for the primers and probe were synthesized by Sigma Genosys (The Woodlands, TX) (Sequences; Table 1; Fig. 1). DNA purification and plasmid cloning of flagellin gene sequence. Primers for the B. lonestari flagellin gene (959bp) were developed with Beacon Designer 4.0 by choosing common regions after alignment of similar sequences and were synthesized by MWG (High Point, NC) (Table 1; Fig. 1). Primer efficiency was tested by nested PCR and verified by agarose gel electrophoresis. QIAquick PCR Purification Kit (Qiagen; Valecia, CA) was used to purify DNA. The purified PCR product was then cloned using TOPO XL PCR Cloning Kit (Invitrogen; Carlsbad, CA). REFERENCES 1)Moore IV, Victor A., et al. "Detection of Borrelia lonestari, Putative Agent of Southern Tick-Associated Rash Illness, in White-Tailed Deer (Odocoileus virginianus) from the Southeastern United States." Journal of Clinical Microbiology 41.1 (Jan. 2003): 424-427. 2)2) Varela, Andrea S., et al. "First Culture Isolation of Borrelia lonestari, Putative agent of Southern Tick-Associated Rash Illness." Journal of Clinical Microbiology 42.3 (Mar. 2004): 1163-1169. 3) 3) B. lonestari flagellin sequence accession codes: AY850063 CONCLUSION Primers and probe were constructed to create a specific and sensitive B. lonestari qPCR assay. The assay detected up to 1 copy/μL; consistently detected 10 copies/μL of B. lonestari target DNA. The assay is specific since only B. lonestari DNA was detected and not 30 related and unrelated bacterial DNA preparations. Future research will include testing of clinical samples with this qPCR to determination the relationship of B. lonestari with disease and potentially reduce diagnosis time and increase diagnosis accuracy of B. lonestari infections among STARI patients so as to expedite proper treatment. RESULTS/DISCUSSION Assay Primers and probe. The synthesized primers and probe were tested on two unknown concentrations of B. lonestari samples and the results for both were positive verifying the assay’s ability to detect B. lonestari (Fig. 2). DNA purification and plasmid cloning of full gene sequence. All five bacteria colonies obtained from DNA cloning of B. lonestari flagellin gene demonstrated positive results when assayed by B. lonestari qPCR (Fig. 3). Assay Sensitivity. Assay sensitivity demonstrated the ability to detect 10 0 copies/μL (data not shown), and consistently detected 10 1 copies/μL (Fig 4). Assay specificity. All Borrelia related and unrelated genomic DNA samples were shown to be negative (Table 3). ACKNOWLEDGEMENTS Dr. Allen L. Richards, Director Rickettsial Diseases Department Dr. Ju Jiang, Navy Medical Research Center Dr. Barbara Wood, Thomas Jefferson High School for Sci. & Tech. Joey Flyer, University of Rochester Science & Engineering Apprentice Program, NMRC TJHSST Mentorship Program TABLE 1. Designed Borrelia lonestari assay and full-gene sequences. The bolded FAM base pairs are complimentary sequences in the Beacon hairpin structure. FIG 4. Standard curve and FAM log with Sample ID values as powers of 10. FIG 1. Primers and probes for the 990bp B. lonestari flagellin gene FIG. 3. Positive results for all five transfomant bacteria colonies. B1, B4, and B5 showed a decrease in fluorescence after crossing over the threshold, caused by over-abundance in amplification. TABLE 2. Optimal final conditions for qPCR FIG 2. Initial testing of assay primers and probe *B. coriaceae showed a weak reaction at the annealing temperature of 55˚C, however at 66˚C the results were negative. TABLE 3. Bacteria sequences tested for B. lonestari assay specificity were all tested negative. 11F Primer 594F Primer 655 Probe 719R Primer 970R Primer MATERIALS AND METHODS (continued) After lysing the resulting bacterial cells via boiling for 10 min of 10μL sample, both qPCR and conventional PCR were employed to test for presence of B. lonestari flagellin sequence. The QIAprep Spin Miniprep Kit (Qiagen; Valencia, CA) was used to extract two of the five bacterial samples tested. The DNA concentrations of those two samples were determined using the Eppendorf BioPhotometer (Eppendorf; Westbury, NY). Optimization tests. Optimization tests were performed with qPCR for conditions of the assay primers (range 0.1μM – 0.7μM), assay probe (range 0.2μM – 0.7μM), MgCl 2 (ranges 3mM – 7mM and 3.5mM – 7.5mM), and annealing temperature (range 56˚C - 66˚C). For each test, 10 1 copies/μl and 10 2 copies/μl of DNA templates were used. Optimal conditions were determined based on the lowest cycle threshold values of logarithmic fluorescence using the Smart Cycler machines (Cepheid; Sunnyvale, CA) (Table 2). Thermal cycling parameters included a pre- hold of 50˚C for 2min, a hold at 95˚C for 2min, followed by 50 two-step cycles of 94˚C/5secs and 60˚C/30secs. Assay Sensitivity/Specificity. To determine the assay sensitivity, cloned B. lonestari flagellin gene target DNA was chosen for use in standard dilutions based on its concentration (~6.93x10 10 copies/μL). Serial ten- fold and half-log dilutions of the sample were performed using TE buffer. The specificity of the assay was tested using seven related Borrelia spp, and twenty-three non-related bacterial genomic DNA preparations (Table 3).