Final Report for the 2015 MEG Entitled: Development of Real-Time Polymerase Chain Reaction (q-PCR) Assays for the Detection and Identification of Human Tick-borne Pathogens

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PI: Richard Robison

The Specific aims for the project were as follows:

  1. Develop singleplex q-PCR assays to identify the tick-borne pathogens Borrelia burgdorferi, Borrelia hermsii, Bartonella henselae, and Babesia microti. Primer generation software will be used to design primer and probe sequences that will theoretically perform optimally in PCR reactions. All sequences will be subjected to BLAST searches to confirm their specificity for a particular organism. Once primer sequences have been obtained, they will be synthesized by Integrated DNA Technologies, Inc. (IDT). These primers will be evaluated in q-PCR reactions using target DNA from multiple isolates and SyberGreen to detect nucleic acid amplification. Parameters for each reaction will be optimized using these same tools. Once a reaction has been optimized, a specific labeled probe will be synthesized which contains both a fluorescent dye and its specific quencher. This will be used in a Taqman® PCR reaction to detect specific target sequence amplification. The assays will be validated using multiple isolates of each species mentioned above, along with several near-neighbors, in order to determine the specificities and sensitivities of each assay.
  2. Develop a multiplexed q-PCR assay to simultaneously identify and distinguish these tick-borne pathogens in a single assay. Optimized singleplex assays will be combined in one multiplexed assay, which will be re-optimized. Typically, individual primer and probe concentrations have to be adjusted (sometimes significantly) when combined in a multiplex format. The multiplexed assay will be validated using genomic DNAs from several isolates of each species mentioned above, both singly and in various combinations.

Accomplishments to date related to the specific aims that were proposed:

  1. We successfully created q-PCR assays that accomplished our objectives in specific aim 1 above. Four specific singleplex assays for these tick-born infectious agents are working well.
  2. We are still working to create a working quadraplexed assay using the four singleplex assays described above. The students that were working on this project have graduated, and we are transitioning this work to a new team this semester. We hope to be able to complete this work by the end of 2017. We have finished work on several other PCR assays that are related to the work in this proposal. These have been presented and/or published as evidenced in the references below:

Presentations:
March, J.K., D. Li, B.C. Holt, C.E. Wilson, W. Lowe, H.D. Tolley, M.L. Lee, and R.A. Robison. GC/MS method for rapid identification and differentiation of Burkholderia pseudomallei, Burkholderia mallei, Burkholderia thailandensis, and several members of the Burkholderia cepacia complex. General Meeting of the American Society for Microbiology, Session 16, Abstract # 118, p. 105. May 18-21, 2013. Denver, CO.

Lowe, W., B.A. Satterfield, D.B. Nelson, M.J. Heder, J.K. March, A.J. Bunnell, K.L. O’Neill, and R.A. Robison. Quadruplex real-time qPCR assay for the rapid detection and differentiation of the B. pseudomallei complex: B. mallei, B. pseudomallei, and B. thailandensis. ASM Biodefense and Emerging Diseases Research Meeting, Abstract # 080, p. 46. January 27-29, 2014. Washington, D.C.

Publications:
Lowe, W., J.K. March, A.J. Bunnell, K.L. O’Neill, and R.A. Robison. 2013. PCR-based methodologies used to detect and differentiate the Burkholderia pseudomallei complex: B. pseudomallei, B. mallei, and B. thailandensis. Current Issues in Molecular Biology 16: 23-54.

Gunnell, M.K., Adams, B.J., and Robison, R.A. 2015. The genetic diversity and evolution of Francisella tularensis with comments on detection by PCR. Current Issues in Molecular Biology. 18: 79-92.

Gunnell, M.K., Robison, R.A., and Adams, B.J. 2016. Natural selection in virulence genes of Francisella tularensis. Journal of Molecular Evolution. DOI 10.1007/s00239-016-9743-y, 1-15.

Lowe, C-W, Satterfield, B.A., Nelson, D.B., Thiriot, J.D., Heder, M.J., March, J.K., Drake, D.S., Lew, C.S., Bunnell, A.J., Moore, E.S., O’Neill, K.L., and Robison, R.A. 2016. A quaduplex real-time assay for the rapid detection and differentiation of the most relevant members of the B. pseudomallei complex: B. mallei, B. pseudomallei, and B. thailandensis. PLoS ONE. 11(10): e0164006. doi:10.1371/journal. pone.0164006, 1-20.

