Eldon Prince and Dr. Richard Robison, Microbiology & Molecular Biology
Goal/Purpose
Develop a real-time PCR assay to uniquely detect S. pyogenes on all three major real-time PCR platforms.
Importance of Project
A faster clinical test would enable doctors to more quickly diagnose and treat group A streptococcal infections. The test we developed can be run from start to finish in 25 minutes using real-time PCR.
Background and Results
Streptococcus pyogenes is the causative agent of group A streptococcal (GAS) infections such as pharyngitis, impetigo, and life-threatening necrotizing fasciitis and sepsis. The most common GAS infection, pharyngitis, is diagnosed by performing a throat swab from the patient and culturing the bacteria, which takes 2-3 days.
Culturing S. pyogenes is the most accurate and reliable method of diagnosing GAS, but it is too slow. Serious streptococcal infections cannot wait 2-3 days before appropriate antibiotics are administered.
Consequently, many doctors now use a rapid strep test that only takes several minutes to perform but is also only 75-80% accurate. If the rapid test is positive, doctors will usually prescribe antibiotics. Conversely, if the rapid test is negative, a throat swab and bacterial culture are necessary due to the inaccurate nature of the test.
The rapid test addresses the need for a faster diagnostic test but lacks needed accuracy. A test that combines the accuracy of the bacterial culture and the speed of the protein-based rapid test is needed. We developed a real-time PCR test that rivals the accuracy of culturing the bacteria and only takes 25 minutes to perform.
To increase the accuracy of the test, we utilized real-time PCR to detect S. pyogenes DNA instead of testing for proteins associated with S. pyogenes, as in the rapid test. The power of our test lies in the high specificity and sensitivity of real-time PCR to accurately detect even the smallest concentrations of DNA. Our test can detect as few as 16 genomes of GAS, or 28 femtograms (10-15 g) of DNA.
Our primer set and probe uniquely identify the speB gene, which is responsible for B exotoxin in S. pyogenes. The speB gene is unique to S. pyogenes and serves as a perfect target to identify virulent GAS.
We successfully diagnosed 281 samples from the Timpview Regional Hospital, IHC in Draper, and the BYU Student Health Center. The large quantity of samples successfully diagnosed reinforces the validity of our assay. To further validate our assay, a previous lab member attending medical school in Alabama is conducting a large-scale evaluation at a hospital there.
We also successfully optimized our assay on all three real-time PCR platforms (Applied Biosystems, Cepheid, and Roche) to make it as widely accessible as possible. This ensures that hospitals can utilize our test regardless of what type of real-time PCR machines they may have.
In conclusion, our real-time PCR test has the potential to quickly and accurately diagnose patients with GAS infections so that physicians can prescribe the appropriate antibiotics as soon as possible.
Academic Outcomes
We intend to publish our work as well as present it at the American Society for Microbiology (ASM) meetings. We have not yet done either because we are in the process of discussing options with technology transfer to possibly market and sell this assay.
As we developed our assay I applied a unique design of experiments to better optimize our test. This unique contribution, specifically the application of fractional factorial designs to real-time PCR optimization, is the subject of a scholarly publication I am currently writing. We hope to publish this paper later this year.
Acknolwedgements
I would like to thank my wife Stephanie for her support and patience. I am grateful for Dr. Robison’s advice and mentoring that made this project possible. I would also like to thank Dr. Lawson from the statistics department for his mentoring with the design of experiments.
Scholarly Sources
- Ferretti, J. et al. (2001) Complete genome sequence of an M1 strain of Streptococcus pyogenes. PNAS
98: 4658-4663. - Harbeck, R., Teague, J., Crossen, G., Maul, D., and Childers, P. (1993) Novel, Rapid Optical Immunoassay Technique for Detection of Group A Streptococci from Pharyngeal Specimens: Comparison with Standard Culture Methods. Journal Clin. Microbiology 31: 839-844.
- Hasenbein, M., Warner, J., Lambert, K., Cole, S., Onderdonk, A., McAdam, A. (2004) Detection of Multiple Macrolide- and Lincosamide-Resistant Strains of Streptococcus pyogenes from Patients in the Boston Area. Journal Clin. Microbiology 42: 1559-1563.
- Hytonen, J., Haataja, S., Gerlach, D., Podbielski, A., and Finne, J. (2001) The SpeB virulence factor of Streptococcus pyogenes, a multifunctional secreted and cell surface molecule with strepadhesin, laminin-binding and cysteine protease activity. Mol. Microbiology 39: 512-519.
- Twagira, M., Wade, J., Eltringham, I., Smith, M. (2005) Development of a real-time PCR assay on the Roche Light-Cycler for the detection of erm and mef erythromycin resistance genes in b-haemolytic streptococci. Journal Antimicrobial Chemotherapy 26 August 2005 Advanced Access Publication.