The Development and Optimization of a Sensitive and Specific quantitative PCR Assay for Borrelia lonestari HaoQi (Esther) Li Naval Medical Research Center, Infectious Diseases Department, Rickettsial Diseases Department Mentors: Dr. Ju Jiang, Dr. Allen Richards 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 becomes very challenging. Previous methods to detect B. lonestari include polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. 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 (1010-100 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, MgCl2, and changing the annealing temperature, an optimal condition was established. The limit of detection of the assay was determined to be 1 copy/uL. Seven related Borrelia spp. and twenty-three non-related bacterial genomic DNA were used to verify the specificity. The assay only responded positively to B. lonestari thus demonstrating that our assay is indeed specific. Therefore, the newly developed qPCR assay has proven to be a sensitive and specific tool for detecting B. lonestari. INTRODUCTIONBacteria Borrelia lonestari belongs to the same genus as Borrelia burgdorferi, the causative agent of the potentially fatal Lyme disease. B. lonestari itself is vectored by the Lone Star tick Amblyomma americanum and causes the infectious southern tickassociated 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 from 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 the variation of the sequences 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 gene 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 (BHQ-1) on the 3’ end. Oligonucleotides of primers and probe (Table 1; Fig. 1) were synthesized by Sigma Genosys (The Woodlands, TX). Cloning of B. lonestari flagellin gene. Primers for the full gene sequence of B. lonestari flagellin were designed 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). Using this pair of primers, a 960 bp amplicon was amplified by nested PCR and verified by agarose gel electrophoresis. QIAquick PCR Purification Kit (Qiagen; Valencia, CA) was used to purify the PCR product according to manufacturer’s instructions. The purified PCR product was then cloned using TOPO XL PCR Cloning Kit (Invitrogen; Carlsbad, CA) as per manufacturer’s instructions. After lysing the resulting bacterial cells via boiling for 10 min of 10μL culture, both qPCR and conventional PCR were employed to identify the positive clones . Following manufacturer’s instructions, the QIAprep Spin Miniprep Kit (Qiagen; Valencia, CA) was used to isolate the plasmid from two of the five positive bacterial cultures. The concentrations of those two plasmid were measured 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), MgCl2 (ranges 3mM – 7mM and 3.5mM – 7.5mM), and annealing temperature (range 56˚C - 66˚C). For each test, 101 copies/μl and 102 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 (Cepheid; Sunnyvale, CA) (Table 2). Thermal cycling parameters include 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, the plasmid with target B. lonestari flagellin gene was chosen for use in standard dilutions based on its concentration (~6.93x1010 copies/μL). Serial ten-fold and half-log dilutions of the plasmid were made with TE buffer. The specificity of the assay was tested using seven related Borrelia spp, and twenty-three non-related bacterial genomic DNA (Table 3). 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). cloning of B. lonestari flagellin gene. All five bacteria cultures obtained from DNA cloning of B. lonestari flagellin gene demonstrated positive results when assayed by B. lonestari qPCR (Fig. 3). Assay Sensitivity. A serial dilutions of the plasmid range from 107 to 100 were test eight times,assay sensitivity demonstrated the ability to detect 1 copy/μL (Ct value = 40.40) however consistently only detected 101 copy/μL (Ct value = 37.88). The R2 value of the standard curve obtained was 0.986 (Fig. 4). Assay specificity. All seven Borrelia related and twenty-three unrelated genomic DNA samples were shown to be negative (Table 3). Conclusion: In this experiment, primers and probe are developed to create a specific and sensitive B. lonestari qPCR assay. Through qPCR, the sensitivity threshold of the assay is shown to be 102.5, or 316, copies/μL. Testing against 30 related bacterial sequences producing negative results verifies specificity of assay. Future research should include testing of clinical samples by B. lonestari qPCR in order to further support conclusions made in this study. Utilizing this qPCR assay will hopefully reduce diagnosis time and increase diagnosis accuracy of B. lonestari infection in STARI patients so as to expedite proper treatment process. References 1) Moore IV, Victor A., et al. "Detection of Borrelia lonestari, Putative Agent of Southern TickAssociated Rash Illness, in White-Tailed Deer (Odocoileus virginianus) from the Southeastern United States." Journal of Clinical Microbiology 41.1 (Jan. 2003): 424-427. 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) B. lonestari flagellin sequence accession codes: AY850063 ACKNOWLEDGEMENTS Dr. Allen L. Richards, Director Rickettsial Diseases Department Dr. Ju Jiang, Navy Medical Research Center Dr. Wood, Research Advisor, TJHSST Mr. Pearce, Mentorship Director, TJHSST Science & Engineering Apprentice Program, NMRC TJHSST Mentorship Program TABLE 1. Designed Borrelia lonestari primers and probe sequences. Oligonucleotide Name Purpose Sequence (5’ – 3’) B. lon-655FAM Assay Beacon probe B. lon-594F Assay forward primer Assay reverse primer Full-gene B. lon-719R B. lon-11F [FAM]-CGC GAC CAG CTC CAG CTC AAG GTG GGA TTA GTC GCG-[BHQ1]* TGG TGG AGA AGG TGT TCA AG GCA TTA GCA TCA ATA GCA GTT G ATC ATA ATA CGT CAG CTA TAA ATG C B. lon-970R forward primer Full-gene reverse primer ATA CAT ATT GAG GCA CTT GAT TTG * The bolded FAM base pairs are complimentary sequences in the Beacon hairpin structure. FIG 1. Developed primers and probes on the 990bp B. lonestari flagellin gene 11F Primer 594F Primer 655 FAM 719R Primer 970R Primer TABLE 2. Optimal final conditions for qPCR Reagent Volume or Concentration Volume Template 1μl Volume Reaction 25μl B. lon 594 Forward Primer 0.4μM B. lon 719 Reverse Primer 0.4μM FAM Probe 0.3μM DNTP (contained in supermix) 0.2mM MgCl2 (3mM also in supermix) 4.5mM Platinum Taq (contained in supermix) 0.75 U The 2X Super Mix-UDG with no ROX and H2O were used but optimization was not needed [in Materials/Methods] TABLE 3. Bacteria sequences tested for B. lonestari assay specificity were all tested negative. No. Borrelia Bacterial DNA 1 B. recurrentis 2 B. coriaceae* 3 B. burgdorferi 4 B. afzelii 5 B. hermsii 6 B. garinii 7 B. duttoni 8 9 10 11 12 13 14 15 16 Non-Borrelia Bacterial DNA R. prowazekii Breinl R. typhi W R. canada R. rickettsii VR 891 R. conorii R. parkeri R. montana R. slovaca R. sibirica R. japanica R. akari Escherichia coli Proteus mirabilis OXK Salmonella enterica Legionella pneumophila Francisella persica 17 Bartonella quintana 18 Bartonella vinsonii 19 Neorickettsia sennetsu 20 Neorickettsia riticii 21 Orientia tsutsugamushi 22 Staphylococcus aureus 23 Corynebacterium sp *B. coriaceae showed a weak reaction at the annealing temperature of 60˚C, however at 66˚C the results were negative. FIG 2. Initial testing of assay primers and probe 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. FIG 4. Standard curve and FAM log with Sample ID values as powers of 10.