Development of Real-Time Polymerase Chain Reaction (q-PCR) Assays for the Specific Detection and Characterization of Select Bacterial Pathogens

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Dr. Richard Robison, Department of Microbiology & Molecular Biology

The Specific aims for the project were as follows

Develop q-PCR reactions for specific target genes of bacterial pathogens

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 following q-PCR reactions will be developed:

Organism Target
Burkholderia thailandensis chromosome-specific
Strptococcus pyogenes chromosome-specific
Francisella tularensis chromosome-specific
Coccidioides immitis chromosome-specific
Coccidioides posadasii chromosome-specific

Multiplex the above q-PCR reactions which involve the same genus

Up to four q-PCR reactions can be multiplexed within a single reaction, provided four different fluorescent dyes are used. These dyes are detected in the four different optical channels of the instrument. In addition, primers and target sequences must be different enough not to interfere with each other during annealing and amplification steps. Multiplexing several reactions is time-consuming. However, once developed, a multiplexed reaction can provide a great deal of information about a particular pathogen, in a short amount of time. Each q-PCR assay described above will be multiplexed within a bacterial agent. For example, the Burkholderia-multiplexed assay will be designed to distinguish between the two species that cause human disease and the non-pathogenic B. thailandensis. The Coccidioides assays will be used to identify and distinguish the similar species C. immitis and C. posadasii.

Accomplishments to date related to the specific aims that were proposed

  • Burkholderia-species specific q-PCR assays have been developed for B. mallei and B. pseudomallei, and were used to characterize over 200 of these Burkholderia isolates in our select agent archive. We have also developed another assay for the non-pathogenic B. thailandensis that occupies a similar environmental niche and is very closely related to B. pseudomallei. This was a very difficult assay to develop because of the high genetic similarities of B. pseudomallei and B. thailandensis. Now that the B. thailandensis assay is in place, we are working to triplex the three individual assays into a single assay with three different probes capable of differentiating these three species.
  • We have developed a novel q-PCR assay for Streptococcus pyogenes (Group A Strep). This assay is meant to be used as a rapid test for group A streptococci present in clinical samples. It has been evaluated in pilot studies at both the BYU Student Health Center and at a clinic associated with the medical school in Birmingham, Alabama. It is extremely sensitive, detecting as few as 10 genome equivalents, and can provide results in as little as ten minutes. A patent application from this work has been filed and the rights to this assay were licensed earlier to a commercial company by the Office of Technology Transfer (OTT). However, that company did not honor their obligations and we are currently looking to license the technology to other interested parties.
  • We also finished the development of our triplexed Francisella tularensis assay which distinguishes between the four types of Francisella: subspecies tularensis Type A.I, subspecies tularensis Type A.II, subspecies holarctica, and subspecies novicida. Mark Gunnell, a graduate student, presented this work at both the 8th Annual ASM Biodefense and Emerging Disease Research Meeting and at the 3rd Annual Biothreat Agent Workshop in February and March of 2010, respectively (see references below).
  • The Coccidioides assays are still being developed. We hope to make significant progress on these assays in the near future.
  • Our quadraplexed q-PCR assay for Clostridium botulinum toxin types A, B, E, and F, which are the four types of toxin that cause disease in humans, was published in the Journal of Medical Microbiology (see reference below). A patent application has also been filed by OTT. We are also looking to license this technology to an interested commercial entity.

Accomplishments to date related to the Robison lab mentoring environment

We successfully conducted the Pathogenesis Journal Club (MMBIO 551R) during Winter and Fall semesters in 2010, and winter semester of 2011. We also offered MMBIO 518 (Pathobiology of CDC Select agents) during Fall semester of 2011. A total of 54 undergraduate and graduate students participated in these courses. The undergraduate mentoring courses (MMBIO 494R) assigned to my laboratory enrolled a total of 29 undergraduate students during 2010, and 30 undergraduate students in 2011. We have also continued to hold weekly lab meetings and have had eight lab parties during the past two years. In addition, I have paid 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 at scientific meetings in 2010 and 2011. Names of undergraduate or graduate students that I personally mentored are shown in bold.

