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Development and Optimization of a Sensitive Real-Time PCR Assay for Borrelia lonestari

This project aimed to create a sensitive and specific quantitative real-time PCR assay for Borrelia lonestari, a newly discovered bacteria carried by lone-star ticks causing Southern Tick-Associated Rash Illnesses. The development process involved designing primers and probe, optimization tests, sensitivity, and specificity testing using qPCR. The results demonstrated a specific and sensitive assay for B. lonestari. Future research and clinical applications are promising based on the assay's performance.

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Development and Optimization of a Sensitive Real-Time PCR Assay for Borrelia lonestari

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  1. Development & Optimization of a Sensitive & Specific Quantitative Real-Time PCR Assay for Borrelia lonestari HaoQi (Esther) Li 06/21/06 – 08/11/06

  2. U.S. Naval Medical Research Center • Infectious Diseases Directorate • Rickettsial Diseases Department • 503 Robert Grant Avenue Silver Spring, MD 20910

  3. Dr. Allen Richards Former Director of Rickettsial Diseases Department Dr. Ju Jiang Naval Medical Research Center Scientist Mentors

  4. Organization • My role: science researcher • Working environment: • Clean room • Dirty hood • Smart cyclers • Safety

  5. Problem/Rationale • Borrelia lonestari • Newly discovered • not related to Rickettsia • Southern Tick-Associated Rash Illnesses (STARI) • Resembles Lyme disease • Not Borrelia burgdorferi • Diagnosis problem

  6. Purpose/Goal To find a molecular probe sensitive and specific for Borrelia lonestari flagellin gene

  7. Borrelia lonestari Bacteria • Spirochete is spiral shaped • Carried by lone-star ticks The Borrelia burgdorferi spirochete:the agent of Lyme disease http://www.marvistavet.com/html/body_lyme_disease.html http://www.health.state.ok.us/program/cdd/STARI.htm

  8. Infection of Bacteria • Rash Illnesses • Location Patient with a classic erythema migrans; 1) site of tick bite, 2) red, radial, expanding edge of rash. 3) central clearing. http://newsletter.mydna.com/health/diseases/lyme/othertick/staritick.html

  9. Previous Research • Cultured isolation • PCR-RFLP • Deer samples http://www.policlinicagipuzkoa.com/GeneticaMolecular/imagenes/PROTOMBINA2.jpg

  10. Beacon Probe + qPCR • FAM reporter and Black Hole Quencher 1 • Quantitative real-time PCR http://www.marine.usf.edu/microbiology/nasba.shtml http://bio.takara.co.jp/catalog/catalog_d.asp?C_ID=C1322

  11. Assays • Find unique sequence of B. lonestari • Use full-gene primers to clone the gene 11F Primer594F Primer 655 FAM 719R Primer 970R Primer

  12. Assay Materials • NCBI GenBank – find • Basic Local Alignment Search Tool – relate • ClustalW – align • GeneDoc – color unique bp

  13. Optimization Tests • Primers • Probe • MgCl2 • Annealing Temperature • Concentration variations testing all done by qPCR

  14. Sensitivity Tests • Full gene PCR • Concentration calculation • qPCR standard tests

  15. Specificity Tests • Types • 7 related Borrelia spp. • 23 non-related bacteria DNA • Hypothesis • qPCR

  16. Results: Initial Testing of Assay

  17. Results: Transformant DNA

  18. Results: Assay Sensitivity

  19. Results: Assay Sensitivity Line

  20. Results: Assay Specificity • Negative results • Implications

  21. Conclusions • Primers and probe • create a specific and sensitive B. lonestari qPCR assay. • The assay • Sensitive • Specific • Future research • clinical samples

  22. Reflections • Wonderful learning experience • Patient, informative mentors • Relaxed environment • Long drive, but worth the effort • http://www.nmrc.navy.mil/nmrc_stdt_main.htm • http://www.asee.org/SEAP/index.cfm

  23. Acknowledgements • Dr. Allen L. Richards, Director Rickettsial Diseases Department • Dr. Ju Jiang, Navy Medical Research Center • Dr. Barbara Wood, Thomas Jefferson High School for Sci. & Tech. • Mr. Fred Lampazzi, Thomas Jefferson High School for Sci. & Tech. • Joey Flyer, University of Rochester • Science & Engineering Apprentice Program, NMRC • TJHSST Mentorship Program

  24. Literature Cited • Burkot, T. R., Mullen, G. R., Anderson, R., Schneider, B. S., Happ, C. M., & Zeidner, N. S. (2001, May/June). Borrelia lonestari DNA in adult Amlyomma americanum ticks, Alabama. Emerging Infectious Diseases, 7(3), 471-473. • Fukunaga, M., Okada, K., Nakao, M., Konishi, T., & Sato, Y. (1996, October). Phylogenetic analysis of Borrelia species based on flagellin gene sequences and its application for molecular typing of lyme disease Borreliae. International Journal of Systematic Bacteriology, 46(4), 898-905. • Jiang, J., Chan, T.-C., Temenak, J. J., Dasch, G. A., Ching, W.-M., & Richards, A. L. (2004). Development of a Quantitative Real-Time Polymerase Chain Reaction assay specific for Orientia tsutsugamushi. American Society of Tropical Medicine and Hygiene, 70(4), 351-356. • Jiang, J., Temenak, J. J., & Richards, A. L. (2003). Real-time PCR duplex assay for Rickettsia prowazekii and Borrelia recurrentis. In K. E. Hechemy, T. Avsic-Zupanc, J. E. Childs, & D. A. Raoult (Eds.), Rickettsiology present and future directions (pp. 302-310). New York: New York Academy of Sciences. From Rickettsiology Present and Future Directions. • Moore IV, V. A., Varela, A. S., Yabsley, M. J., Davidson, W. R., & Little, S. E. (2003, January). 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), 424-427.

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