Detection of Waterborne Pathogens by Polymerase Chain Reaction- Reverse Blot Hybridization Assay Yeonim Choi The Graduate School Yonsei University Department of Biomedical Laboratory Science Detection of Waterborne Pathogens by Polymerase Chain Reaction- Reverse Blot Hybridization Assay A Dissertation Submitted to the Department of Biomedical Laboratory Science and the Graduate School of Yonsei University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Yeonim Choi July 2011 G This certifies that the dissertation of Yeonim Choi is approved. ๤๤๤๤๤๤๤๤๤๤๤๤๤๤๤๤๤๤๤ Thesis Supervisor : Hyeyoung Lee ๤๤๤๤๤๤๤๤๤๤๤๤๤๤๤๤๤๤๤ Ok Doo Awh : Thesis Committee Member ๤๤๤๤๤๤๤๤๤๤๤๤๤๤๤๤๤๤๤ Tae Ue Kim : Thesis Committee Member ๤๤๤๤๤๤๤๤๤๤๤๤๤๤๤๤๤๤๤ Jong Bae Kim : Thesis Committee Member ๤๤๤๤๤๤๤๤๤๤๤๤๤๤๤๤๤๤๤ Yong Serk Park : Thesis Committee Member The Graduate School Yonsei University July 2011 G G Dedicated to my family and my friends, who have encouraged me. G G CONTENTS LIST OF FIGURES AND TABLES ------------------------------------------------ iv ABBREVIATIONS ------------------------------------------------------------------- ix ABSTRACT IN ENGLISH ----------------------------------------------------------- x I. INTRODUCTION ------------------------------------------------------------- 1 II. MATERIALS AND METHODS -------------------------------------------- 9 1. Development of PCR-REBA targeting waterborne pathogens -------- 9 Bacterial reference strains and cultivation ------------------------------- 9 Genomic DNA extraction from reference strains --------------------- 11 Preparation of PCR primers for 16S rRNA gene --------------------- 12 PCR amplification for waterborne pathogens and other opportunistic pathogen -------------------------------------------------- 14 Analysis of sensitivity for nested PCR --------------------------------- 15 Preparation of oligonucleotide probes for REBA -------------------- 16 Covalent coupling of probe to the membrane ------------------------- 19 Reverse blot hybridization assay (REBA) ----------------------------- 22 Sensitivity comparison of PCR and REBA ---------------------------- 23 2. Application of REBA for detection of waterborne pathogens in diverse water samples ---------------------------------------------------- 25 Site description and water sample collection -------------------------- 25 Microbiological characteristics of water samples -------------------- 28 Decontamination and filtration of water samples for G G cultural detection of nontuberculous mycobateria (NTM) ---------- 28 Bacterial culture of mycobacteria in water samples for ------------- 29 Identification of mycobacteria by sequence analysis 16S rRNA gene ----------------------------------------------------------------- 30 Enumerating process for waterborne pathogens ---------------------- 31 DNA purification from waterborne pathogens and other opportunistic pathogen -------------------------------------------------- 35 Application of PCR and REBA for detection of waterborne pathogens ------------------------------------------------------------------- 35 III. RESULTS 1. Development of REBA targeting waterborne pathogens ------------- 36 Efficiency of 16S rRNA gene PCR primer for detection of waterborne pathogens ---------------------------------------------------- 36 Sensitivity of PCR amplification from reference strains ------------ 39 Development of REBA for detection of waterborne pathogens ---- 41 Sensitivity analysis of REBA ------------------------------------------- 44 Comparison of sensitivities of PCR and REBA ---------------------- 46 2. Application of REBA for detection of waterborne pathogens in diverse water samples ---------------------------------------------------- 49 Microbiological data for water quality parameters ------------------- 49 Comparison of decontaminant efficiencies in isolation of mycobacteria from water samples -------------------------------------- 52 Optimization of CPC concentration for isolation of G G mycobacteria from water samples ------------------------------------- 54 Identification of mycobacteria using culture methods and 16S rRNA gene sequence analysis ------------------------------------------ 57 Detection of waterborne pathogens and other opportunistic pathogen using nested PCR ---------------------------------------------- 61 Application of REBA and comparison to culture methods for identification of waterborne pathogens -------------------------------- 61 IV. DISCUSSION ---------------------------------------------------------------- 65 V. REFERENCES ---------------------------------------------------------------- 75 SUPPLEMENTAL DATA ---------------------------------------------------------- 89 ABSTRACT IN KOREAN--------------------------------------------------------- 106 G