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.
Introduction/Background
Bacteria Borrelia lonestari, belongs to the same genus as B. burgdorferi, the vector of the
potentially fatal Lyme disease. B. lonestari itself causes the infectious southern tick-associated
rash illness (STARI), which exhibits symptoms linked 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 the vector of Borrelia lonestari, the
Lone Star tick Amblyomma americanum (2), is found throughout south and southeastern United
States, the development of an assay for this bacteria will be very practical.
Fortunately, in recent years qPCR has proved to be an efficient and accurate method of detecting
bacterial DNA. By developing a qPCR assay (i.e. forward and reverse primers with a probe in
between) that is sensitive and specific for B. lonestari, it will provide an expedient, reliable
method for diagnosing STARI and treating patients.
MATERIALS AND METHODS:
Assay primers and probe. The B. lonestari flagellin gene sequence (3) was obtained using
National Center for Biotechnology Information's 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 eighteen-base-pair region between 667-668bp was
deleted only in the B. lonestari flagellin sequence. Targeting around this area, the program
Beacon Designer 4.0 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, which fluoresces if the original
Beacon hairpin structure anneals onto the target DNA. The Black Hole Quencher 1 (DBH1),
with a quench range of 480-580nm, is attached on the 3’ end of the probe to minimize
interference of the fluorescent. Adding five extra complimentary base pairs on either side of the
probe, the double-stranded handle of the hairpin can be formed. Oligonucleotides of primers and
probe were synthesized by Sigma Genosys (The Woodlands, TX) (Table 1).
DNA purification and plasmid cloning of entire gene sequence. The primers for the entire
gene sequence were developed with Beacon Designer 4.0 by choosing common regions after the
sequences realignment. These oligonucleotides were synthesized by MWG (Table 2).
Primer efficiency was tested by nested PCR and verified by agarose gel electrophoresis (Figure
1). 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. lon.
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 samples
#2 and #4 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 flouresence using the Smart Cycler
machines (Cepheid; Sunnyvale, CA) (Table 3).
Assay Sensitivity/Specificity. A 1010 – 100 serial dilution was performed on sample #2 and
tested by qPCR to determine assay sensitivity. The specificity of the assay was tested using
seven related Borrelia spp, and twenty-three non-related bacterial genomic DNA (Table 4).
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 (Figure 2).
DNA purification and plasmid cloning of entire gene sequence. The five bacteria colonies
extracted all showed positive results for B. lonestari (Figure 3).
The Miniprep Sample #4 had a calculated concentration about 5.91 x 1010 copies/μl, but sample
#2, with a calculated concentration of about 6.93 x 1010 copies/μl, was chosen for the standard
curve, with samples diluted from TE buffer.
Assay Sensitivity. A standard curve for sensitivity is ran for B. lonestari DNA concentrations
107, 106, 105, 104, 103, 102.5, 102, 101.5, 101, 100.5, and 100. The best standard curve and its
corresponding FAM log and results table obtained are in Figure 4. The assay was sensitive
enough for 101 copies/μl, but 102 failed to show due to experimental errors. However with the
standard curve 4-squared value so close to 1, the assay is nevertheless very reliable.
Assay specificity. The specificity of the assay is tested on the bacteria sequences listed in Table
4 and all were shown to be negative, except that B. coriaceae was almost positive. However, at
the annealing temperature of 66˚C, B. coriaceae results in negative. Thus using higher annealing
temperatures may reduce the chance of erroneous data.
Conclusion:
The developed assay is shown to be sensitive to 10copies/μl and unique to B. lonestari. More
research is needed to find out the reason for the almost-positive result of B. coriacecae, whether
a human error had occurred or whether the sequence of B. coriaceae itself is inclined to the assay
developed.
Furthermore, the assay still needs to be tested on real samples from ticks and other animals,
including humans.
With the information from this study, scientists and doctors may utilize this newly synthesized
molecular Beacon assay, which is highly specific for the detection of B. lonestari’s flagellin
gene, as a more efficient tool to diagnose the Lyme disease-like illness so that proper treatments
can be given to the patients.
Summarizing of Materials and Methods:
Using programs GenBank, ClustalW, and GeneDoc, the flagellin gene sequence of B. lonestari
will be aligned and compared to the sequences of other Borrelia strands. Manually, areas highly
unique for B. lonestari will be selected for the design of molecular Beacon probe and primers.
The feasibility of the proposed probes will be tested with Beacon Designer 4.0, the program used
to optimize the lengths and locations of the primers. Using the online BLAST (Basic Local
Alignment Search Tool) program, the targeted probe is compared to all other sequences in the
gene bank to ensure its uniqueness. A request will be sent to Sigma-Genosys, a company that
will synthesizes the probe and primers. The Invitrogen TOPO XL PCR Cloning kit will be used
to make clones of B. lonestari flagellin gene in order to determine the assay’s sensitivity.