  • Gunnell, M.K., C.D. Carter, W.S. Jonas, B.A. Satterfield, E.A. Moore, K.L. O’Neill, and R.A. Robison. A multiplex real-time PCR assay for the detection and differentiation of Francisella tularensis subspecies. 8th Annual ASM Biodefense and Emerging Disease Research Meeting. February 21-24, 2010. Baltimore, MD.
  • Gunnell, M.K., C.D. Carter, W.S. Jonas, B.A. Satterfield, E.A. Moore, K.L. O’Neill, and R.A. Robison. A multiplex real-time PCR assay for the detection and differentiation of Francisella tularensis subspecies. 3rd Annual Biothreat Agent Workshop. March 15, 2010. Charlotte, NC.
  • Bills, T.M., Q.C. Shepherd, A.J. Blam, E.A. Moore, J. Gardner, B. Schaalje, K.L. O’Neill, and R.A. Robison. Improved recovery of spores from coated carriers used in the AOAC Sporicidal Activity of Disinfectants (Method II) spore enumeration method, by the addition of a surfactant to the rinse solution. Intermountain Branch of the American Society for Microbiology Annual Meeting. Abstract #39, p.12, April 10, 2010. Provo, UT. (Note: T.M. Bills won first place in the student poster presentation competition.)
  • Hendriksen, B.S., R.A. Enz, R.A. Robison, and K.L. O’Neill. Migration and polarization of macrophages may be simultaneously affected by macrophage migration inhibition factor (MIF). Intermountain Branch of the American Society for Microbiology Annual Meeting. Abstract #40, p.12, April 10, 2010. Provo, UT.
  • Estevez, D., R. Whitehurst, J.D. Evans, L.T. Peavler, M. Tovar, R.A. Robison, and K.L. O’Neill. The potential of TK1 as a molecular target for cancer treatment. Intermountain Branch of the American Society for Microbiology Annual Meeting. Abstract #41, p.13, April 10, 2010. Provo, UT.
  • Griffin, D., B. Hansen, B.R. Hendricks, R.A. Robison, and K.L. O’Neill. Could macrophage phagocytosis be regulated by the tumor microenvironment? Intermountain Branch of the American Society for Microbiology Annual Meeting. Abstract #42, p.13, April 10, 2010. Provo, UT.
  • Chauca, J.A., A.R. Garrett, A.D. Martinez, E. Giraldez, J. Loong, J. Whitaker, J.M. Pena, R. A. Robison, and K.L. O’Neill. Oxidative stress promotes increased antioxidant uptake in a cellular model. Intermountain Branch of the American Society for Microbiology Annual Meeting. Abstract #43, p.13, April 10, 2010. Provo, UT.
  • Hardy, M., M. Tovar, M. Standing, R.A. Robison, and K.L. O’Neill. Immunolabeling transmission electron microscopy: and effective method for examining location of thymidine kinase 1 in human lymphoma cells. Intermountain Branch of the American Society for Microbiology Annual Meeting. Abstract #44, p.14, April 10, 2010. Provo, UT.
  • Loong, J.K., E. Giraldez, J. Whitaker, R.A. Robison, and K. L. O’Neill. Can drinking tea prevent cancer? 101st Annual Meeting of the American Association for Cancer Research. April 17, 2010. Washington, DC.
  • Elera, G.G., J. Whitaker, J. Chauca, R. Robison, and K. O’Neill. Antioxidant studies on the Peruvian cherimoya fruit give an insight into the healthy lifestyle of the Incas. 101st Annual Meeting of the American Association for Cancer Research. April 17, 2010. Washington, DC.
  • Whitaker, J., E. Giraldez, J. Loong, J. Wagstaff, R. Robison, and K.L. O’Neill. Changes in antioxidant capacities of isolated radish components during growth. 101st Annual Meeting of the American Association for Cancer Research. April 17, 2010. Washington, DC.
  • Enz, R.A., C. Knechtel, A.D. Enz, B. Swanson, E. Grialdez, R. Robison, and K.L. O’Neill. Inhibition of M1 macrophages may aid the conversion process to M2, which may aid tumor survival. 101st Annual Meeting of the American Association for Cancer Research. April 17, 2010. Washington, DC.
  • Hendriksen, B.S., R.A. Enz, C. Knechtel, A.D. Enz, B. Swanson, R. Robison, and K.L. O’Neill. Release of MIF by aggressive adenocarcinoma cell lines may be an initial step in the polarization of macrophages. 35th Annual Biological Sciences Undrgraduate Research Conference. April 24, 2010. Santa Clara, CA.
