Download The Development and Optimization of a Sensitive and Specific

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.
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-text). * 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.
*The master mixes were prepared for the qPCR assays in a separate clean room (DNA-free).
Three negative controls (Gibco ultrapure DNA-free distilled water, Invitrogen Corp., Grand
Island, NY), one produced in the clean room and two in a biosafety cabinet in another laboratory
where the DNA templates were added, were included in each run.
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) and PCR(Fig [7/20]).
Assay Sensitivity. A serial dilutions of the plasmid range from ? to ? were test eight times,assay
sensitivity demonstrated the ability to detect 10 copies/μL (Ct value = 40.40) however
consistently only detected ?102.5 copies/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.
////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 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.
[Add: B. lonestari qPCR [all negative]
No.
Borrelia Bacterial DNA
Non-Borrelia Bacterial DNA
B. lonestari
1 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. parkeri
7 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 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.