Primers, probe, and magnesium concentrations will be optimized with the Smart Cycler 2.0d
qPCR machine, testing assay reactions within reasonable ranges to locate the best conditions and
concentrations produced by the lowest concentrations are found. Finally using the optimized
concentrations to determine the sensitivity of the assay by running a standard dilution from 107 –
100 and the specificity of the assay by using DNA preparation from seven related Borrelia spp.
and twenty-three non-related bacterial genomic DNA.


The positive results are the logarithmic curves that have crossed the threshold.
The negative results are the logarithmic curves that have remained under the threshold.
Analysis:
The logarithmic curves of the fluorescence detection will be analyzed and if everything goes
according to the plan and the assay is specific, only the positive sample that contains B. lonestari
will have a curve over the threshold. The threshold is arbitrarily set at 30 fluorescence units.
The logarithmic curve above the threshold level is a strong indication of a positive B. lonestari
DNA sample.
The sensitivity of the assay can be determined based on the minimum concentration for the
fluorescent logarithmic curve to cross the threshold. Thus a more sensitive assay with lower
DNA concentrations would still cross the threshold line. A standard curve is then constructed
after compiling the data for “concentration of samples vs. cycles for threshold crossing”.
If the assay will be specific and sensitive, then future scientists can use the assay designed in this
experiment for medical purposes of detecting Lyme-like disease caused by the lone star tick.
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): 424427.
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 code: AY850063 and AY850064
Table 1
Designed Borrelia lonestari assay sequences
Oligonucleotide Name
Purpose
Sequence (5’ – 3’)
B. lon-655FAM
Assay Beacon
[DFAM]-CGC GAC CAG CTC CAG CTC
probe
AAG GTG GGA TTA GTC GCG-[DBH1]
B. lon-594F
Assay forward
TGG TGG AGA AGG TGT TCA AG
primer
B. lon-719R
Assay reverse
GCA TTA GCA TCA ATA GCA GTT G
primer
Table 2
Designed Borrelia lonestari entire flagellin gene primers sequences
Oligonucleotide Name
Purpose
Sequence (5’ – 3’)
B. lon-11F
Entire-gene
ATC ATA ATA CGT CAG CTA TAA ATG C
forward primer
B. lon-970R
Entire-gene
ATA CAT ATT GAG GCA CTT GAT TTG
reverse primer
Figure 1.
The results of the nested PCR to show that the primers for the entire B. lon flagellin are
functioning correctly.
Lane Name
100bp Ladder
F2R1
F1R2
F1R1
Probes
Length
594F, 970R
11F, 719R
11F, 970R
377 bp
709 bp
9960 bp
12-Jul-2006 15:17:24 Low=5 High=179 Gamma=1.0 Exposure=0.10 secs
The standard lane have bands in intervals of 100bp, with a bright double-banded 600bp.
Located in the right position, the longest 960 bp band of interest was also the faintest band since
more energy is required to amplify a longer band.
Table 3
Optimal Final Conditions
Volume Template
Volume Reaction for SmartCycler Tube
B. lon 594 Forward Primer
B. lon 719 Reverse Primer
FAM Probe
DNTP (contained in supermix)
MgCl2 (3mM already in supermix)
Platinum Taq (contained in supermix)
1μl
25μl
0.4μM
0.4μM
0.3μM
0.2mM
4.5mM
0.75 U
The 2X Super Mix-UDG with no ROX and H2O were used but not necessary to be optimized.
Pre-hold
Hold
50 Cycles of:
qPCR temperature conditions
50˚C for 2 min
95˚C for 2 min
94˚C for 5 sec
60˚C for 30 sec – annealing temperature
Table 4.
Bacteria sequences tested for B. lonestari assay specificity
No.
Bacteria
1. 1B. recurrentis
2. 2B. coriaceae
3. 3B. burgdorferi
4. 4B. afzelii
5. 5B. hermsii
6. 6B. garinii
7. 7B. duttoni
8. 1R. prowazekii Breinl
9. 3R. typhi W
10. 4R. canada
11. 5R. rickettsii VR 891
12. 7R. conorii
13. 8R. parkeri
14. 9R. montana
15. 0R. slovaca
16. 1R. sibirica
17. 2R. japanica
18. 3R. akari
19. 1Escherichia coli
20. 2Proteus mirabilis OXK
21. 3Salmonella enterica
22. 4Legionella pneumophila
23. 5Francisella persica
24. 6Bartonella quintana
25. 7Bartonella vinsonii
26. 8Neorickettsia sennetsu
27. 9Neorickettsia riticii
28. 0Orientia tsutsugamushi
29. 1Staphylococcus aureus
30. 2Corynebacterium sp
Figure 2.
Initial testing of assay primers and probe
Figure 3.
Positive results for all five transfomant bacteria colonies
B1, B4, and B5 showed a decrease in fluorescence after crossing over the threshold. According
to the Smart Cycler instruction manuals, this was caused by over-abundance in amplification.
Figure 4. Standard curve and FAM log
The values in Sample ID are powers of 10.