  • Estevez, D.E., R. Whitehurst, J.D. Evans, M. Tovar, R.A. Robison, and K.L. O’Neill. A novel target for cancer immunotherapy. 35th Annual Biological Sciences Undrgraduate Research Conference. April 24, 2010. Santa Clara, CA.
  • Bills, T.M., Q.C. Shepherd, A.J. Blam, E.A. Moore, J. Gardner, B. Schaalje, K.L. O’Neill, and R.A. Robison. Improved recovery of spores from coated carriers used in the AOAC Sporicidal Activity of Disinfectants (Method II) spore enumeration method, by addition of a surfactant to the rinse solution. General Meeting of the American Society for Microbiology, Session 329/Q, Abstract # Q-3097/176, p. 218. May 27, 2010. San Diego, CA.
  • Meyers, J.M., E. Ryndock, M.J. Conway, K.L. O’Neill, C.M. Meyers, and R.A. Robison. Susceptibility of native and synthetic HPV16 virions to clinical disinfectants. General Meeting of the American Society for Microbiology, Session 329/Q, Abstract # Q-3111/190, p. 218. May 27, 2010. San Diego, CA.
  • Meyers, J., E. Ryndock, M.J. Conway, C. Meyers, and R. Robison. The susceptibility of HPV16 native and quasivirus particles to clinically relevant disinfectants. 26th International Papillomavirus Conference, Poster P-109, p.115. July 3-8, 2010. Montreal, Qc, Canada.
  • Truong, T.V., D. Li, D.N. VanDerwerken, J.R. Williams, C.W. Taylor, R.A. Robison, H.D. Tolley, A.D. Rands, C.R. Bowerbanks, S.A. Lammert, and M.L. Lee. Sampling and sample treatment methods for on-site analysis of bacteria using gas chromatography-mass spectrometry. The International Chemical Congress of Pacific Basin Societies. December 15-20, 2010. Honolulu, Hawaii.
  • Jensen, N.R, K.L. O’Neill, and R.A. Robison. CRISPR loci and bacteriophage: a mutually exclusive relationship in pathogenic bacteria. Intermountain Branch of the American Society for Microbiology Annual Meeting. Abstract #A-1, p.4, April 9, 2011. Ogden, UT.
  • Engle, J.M. and R.A. Robison. Phenotypic analysis of M. smegmatis strains resistant to infection by C1 mycobacteriophage “Charlie Brown” provides evidence for identification of glycopeptidolipids as phage receptors. Intermountain Branch of the American Society for Microbiology Annual Meeting. Abstract #P-1, p.12, April 9, 2011. Ogden, UT.
  • Grooms, A.J., T.S. Bluemel, M.Q. Sanderson, J.R. Jensen, A.R. Garrett, R.A. Robison, and K.L. O’Neill. Cancer patients possess lower serum antioxidant levels than age matched healthy subjects. Intermountain Branch of the American Society for Microbiology Annual Meeting. Abstract #B-1, p.7, April 9, 2011. Ogden, UT.
  • Michaelis, T.C., A.D. marinez, A.R. Garrett, J.M. Pena, R.A. Robison, and K.L. O’Neill. Power juices: the role of exotic anti-oxidant fruit juices in preventing cancer. Intermountain Branch of the American Society for Microbiology Annual Meeting. Abstract #B-2, p.7, April 9, 2011. Ogden, UT.
  • Kraus, R.D., J. Jensen, A.R. Garrett, R.A. Robison, and K.L. O’Neill. Antioxidant levels in blueberries: are organic better than conventional? Intermountain Branch of the American Society for Microbiology Annual Meeting. Abstract #B-3, p.8, April 9, 2011. Ogden, UT. (Note: R. D. Kraus won the award for the best student oral presentation in his session.)
  • Quinton, R.J., D. Griffin, B. Hendricks, B. Hansen, A. El Naggar, M. Alegre, R.A. Robison, and K.L. O’Neill. Macrophage phagocytic activity in the tumor microenvironment of multiple cancer cell lines. Intermountain Branch of the American Society for Microbiology Annual Meeting. Abstract #B-4, p.9, April 9, 2011. Ogden, UT.
  • Robison, R.A., T. M. Bills, D. Li, B. Holt, H.D. Tolley, and M.L. Lee. Investigation of potential biomarkers of Burkholderia species by thermochemolysis and GC-MS. Burkholderia Workshop hosted by Northern Arizona University and the US Department of Homeland Security, July 27, 2011. Flagstaff, AZ.

