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 10 copies/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.
INTRODUCTION/BACKGROUND
Bacteria 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 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 of primers and probe were synthesized by Sigma Genosys (The Woodlands, TX) (Sequences; Table 1; Fig. 1).
DNA purification and plasmid cloning of entire gene sequence. Primers for the full gene sequence of B. lonestari flagellin 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).
Full gene primer efficiency was tested by nested PCR and verified by agarose gel electrophoresis. QIAquick PCR Purification Kit (Qiagen; Valencia, CA) was used to purify DNA 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 1- min of 10μL sample, both qPCR and conventional PCR were employed to test for presence of B. lonestari.
Following manufacturer’s instructions, 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 found 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 machines (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, cloned B. lonestari flagellin gene target DNA was chosen for use in standard dilutions based on its concentration (~6.93x1010 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 (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).
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 101 copies/μL (Ct value = 40.40) however consistently only detected 102.5 copies/μL (Ct value = 37.88). The R2 value obtained was 0.986 (Fig. 4).
Assay specificity. All Borrelia related and 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.
////For poster:
· Primers and probe are developed to create a specific and sensitive B. lonestari qPCR assay.
· The sensitivity threshold of the assay is shown consistently to be 101 copies/μL and up to 100, or 1, copy/μL.
· The assay is specific since only B. lonestari DNA was detected and not 30 related bacterial sequences
· 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 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) 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 assay and full-gene sequences. The bolded FAM base pairs are complimentary sequences in the Beacon hairpin structure.
Oligonucleotide Name / Purpose / Sequence (5’ – 3’)B. lon-655FAM / Assay Beacon probe / [DFAM]-CGC GAC CAG CTC CAG CTC AAG GTG GGA TTA GTC GCG-[DBH1]
B. lon-594F / Assay forward primer / TGG TGG AGA AGG TGT TCA AG
B. lon-719R / Assay reverse primer / GCA TTA GCA TCA ATA GCA GTT G
B. lon-11F / Full-gene forward primer / ATC ATA ATA CGT CAG CTA TAA ATG C
B. lon-970R / Full-gene reverse primer / ATA CAT ATT GAG GCA CTT GAT TTG
FIG 1. Developed primers and probes on the 990bp B. lonestari flagellin gene
TABLE 2. Optimal final conditions for qPCR
Reagent / Volume or ConcentrationVolume Template / 1μl
Volume Reaction for SmartCycler Tube / 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
TABLE 3. Bacteria sequences tested for B. lonestari assay specificity were all tested positive.
No. / Borrelia Bacterial DNA / Non-Borrelia Bacterial DNA1 / B. recurrentis / R. prowazekii Breinl
2 / B. coriaceae* / R. typhi W
3 / B. burgdorferi / R. canada
4 / B. afzelii / R. rickettsii VR 891
5 / B. hermsii / R. conorii
6 /
B. garinii
/ R. parkeri7 / B. duttoni / R. montana
8 / R. slovaca
9 / R. sibirica
10 / R. japanica
11 / R. akari
12 / Escherichia coli
13 / Proteus mirabilis OXK
14 / Salmonella enterica
15 / Legionella pneumophila
16 / 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 55˚C, however at 66˚C the results are 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.