Accomplishments to date related to the Robison lab mentoring environment:

We successfully conducted the Pathogenesis Journal Club (MMBIO 528R) during Winter and Fall semesters each year. We also offered MMBIO 518 (Pathobiology of CDC Select agents) during each odd Fall semester. Over 50 undergraduate and graduate students participated in these courses. The undergraduate mentoring courses (MMBIO 494R) assigned to my laboratory enrolled an average of 6 students each semester. We have also continued to hold weekly lab meetings and have had six lab parties during the past two years. In addition, I have continued to pay the registration fees for each student who works in my lab to be a student member of the American Society for Microbiology under ASM’s mentoring program.

Presentations:
Our lab participated in the following presentations since 2015.

  1. Weagel, E.G., Liu, P.G., Meng, W., Smith, C.D., Robison, R.A., and O’Neill, K.L. How does the tumor microenvironment affect macrophage aggressiveness? American Association for Cancer Research Annual Meeting. April, 2015. Philadelphia, PA.
  2. Brog, R., Townsend, M., Monroe, J., Pagaduan, J., Weagel, E., Robison, R., and O’Neill, K. Thymidine kinase 1, a possible target for immunotherapy. American Association for Cancer Research Annual Meeting. April, 2015. Philadelphia, PA.
  3. Jaco, E.I., Weagel, E., Anderson, N., Meng, W., Davis, J., Robison, R., and O’Neill, K. Are cancer cells redefining macrophages’ aggressiveness? American Association for Cancer Research Annual Meeting. April, 2015. Philadelphia, PA.
  4. Weagel, E., Anderson, N., Meng, W., Davis, J., Robison, R., and O’Neill, K. Prostate cancer microenvironment modulates macrophage phagocytosis. American Association for Cancer Research Annual Meeting. April, 2015. Philadelphia, PA.
  5. Weagel, E., Martinez, A., El Naggar, A., Liu, P.G., Robison, R., and O’Neill, K. Differences in cellular antioxidant activity in Burkitt’s lymphoma and normal human lymphocytes. American Association for Cancer Research Annual Meeting. April, 2015. Philadelphia, PA.
  6. Abbott, D., Shrestha, G., St Clair, L., Robison, R., and O’Neill, K. Usnic and vulpinic acids, lichen-derived metabolites, induce apoptosis in SW620 colon cancer cell line independent of caspase-3. American Association for Cancer Research Annual Meeting and 10th Annual Student Caucus and Poster Competition. April, 2015. Philadelphia, PA.
  7. Ryndock, E., Burdach, J., Weinberger, R., Robison, R., and Meyers, C. The efficacy of an automated ultrasound probe disinfector against high-risk human papillomavirus type 16 and 18. 30th International Papillomavirus Conference. September 17-21, 2015. Lisbon, Portugal.
  8. Weagel, E.G., Meng, W., Robison, R.A., and O’Neill, K.L. Prostate cancer exosomes affect macrophage polarization and phagocytosis. Midwinter Conference of Immunologists Annual Meeting. January, 2016. Pacific Grove, California.
  9. Townsend, M.H., Weagel, E.G., Meng, W., Brog, R.A., Valezquez, E.J., Robison, R.A., and O’Neill, K.L. Macrophage polarization in the colon cancer tumor microenvironment. Midwinter Conference of Immunologists Annual Meeting. January, 2016. Pacific Grove, California.
  10. Brog, R.A., Weagel, E.G., Meng, W., Townsend, M.H., Valezquez, E.J., Robison, R.A., and O’Neill, K.L. Breast cancer cells cause decrease in macrophage aggressiveness. Midwinter Conference of Immunologists Annual Meeting. January, 2016. Pacific Grove, California.
  11. Meng, W., Weagel, E.G., Brog, R.A., Townsend, M.H., Valezquez, E.J., Robison, R.A., and O’Neill, K.L. The effect of prostate tumor microenvironment on macrophage aggressiveness. Midwinter Conference of Immunologists Annual Meeting. January, 2016. Pacific Grove, California.
