Project Proposal draft 3
June 30, 2006
Title of Project: Developing an Optimized qPCR for Borrelia lonestari
Name: HaoQi (Esther) Li
Objective:
The purpose of this project is to find and optimize the probe for detecting the Lyme disease-like illness caused by
bacteria Borrelia lonestari.
Justification:
Lyme disease is caused by bites from Borrelia burgdorfeir-bacteria-carrying ticks. The symptoms, such as rash,
fever, fatigue, etc. resemble those of common ailments, thus making the diagnosis of Lyme disease more
challenging.
Borrelia lonestari is a recently discovered Lyme disease-like causing spirochete, i.e. a spiral shaped bacterium. Its
vector is the lone star tick, Amblyomma americanum, commonly found in southeastern United States. Like the other
strands of the Gram-negative Borrelia, B. lonestari has two flagella that can facilitate the corkscrew movements of
the spirochete, making the disease more easily to be spread in a host’s body.
If this experiment shall succeed, the synthesized molecular Beacon probe will be very specific for the detection of B.
lonestari’s flagellin gene, providing a more efficient tool to diagnose the Lyme disease-like illness.
Description:
Testing Design:
Using programs 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. A request will be sent to
Sigma-Genosys, a company that will synthesizes the probe and primers. The Invitrogen TOPO TA Cloning kit will
be used to make clones of B. lonestari flagellin gene in order to determine the assay’s sensitivity. Each reagent
concentration is optimized in preparation for the test. Finally test the probe and primers using real-time PCR, i.e.
quantitative PCR (qPCR), to determine the sensitivity of the assay. If the results are not desired, then draw
conclusions and modify the probe on Beacon Designer and repeat the process again.
Controls: Negative control = no DNA. Positive control = B. lonestari flagellin DNA positive
1. Clean room (no DNA allowed) negative control
2. Dirty hood negative control
3. Positive control
The sensitivity of the assay is determined by using the positive control with known concentrations of B. lonestari
flagellin gene and the specificity of the array will be determined using DNA preparations from 31 other Borrelia,
Rickettsia, and other bacteria:
No.
1.
2.
3.
4.
5.
6.
7.
8.
9.
Bacteria
1B. recurrentis
2B. coriaceae
3B. burgdorferi
4B. afzelii
5B. hermsii
6B. garinii
7B. duttoni
1R. prowazekii Breinl
2R. prowazekii Ananiev
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
3R. typhi Musibov
4R. canada
5R. rickettsii R
6R. rickettsii 364-D
7R. conorii
8R. parkeri
9R. montana
0R. slovaca
1R. sibirica
2R. japanica
3R. akari
1Escherichia coli
2Proteus mirabilis OXK
3Salmonella enterica
4Legionella pneumophila
5Francisella persica
6Bartonella quintana
7Bartonella vinsonii
8Neorickettsia sennetsu
9Neorickettsia riticii
0Orientia tsutsugamushi
1Staphylococcus aureus
2Corynebacterium sp
Using the online BLAST (Basic Local Alignment Search Tool) program, the targeted probe is compared to all other
sequences in the gene bank to insure its uniqueness. And indeed, this expectation has been proven true.
Trials: 3 trials
Quantitative goal:
The probe should detect at least 10 copies of Borrelia lonestari flagellin target gene per μl. In addition the probe
should not detect any copies of Borrelia lonestari in the non-B. lonestari samples.
Materials:
In addition to the basic material such as test tubes, micropipettors, lab coats, gloves, cleaning liquid, biohazard bags,
test tube racks, micropipettor tips, dNTP, MgCl2, qPCR buffers, H2O etc.
The programs ClustalW and GeneDoc will be used for sequence alignment and comparison.
The program Beacon Designer 4.0 will be used for optimizing the possible probe and primers.
Company Sigma-Genosys will synthesize the Beacon probe and primers
Invitrogen TOPO TA Cloning kit will be used for DNA cloning via bacteria.
The program and machines of Smart Cycler 2.0d will be used for running the qPCR.
Costs:
The estimated coast for each sample reaction is $1.3 for the buffer, the tube, the micropipette tips, etc.
The cost of the probe synthesis by Sigma-Genosys after a discount will be $150.
Procedures:
Preparation for qPCR analysis in the clean room (i.e. no DNA)
 Safety: Put on lab coats and gloves, set up biohazard bags in the plastic enclosed workspace.
 Prepare test tubes, test tube racks, micropipetters (10μl, 20μl and 100μl), micropipetter tips of different
sizes

Vortex and centrifuge the reagents and place them in a test tube rack and start transferring materials by
specific μl amounts.
 Clean up the workplace with DNase and RNase cleaning solution
Continued in the dirty hood…
 Add specific concentration of each DNA sample into special qPCR tubes
 After vortex and centrifuging, place the samples inside SmartCycler II qPCR machine and start the
SmartCycler computer program to receive data from the machine.
 Clean up workspace and wait for the results on the computer.
Testing:
 For cross reactivity testing, 100pg of DNA is used in each reaction, which is about 10 4 – 105 copies in each
reaction
 For the positive control, 103 copies of DNA is used in the reaction
 Standard curve with “Crossing Threshold vs. Concentration or Copy Number” to determine the sensitivity
of the assay
Results
 Data measured and collected by the SmartCycler 2.0d will display in a window resembling:
Analysis:
I would look at the logarithmic curve of the fluorescence detected and if everything goes according to the plan and
the probe is specific, only the positive sample that contains B. lonestari would 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 probe can be detected based on the minimum concentration required 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 probe will be specific and sensitive, then future scientists can use the probe designed in this experiment for
medical purposes of detecting Lyme disease caused by the lone star tick.
Limitations:
The biggest concern, besides limiting the probable contamination, is that there will be some undesired binding by
the probe to unpredicted DNA sites of other organisms by the Beacon Designer 4.0 program so that the experiment
fails to run.
If time will be limited, not all 32 strands of DNA will be tested, however, it is crucial that all the other Borrelia
sequences are tested before the experiment is over.
Mentor signature:
References
Cutler, S. J., Moss, J., Fukunaga, M., Wright, D. J., Fekade, D., & Warrell, D. (1997, October). Borrelia recurrentis
characterization and comparison with relapsing-fever, lyme-associated, and other Borrelia spp.
International Journal of Systematic Bacteriology, 47(4), 958-968.
Similar methods
Jiang, J., Temenak, J. J., & Richards, A. L. (2003, July). Real-time PCR duplex assay for Rickettsia prowazekii and
Borrelia recurrentis. Rickettsiology: Present and Future Directions, 990, 302-310.
Kelly, D. J., Richards, A. L., Temenak, J., Strickman, D., & Dasch, G. A. (2002, June). The past and present threat
of Rickettsial diseases to military medicine and international public health. Clinical Infectious Diseases,
34(Suppl. 4), S145-S169.
Meri, T., Cutler, S. J., Blom, A. M., Meri, S., & Jokiranta, T. S. (2006, July). Relpsing fever spirochetes Borrelia
recurrentis and B. duttonii acquire complement regulators c4b-binding protein and factor H. Infection and
Immunity, 74(7), 4157-4163.
Nice introduction on Borrelia
Probe based quantitative PCR. (2006). Quantitative PCR. Retrieved June 21, 2006, from Sigma-Aldrich Co. Web
site:
http://www.sigmaaldrich.com/Area_of_Interest/Life_Science/Molecular_Biology/PCR/Quantitative_PCR.h
tml
What is lyme disease? (2006). Lyme disease. Retrieved June 29, 2006, from American Lyme Disease Foundation
Web site: http://www.aldf.com/lyme.shtml