G LIST OF FIGURES AND TABLES Figure 1. The principle of the Reverse blot hybridization assay--------- 21 Figure 2. The locations of the six water sampling sites in Korea ------- 27 Figure 3. Schematic illustration of bacteria species identification ------ 33 Figure 4. Identification of microorganisms in diverse water samples used in this study using conventional tests, selective media, and API® strips --------------------------------- 34 Figure 5. PCR amplification of waterborne pathogens using 16S rRNA gene primers ------------------------------------------------ 38 Figure 6. Sensitivity of the PCR using 16S rRNA gene primers and genomic DNA of E. coli O157:H7 ATCC 43894 -------------- 40 Figure 7. Specificity of the REBA from reference strains of waterborne pathogens ---------------------------------------------- 43 Figure 8. Determination of REBA sensitivity using reference strains of waterborne pathogens ---------------------------------- 45 Figure 9. Comparison of sensitivities of PCR and REBA using RNA of E. coli O157:H7 ATCC 43894 ------------------------- 48 Figure 10. Microbiological data for water quality parameters in treated wastewater -------------------------------------------------- 51 G G Figure 11. An application example in REBA for detection of waterborne pathogens ---------------------------------------------- 64 Table 1. Examples of waterborne pathogens ------------------------------- 2 Table 2. Bacterial strains used in this study ------------------------------- 10 Table 3. Oligonucleotide sequence used to amplify 16S rRNA gene ------------------------------------------------------------------- 13 Table 4. List of oligonucleotide probes for identification of waterborne pathogens ---------------------------------------------- 18 Table 5. Effects of different concentrations of CPC and NaOH on microbial growth isolated from the surface water of lake and underground water -------------------------------------------- 53 Table 6. Effects of different concentration of CPC on microbial growth isolated from diverse water samples -------------------- 56 Table 7. Identification of non-mycobacterial isolates based on 16S rRNA gene sequence analysis ------------------------------------ 59 Table 8. Identification of mycobacterial isolates based on 16S rRNA gene sequence analysis ------------------------------------ 60 Table 9. Detection of waterborne pathogens using REBA in treated water samples ------------------------------------------------------- 63 G G Supplemental data 1. Water quality parameters in treated wastewater (November 2006 and January 2007) --------------- 89 Supplemental data 2. Water quality parameters in treated wastewater (March 2007 and May 2007) ----------------------- 90 Supplemental data 3. Water quality parameters in treated wastewater (July 2007 and September 2007) ------------------- 91 Supplemental data 4. Water quality parameters in treated wastewater (November 2007 and January 2008) --------------- 92 Supplemental data 5. Water quality parameters in treated wastewater (March 2008 and May 2008) ------------------------- 93 Supplemental data 6. Water quality parameters in treated wastewater (July 2008) -------------------------------------------- 94 Supplemental data 7. Comparison of microorganisms detected in this study using culture methods and REBA (November 2006) ----------------------------------------------------------------- 95 Supplemental data 8. Comparison of microorganisms detected in this study using culture methods and REBA (January 2007) ----------------------------------------------------------------- 96 Supplemental data 9. Comparison of microorganisms detected in this study using culture methods and REBA (March G G 2007) ----------------------------------------------------------------- 97 Supplemental data 10. Comparison of microorganisms detected in this study using culture methods and REBA (May 2007) ---- 98 Supplemental data 11. Comparison of microorganisms detected in this study using culture methods and REBA (July 2007) ----- 99 Supplemental data 12. Comparison of microorganisms detected in this study using culture methods and REBA (September 2007) --------------------------------------------------------------- 100 Supplemental data 13. Comparison of microorganisms detected in this study using culture methods and REBA (November 2007) --------------------------------------------------------------- 101 Supplemental data 14. Comparison of microorganisms detected in this study using culture methods and REBA (January 2008) --------------------------------------------------------------- 102 Supplemental data 15. Comparison of microorganisms detected in this study using culture methods and REBA (March 2008) ---------------------------------------------------------------