Nucleic acid based detection and separation of bacteria causing nosocomial infections using novel types of polynucleotide probes [Elektronische Ressource] / Natalie Anna Ludyga

Natalie.Ludyga

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• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 18
• Langue: Deutsch
• Poids de l'ouvrage: 11 Mo

Extrait

Lehrstuhl für Mikrobiologie
der Technischen Universität München



Nucleic Acid Based Detection and Separation
of Bacteria Causing Nosocomial Infections
Using Novel Types of Polynucleotide Probes

Natalie Anna Ludyga

Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum
Weihenstephan für Ernährung, Landnutzung und Umwelt
der Technischen Universität München
zur Erlangung des akademischen Grades eines

Doktors der Naturwissenschaften

genehmigten Dissertation.


Vorsitzender: Univ.-Prof. Dr. W. Höll

Prüfer der Dissertation: 1. Univ. Prof. Dr. K.-H. Schleifer, i.R.
2. Univ. Prof. Dr. S. Scherer




Die Dissertation wurde am 5. April 2007 bei der Technischen Universität
München eingereicht und durch die Fakultät Wissenschaftszentrum
Weihenstephan für Ernährung, Landnutzung und Umwelt am 18. Juli
2007 angenommen.
1 Table of contents
A. Introduction...................................................................................................... 9
A.1. Identification of pathogens using FISH techniques............................... 9
A.2. Clinical diagnostics in hospital laboratories ......................................... 13
A.3. Bacteria associated with nosocomial infections and the
PathoGenoMik project (Genome research on pathogenic bacteria).15
A.4. Aims of this study........................................................................................ 21
B. Material and Methods ................................................................................. 22
B.1. Bacterial strains and sample material..................................................... 22
B.2. Cultivation of organisms............................................................................ 25
B.3. Cell fixation of bacteria from pure culture and sample material with
PFA.................................................................................................................. 26
B.4. Purification of nucleic acids...................................................................... 27
B.4.1. Purification of genomic DNA (Wisotzkey et al., 1990 modified) .............. 27
B.4.2. Purification of plasmid DNA .................................................................... 28
B.5. Photometric measurement of nucleic acid concentration in
solution .......................................................................................................... 29
B.6. Agarose Gel Electrophoresis 29
B.7. Primers........................................................................................................... 31
B.8. Polymerase chain reaction (PCR) ............................................................ 35
B.9. Fluorescence in situ hybridisations (FISH) on slides.......................... 37
B.9.1. FISH using oligonucleotide probes.......................................................... 38
B.9.1.1. Protocol for FISH with Gram-negative bacteria using
oligonucleotide probes................................................................................... 38
B.9.1.2. Protocol for FISH with Gram-positive bacteria using 39
B.9.2. FISH using RNA polynucleotide probes targeting antibiotic resistance
genes - RING-FISH ........................................................................................... 42
B.9.2.1. Protocol for RING-FISH with Gram-negative bacteria using
RING-FISH probes......................................................................................... 42
B.9.2.2. Protocol for RING-FISH with Gram-positive bacteria using 43
B.9.3. FISH using RNA oligo-oligonucleotide probes targeting rRNA................ 44
B.9.3.1. Protocol for FISH with Gram-negative bacteria using
oligo-oligonucleotide probes .......................................................................... 44
B.9.3.2. Protocol for FISH with Gram-positive bacteria using 45
B.9.4. FISH using DNA oligo-oligonucleotide probes targeting rRNA................ 46

1Table of contents
B.10. Generation of labelled polynucleotide probes .................................... 47
B.10.1. Generation of RING-FISH polynucleotide probes via in vitro transcription
.......................................................................................................................... 47
B.10.2. Generation of RNA oligo-oligonucleotide probes via in vitro 49
B.10.3. Generation of DNA oligo- via asymmetric PCR. 56
B.11. Hybridisation in coated microplates for the separation of bacteria
from different biological material ............................................................. 57
B.11.1. Coating of microplates with DNA........................................................... 58
B.11.2. Hybridisation of bacteria in solution....................................................... 59
B.11.2.1. Protocol for hybridisation with Gram-negative bacteria .................. 59
B.11.2.2. Protocol for hybridis-positive bacteria.................... 59
B.11.3. Immobilisation of bacteria in coated microplates................................... 60
B.11.4. Detection of immobilised bacterial cells by PCR 61
B.12. Cloning and Sequencing.......................................................................... 62
®B.12.1. Cloning with TOPO TA Cloning Kit...................................................... 62
B.12.2. Identification of recombinant clones 63
B.12.3. Sequencing according to Sanger et al., ................................................ 64
C. Results ............................................................................................................. 68
C.1. FISH on slides using oligonucleotide probes........................................ 68
C.2. Construction and application of RNA oligo-oligonucleotide probes
for the identification of pathogens causing nosocomial infections.73
C.2.1. Design and generation of monospecific RNA oligo-oligonucleotide
probes ............................................................................................................... 73
C.2.2. Development and optimisation of the FISH protocol for the detection
of bacteria.......................................................................................................... 77
C.2.2.1. Fixation of cells in different growth phases....................................... 77
C.2.2.2. Pretreatments to ensure the accessibility of the probe to
its target site .................................................................................................. 78
C.2.2.3. Selection of target and non-target organisms................................... 80
C.2.2.4. Sequencing of probe templates and in silico analysis of oligo-
oligonucleotide probes................................................................................... 81
C.2.2.5. Generation of probes containing different spacer sequences .......... 82
C.2.2.6. Hybridisations on slides using monospecific oligo-oligonucleotide
probes............................................................................................................ 83
C.2.3. Adaptations for FISH on slides using monospecific oligo-oligonucleotide
probes for the detection of bacteria from spiked clinical material ...................... 93
C.2.4. Adaptations for FISH on slides ic oa from real clinical material........................... 95
C.2.5. FISH on slides using multispecific oligo-oligonucleotide probes for
the detection of bacteria from pure culture and artificial mixture ....................... 98
2 Table of contents
C.3. Immobilisations of pathogens using monospecific RNA oligo-
oligonucleotide probes............................................................................. 101
C.3.1. Evaluation of the immobilisation procedure .......................................... 101
C.3.1.1. Fixation of cells in different growth phases..................................... 101
C.3.1.2. Pretreatments to ensure the accessibility of the probe to
its target site ................................................................................................ 102
C.3.1.3. Modification of the standard protocol.............................................. 103
C.3.1.4. Application of the time-reduced protocol ........................................ 103
C.3.1.5. Immobilisations using monospecific oligo-oligonucleotide probes.. 104
C.3.2. Immobilisation results obtained from spiked clinical material................ 106
C.3.3. Immobilisation refrom real clinic .................... 108
C.3.4. Cloning and sequencing of PCR products from immobilisations of
real clinical samples ........................................................................................ 112
C.4. FISH on slides using DNA oligo-oligonucleotide probes.................. 113
C.5. FISHs using RING-FISH probes for the detection of
antibiotic resistance genes harboured by pathogens ....................... 114
C.5.1. Optimisation of the RING-FISH protocol for FISH on slides.................. 114
C.5.1.1. Sequencing of the templates and in silico analysis of RING-FISH
probes...................