1 / 24

HaoQi (Esther) Li 06/21/06 – 08/11/06

Development & Optimization of a Sensitive & Specific Quantitative Real-Time PCR Assay for Borrelia lonestari. HaoQi (Esther) Li 06/21/06 – 08/11/06. U.S. Naval Medical Research Center. Infectious Diseases Directorate Rickettsial Diseases Department 503 Robert Grant Avenue

ellard
Download Presentation

HaoQi (Esther) Li 06/21/06 – 08/11/06

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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.

More Related