Publications

Our lab participated in the following peer-reviewed publications in 2010 and 2011. Names of undergraduate or graduate students that I personally mentored are shown in bold.

  • Mitchell, A.M., G.A. Strobel, E. Moore, R. Robison, and J. Sears. 2010. Volatile antimicrobials from Muscodor crispans, a novel endophytic fungus. Microbiology 156:270-277.
  • Satterfield, B.A., A.F. Steward, C.S. Lew, D.O. Pickett, M.N. Cohen, E.A. Moore, P.F. Luedtke, K.L. O’Neill, and R.A. Robison. 2010. A quadraplexed real-time PCR assay for rapid detection and differentiation of the Clostridium botulinum toxin genes A, B, E and F. Journal of Medical Microbiology 59:55-64. Note: This paper was featured in the ‘Highlights of Hot papers in Recent SGM Journals” section of Microbiology Today, February, 2010, p. 60.
  • Margolis, J.M., S. El-Etr, L.M. Joubert, E. Moore, R. Robison, A. Rasley, A.M. Spormann, and D.M. Monack. 2010. Contributions of Francisella tularensis subsp. novicida chitinases and Sec secretion system to biofilm formation on chitin. Applied and Environmental Microbiology 76:596- 608. Note: The cover photo of this issue was from this article.
  • Truong, T.V., A.N. Nackos, J.R. Williams, D.N. VanDerwerken, J.A. Kimball, J.A. Murray, J.E. Hawkes, D.J. Harvey, H.D. Tolley, R.A. Robison, C.H. Bartholomew, and M.L. Lee. 2010. Differentiation of Bacillus endospore species from fatty acid methyl ester biomarkers. Analytical Methods 2:638-644. Note: The cover of this issue was a composite of photos taken from this article.
  • Dudleenamjil, E., C.Y Lin, D. Dredge, B.K. Murray, R.A. Robison, and F.B. Johnson. 2010. Bovine parvovirus uses clathrin-mediated endocytosis for cell entry. Journal of General Virology 91:3032-3041.
  • Garrett, A.R., B.K. Murray, R.A. Robison, and K.L. O’Neill. 2010. Measuring antioxidant capacity using the ORAC and TOSC assays. Methods in Molecular Biology 594:251-262.
  • Gupta-Elera, G., A.R. Garrett, A. Martinez, R.A. Robison, and K.L. O’Neill. 2011. The antioxidant properties of the cherimoya (Annona cherimola) fruit. Food Research International 44(7):2205-2209.
  • Nackos, A.N., T.V. Truong, T.C. Pulsipher, J.A. Kimball, H.D. Tolley, R.A. Robison, C.H. Bartholomew, and M.L. Lee. 2011. One-step conversion of dipicolinic acid to its dimethyl ester using monomethyl sulfate salts for GC-MS detection of bacterial endospores. Analytical Methods 3:245-258.
  • Gupta-Elera, G., A.R. Garrett, R.A. Robison, and K.L. O’Neill. 2011. The role of oxidative stress in prostate cancer – a review. European Journal of Cancer Prevention. doi: 10.1097/CEJ.0b013e32834a8002.

Written Manuscripts in Final Stages of Preparation

  • Satterfield, B., A. Stewart, M. Cohen, K. O’Neill, and R. Robison. A duplex real-time PCR assay for rapid identification and differentiation of Mycobacterium marinum and Mycobacterium ulcerans. To be submitted to the Journal of Medical Microbiology.
  • Bills, T.M., Q.C. Shepherd, A.J. Blam, E.A. Moore, J. Gardner, B. Schaalje, K.L. O’Neill, and
    R.A. Robison. Improved recovery of spores from coated carriers used in the AOAC Sporicidal Activity of Disinfectants (Method II) spore enumeration method. To be submitted to the Journal of AOAC International.
  • Gunnell, M.K., C.D. Carter, B.A. Satterfield, E.A. Moore, K.L. O’Neill, and R.A. Robison. A multiplex real-time PCR assay for the detection and differentiation of Francisella tularensis subspecies. To be submitted to the Journal of Medical Microbiology
  • Meyers, J.M., E. Ryndock, M.J. Conway, K.L. O’Neill, C.M. Meyers, and R.A. Robison. The susceptibility of HPV16 native and quasivirus particles to clinically relevant disinfectants. To be submitted to Nature Medicine.

Summary

We have made very good progress relative to the specific aims of the MEG proposed in 2010, 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 32% 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 in 2010 and 2011:

  • 25 presentations at scientific meetings
  • 9 peer-reviewed publications
  • 2 patent applications with students as co-inventors
  • 2 technology transfer license agreements

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. Our lab was very well-represented at the Intermountain ASM meetings held at BYU in March of 2010 and at Weber State in 2011. There were 8 students from my laboratory that participated in these meetings, with Teri Bills winning first place in the student poster presentation competition in 2010.