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
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
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)
655 Probe
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
FIG 2. Initial testing of assay primers and probe
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.
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.
ABSTRACT
MATERIALS AND METHODS (continued)
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.
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).
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.
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
• 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
MATERIALS AND METHODS
1) Moore IV, Victor A., et al. "Detection of Borrelia lonestari,
Assay primers and probe. The B. lonestari flagellin gene sequence (3)
Putative Agent of Southern Tick-Associated Rash Illness, in
was obtained using NCBI GenBank (NCBI; Bethesda, MD). NCBI’s Basic
White-Tailed
Deer
(Odocoileus
virginianus)
from
the
Local Alignment Search Tool (BLAST) was used to identify 34 highly
Southeastern United States." Journal of Clinical Microbiology
related sequences. The sequences were aligned using ClustalW (EBI;
41.1 (Jan. 2003): 424-427.
Cambridge, UK) and their base pair differences were colored-coded
using GeneDoc (PSC; Pittsburgh, PA). Regions of uniqueness were
2) Varela, Andrea S., et al. "First Culture Isolation of Borrelia
identified and it was found that an 18bp region between 667-668bp was
lonestari, Putative agent of Southern Tick-Associated Rash
deleted only in the B. lonestari flagellin sequence. Targeting around this
Illness." Journal of Clinical Microbiology 42.3 (Mar. 2004): 1163area, the software program Beacon Designer 4.0 (Premier Biosoft; Palo
1169.
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 3) B. lonestari flagellin sequence accession codes: AY850063
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).
ACKNOWLEDGEMENTS
Dr. A. L. Richards, Director Rickettsial Diseases Department
Dr. J. Jiang, Navy Medical Research Center
Dr. B. Wood, Thomas Jefferson High School for Sci. & Tech.
Mr. F. Lampazzi, Thomas Jefferson High School for Sci. &
Tech.
Ms. J. Flyer, University of Rochester
Science & Engineering Apprentice Program, NMRC
TJHSST Mentorship Program