Title: The Development and Optimization of a Sensitive and Specific quantitative PCR Assay
for Borrelia lonestari
Student: Li, HaoQi
Firm: Naval Medical Research Center, Rickettsial Diseases Department
Mentors: Dr. Richards, Dr. Ju
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 was
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/µL) 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/μL. Seven related Borrelia spp. and
twenty-five 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.
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