Brigham Young University BYU ScholarsArchive All Theses and Dissertations 2013-10-01 A Quadruplex Real-Time PCR Assay for the Rapid Detection and Differentiation of the Burkholderia pseudomallei Complex: B. mallei, B. pseudomallei, and B. thailandensis Chinn-woan Lowe Brigham Young University Follow this and additional works at: https://scholarsarchive.byu.edu/etd Part of the Microbiology Commons BYU ScholarsArchive Citation Lowe, Chinn-woan, "A Quadruplex Real-Time PCR Assay for the Rapid Detection and Differentiation of the Burkholderia pseudomallei Complex: B. mallei, B. pseudomallei, and B. thailandensis" (2013). All Theses and Dissertations. 6247. https://scholarsarchive.byu.edu/etd/6247 This Thesis is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in All Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. A Quadruplex Real-Time PCR Assay for the Rapid Detection and Differentiation of the Burkholderia pseudomallei Complex: B. mallei, B. pseudomallei, and B. thailandensis Chinn-Woan Lowe A thesis submitted to the faculty of Brigham Young University in partial fulfillment of the requirements for the degree of Master of Science Richard A. Robison, Chair Joel S. Griffitts Brian D. Poole Department of Microbiology and Molecular Biology Brigham Young University October 2013 Copyright © 2013 Chinn-Woan Lowe All Rights Reserved ABSTRACT A Quadruplex Real-Time PCR Assay for Rapid Detection and Differentiation of the B. pseudomallei Complex: B. mallei, B. pseudomallei, and B. thailandensis Chinn-Woan Lowe Department of Microbiology and Molecular Biology, BYU Master of Science Methods for the rapid detection and differentiation of the Burkholderia pseudomallei complex comprising B. pseudomallei, B. mallei, and B. thailandensis, have been the topic of recent research due to the high degree of phenotypic and genotypic similarities of these species. B. pseudomallei and B. mallei are the causative agents of melioidosis and glanders, respectively. B. pseudomallei and B. mallei are recognized by the CDC as tier 1 select agents. Although B. thailandensis is generally avirulent in mammals, this species displays very similar phenotypic characteristics to that of B. pseudomallei. Optimal identification of these species remains problematic, due to the difficulty in developing a sensitive, selective, and accurate assay. To date, no real-time, multiplex PCR assay has been developed that can detect and differentiate between B. pseudomallei, B. mallei, and B. thailandensis in a single tube format. Here, we describe the development of such an assay that detects and differentiates the species of the B. pseudomallei complex. A real-time quadruplex qPCR assay, Bcom, was designed to target unique genomic regions of B. pseudomallei, B. mallei, B. thailandensis, and the B. pseudomallei complex that detects and differentiates the three species. A total of 299 isolates within the B. pseudomallei complex was evaluated in this study, as well as 15 near-neighbors and other bacterial species. The results showed that this quadruplex assay was capable of detecting the respective species in a given sample at a sensitivity between 288 fg and 277 pg of genomic DNA. The B. pseudomallei- and B. pseudomallei complex-specific assays tested negative on two presumed B. pseudomallei isolates. In addition, a third presumed B. pseudomallei isolate tested negative by the B. pseudomallei-specific test, but was detected by the B. thailandensis and B. pseudomallei complex-specific assays. After cultural and biochemical characterization, 16s rRNA sequencing, and multiple loci sequencing, it is proposed that B. pseudomallei 34 is B. thailandensis 82172 (Accession No. DQ388536), B. pseudomallei Darwin 175 is Elizabethkingia meningoseptica, and B. pseudomallei 135 is a new strain of B. ubonensis 135. Keywords: B. pseudomallei complex, B. mallei, B. pseudomallei, B. thailandensis, qPCR, PCR, multiplex, quadruplex, detection, differentiation, TaqMan ACKNOWLEDGEMENTS This thesis would not have been possible without the help, support and patience of my principal supervisor/committee chair, Dr. Richard A. Robison, not to mention his advice and unsurpassed knowledge of all things science! In addition, I am grateful for the invaluable advice and support of Dr. Joel S. Griffitts and Dr. Brian D. Poole in further developing the concept of my research; further I thank Dr. Joel S. Griffitts for providing his designed 16s rDNA primers (Primers 1035, F1, and 1036/R1) used to amplify and sequence the 16s rDNA of some bacterial DNA. I thank my fellow lab colleague Benjamin A. Satterfield for introducing me to the Robison lab and for his insight and experience in qPCR development. I thank Jordon K. March for the several bets we made to meet manuscript completion milestones. I thank Annette J. Bunnell for her help in extracting bacterial DNA and processing the Burkholderia isolates by GC-FAME. I thank Daniel B. Nelson, Michael J. Heder, and David S. Drake for their aid in progressing my research. I would also like to thank Derek Houston for his experience and advice in phylogenetic analysis. I want to thank my sister and brother-in-law Tina and Matthew Anthon with their help in formatting and proofing aspects of my manuscript drafts. Lastly, I would like to thank my husband Patrick Lowe, for his aid in proofing my manuscript drafts, and for his general support. TABLE OF CONTENTS TITLE PAGE ................................................................................................................................................. i ABSTRACT .................................................................................................................................................. ii ACKNOWLEDGMENTS ........................................................................................................................... iii TABLE OF CONTENTS ............................................................................................................................. iv LIST OF TABLES ..................................................................................................................................... viii LIST OF FIGURES ..................................................................................................................................... ix CHAPTER I: The Evolution and Development of PCR-Based Methodologies Used to Detect and Differentiate the B. pseudomallei Complex INTRODUCTION ........................................................................................................................................ 1 B. pseudomallei and Melioidosis ............................................................................................... 1 B. mallei and Glanders .............................................................................................................. 2 B. thailandensis ......................................................................................................................... 3 Use as Bioweapons .................................................................................................................... 4 B. pseudomallei Complex .......................................................................................................... 4 Standard Detection Methods for the B. pseudomallei Complex ............................................... 6 Assay Organization ................................................................................................................... 6 Single-Species Differentiation Assays .................................................................................... 31 B. pseudomallei ................................................................................................................. 31 16s rRNA ............................................................................................................. 31 Type three secretion system ................................................................................. 32 Serine metalloprotease ......................................................................................... 35 Additional targets ................................................................................................. 36 B. mallei ............................................................................................................................ 40 16s rRNA ............................................................................................................. 41 Flagellar biosynthesis protein .............................................................................. 41 Burkholderia intracellular motility A .................................................................. 42 Additional targets ................................................................................................. 43 iv B. thailandensis ................................................................................................................. 43 Multi-Species Differentiation Assays ...................................................................................... 44 B. pseudomallei and B. mallei........................................................................................... 44 23s rRNA ............................................................................................................. 44 16-23s rRNA internal transcribed spacers ..........................................................