  12. Valezquez, E.J., Weagel, E.G., Meng, W., Townsend, M.H., Brog, R.A., Robison, R.A., and O’Neill, K.L. Burkitt’s lymphoma cells affect macrophage polarization. Midwinter Conference of Immunologists Annual Meeting. January, 2016. Pacific Grove, California.
  13. Valezquez, E.J., Weagel, E.G., Meng, W., Townsend, M.H., Commonck, A., Chandler, C., Downey, M.R., Robison, R., and O’Neill, K.L. A novel molecular target for lung cancer. American Association for Cancer Research Annual Meeting. April, 2016. New Orleans, LA.
  14. Xiao, M., Chu, R., Pagaduan, J., Robison, R.A., and O’Neill, K.L. Killing cancer one cell at a time: Development and characterization of a novel antibody drug conjugate. American Association for Cancer Research Annual Meeting. April, 2016. New Orleans, LA.
  15. Weagel, E.G., Meng, W., Robison, R.A., and O’Neill, K.L. Prostate cancer exosomes and their effects on macrophage engulfment and polarization. American Association for Cancer Research Annual Meeting. April, 2016. New Orleans, LA.
  16. Weagel, E.G., Brog, R.A., Townsend, M.H., Valezquez, E.J., Becker, T.A., Mejia, C.A., Downey, M.R., Arroyo, J.A., Robison, R.A., and O’Neill, K.L. Breast cancer cells expose thymidine kinase 1 as a new immunotherapy target. American Association for Cancer Research Annual Meeting. April, 2016. New Orleans, LA.
  17. Townsend, M.H., Weagel, E.G., Meng, W., Chandler, C., Robison, R., and O’Neill, K.L. Cancer immunotherapy: Could TK1 be used as a target for colon cancer? American Association for Cancer Research Annual Meeting. April, 2016. New Orleans, LA.
  18. Dunn, A.R., Townsend, M.H., Peck. C., Nichols, T., Meng, W., Heaton, M., Robison, R.A., and O’Neill, K.L. A study of glyphosate carcinogenicity. Eleventh Annual Undergraduate Student Caucus and Poster Competition at the American Association for Cancer Research Annual Meeting. April, 2016. New Orleans, LA.
  19. Peck, C., Weagel, E.G., Robison, R.A., and O’Neill, K.L. The impact of prostate cancer exosomes on macrophage phagocytosis and polarization. Eleventh Annual Undergraduate Student Caucus and Poster Competition at the American Association for Cancer Research Annual Meeting. April, 2016. New Orleans, LA.
  20. Weagel, E.G., Chu, R.P., Meng, W., Brog, R.A., Robison, R.A., and O’Neill, K.L. Thymidine kinase 1 is on the cell surface of prostate cancer cells. Eleventh Annual Undergraduate Student Caucus and Poster Competition at the American Association for Cancer Research Annual Meeting. April, 2016. New Orleans, LA.
  21. Weagel, E.G., Brog, R.A., Meng, W., Townsend, M.H., Valezquez, E.J., Becker, T.A., Mejia, C.A., Downey, M.R., Arroyo, J.A., Robison, R.A., and O’Neill, K.L. Breast cancer cells expose thymidine kinase 1 as a new immunotherapy target. Eleventh Annual Undergraduate Student Caucus and Poster Competition at the American Association for Cancer Research Annual Meeting. April, 2016. New Orleans, LA.
  22. Xiao, M., Chu, R., Pagaduan, J., Robison, R.A., and O’Neill, K.L. Development and characterization of a novel antibody drug conjugate. Eleventh Annual Undergraduate Student Caucus and Poster Competition at the American Association for Cancer Research Annual Meeting. April, 2016. New Orleans, LA.
  23. Weagel, E.G., Meng, W., Robison, R.A., and O’Neill, K.L. The effects of prostate cancer exosomes in macrophage polarization and phagocytosis. 19th Beatson International Cancer Conference. July, 2016. Glasgow, Scotland, UK.

Publications:
Our lab participated in the following peer-reviewed publications since 2015.

