Development & Optimization of a Sensitive & Specific
Quantitative Real-Time PCR Assay for Borrelia lonestari
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 (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
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.
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
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 GA C CAG CTC CAG CTC AAG GTG GGA TTA G TC GCG -[DBH1]
B. lon -594F
B. lon -719R
Assay forward primer
TGG TGG AGA AGG TGT TCA AG
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. Primers and probes for the 990bp B. lonestari flagellin gene
11F Primer
594F Primer
TABLE 2. Optimal final conditions for qPCR
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.
Reagent
Volume Template
Volume Reaction for
SmartCycler
Tube
B. lon
594 Forward
Primer
Volume or Concentration
1μl
25μl
B. lon 719 Reverse Primer
FAM Probe
DNTP (contained in supermix)
0.4μM
0.3μM
0.2mM
4.5mM
MgCl2 (3mM also in supermix)
Platinum Taq (contained in
supermix)
719R Primer 970R Primer
FIG 4. Standard curve and FAM log with Sample ID values as powers of 10.
0.4μM
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 negative.
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.
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
100 copies/μL (data not shown), and consistently detected 101 copies/μL
(Fig 4).
Assay specificity. All Borrelia related and unrelated genomic DNA
samples were shown to be negative (Table 3).
CONCLUSION
FIG 2. Initial testing of assay primers and probe
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 480580nm 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).
655 Probe
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), 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 included a prehold 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 tenfold 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).
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Non-Borrelia Bacterial
Borrelia Bacterial DNA
DNA
B. recurrentis
R. prowazekii Breinl
B. coriaceae*
R. typhi Wilmington
B. burgdorferi
R. canadensis
B. afzelii
R. rickettsii VR 891
B. hermsii
R. conorii
B. garinii
R. parkeri
B. duttoni
R. montanensis
R . slovaca
R. sibirica
R. japonica
R. akari
Escherichia coli
Proteus mirabilis OXK
Salmonella enterica
Legionella pneumophila
Francisella persica
Bartonella quintana
Bartonella vinsonii
Neorickettsia sennetsu
Neorickettsia risticii
Orientia tsutsugamushi
Staphylococcus aureus
Corynebacterium sp
*B. coriaceae showed a weak reaction at the annealing
temperature of 55˚C, however at 66˚C the results were negative.
• 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.
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
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