  1. Castro-Nallar, E., Hasan, N.A., Cebula, T.A., Colwell, R.R., Robison, R.A., Johnson, W.E., and Crandall, K.A. 2015. Concordance and discordance of sequence survey methods for molecular epidemiology. PeerJ. 3:e761; DOI 10.7717/peerj.761.
  2. Shrestha, G., Thompson, A., Robison, R., and St. Clair, L. 2015. Letharia vulpine, a vulpinic acid containing lichen, targets cell membrane and cell division processes in methicillin-resistant Staphylococcus aureus. Pharmaceutical Biology. DOI: 10.3109/13880209.2015.1038754.
  3. Livingston, J.R., Peterson, J.J., Martinez, G.A., Peck, C.J., Garrett, A.R., Uhl, R.A., Thompson, B.H., Shrestha, G., Robison, R.A., and O’Neill, K.L. 2015. The antioxidant and DNA repair activities of resveratrol, Piceatannol, and pterostilbene. Journal of Food Research. 4(5): 9-18.
  4. March, J.K., Pratt, M.D., Lowe, C-W., Cohen, M.N., Satterfield, B.A., Schaalje, G.B., O’Neill, K.L., and Robison, R.A. 2015. The differential effects of heat-shocking on the viability of spores from Bacillus anthracis, Bacillus subtilis, and Clostridium sporogenes after treatment with peracetic acid- and glutaraldehyde-based disinfectants. MicrobiologyOpen 1-10, DOI: 10.1002/mbo3.277.
  5. Weagel, E., Smith, C., Liu, P.G., Robison, R., and O’Neill, K. 2015. Macrophage polarization and its role in cancer. Journal of Clinical and Cellular Immunology. 6(4): 1-8.
  6. Gunnell, M.K., Adams, B.J., and Robison, R.A. 2015. The genetic diversity and evolution of Francisella tularensis with comments on detection by PCR. Current Issues in Molecular Biology. 18: 79-92.
  7. Ryndock, E., Robison, R., and Meyers, C. 2016. Susceptibility of HPV16 and 18 to high level disinfectants indicated for semi-critical ultrasound probes. Journal of Medical Virology. 88:1076-1080.
    8. Steck, R.P., Hill, S.L., Weagel, E.G., Weber, K.S., Robison, R.A., and O’Neill, K.L. 2015. Pharmacologic immunosuppression of mononuclear phagocytosis by caffeine. Pharmacology Research & Perspectives. 3(6): 1-8.
  8. Fudge, J. Dunn, M., Pike, Robison, R., and Steele, F. 2016. The isolation and identification of Pantoea dispersa strain JFS as a non-pathogenic surrogate for Salmonella Typhimurium phage type 42 in flour. International Journal of Food Microbiology. 219: 1-6.
  9. Gunnell, M.K., Robison, R.A., and Adams, B.J. 2016. Natural selection in virulence genes of Francisella tularensis. Journal of Molecular Evolution. DOI 10.1007/s00239-016-9743-y, 1-15.
  10. Pitt, W.G., Alizadeh, M., Husseini, G.A., McClellan, D.S., Buchanan, C.M., Bledsoe, C.G., Robison, R.A., Blanco, R., Roeder, B.L., Melville, M., and Hunter, A.K. 2016. Rapid separation of bacteria from blood – review and outlook. Biotechnology Progress. DOI 10.1002/btpr.2299.
  11. Lowe, C-W, Satterfield, B.A., Nelson, D.B., Thiriot, J.D., Heder, M.J., March, J.K., Drake, D.S., Lew, C.S., Bunnell, A.J., Moore, E.S., O’Neill, K.L., and Robison, R.A. 2016. A quaduplex real-time assay for the rapid detection and differentiation of the most relevant members of the B. pseudomallei complex: B. mallei, B. pseudomallei, and B. thailandensis. PLoS ONE. 11(10): e0164006. doi:10.1371/journal. pone.0164006, 1-20.
  12. Bewley, K.D., Bennallack, P.R., Burlingame, M.A., Robison, R.A., Griffitts, J.S., and Miller, S.M. 2016. Capture of micrococcin biosynthetic intermediates reveals C-terminal processing as an obligatory step for in vivo maturation. PNAS. doi: 10.1073/pnas.1612161113, 1-6.

Summary

We have made very good progress relative to the overall specific aims of the MEG proposed in 2015, and have also fostered and maintained an excellent mentoring environment in which other research projects have been supported. To date, we have spent 100% of the funds awarded with about 45% of this amount expended on undergraduate student wages and travel. The number and quality of our academic deliverables have been excellent.

Undergraduate and graduate students working in the Robison laboratory have participated in the following academic deliverables since 2015:

  • 23 presentations at scientific meetings
  • 13 peer-reviewed publications

We have maintained an excellent mentoring environment with students participating in the pathogenesis journal club, weekly lab meetings, collaborations with other laboratories, and professional scientific meetings.