Development of a rapid diagnostic platform for nucleic acid testing of infectious pathogens

____________________________________________________________ III Zusammenfassung ____________________________________________________ V Abbreviations ________________________________________________________ VII Publications _________________________________________________________ IX Acknowledment ______________________________________________________ XV Background _________________________________________________________ 17 Infectious pathogens ________________________________________________ 17 Molecular diagnostics of infectious diseases ____________________________ 19 Objectives of this Ph.D. thesis ________________________________________ 23 Chapter 1. Integrated microfluidic diagnostic Platform ___________________ 25 Development of a rapid diagnostic platform for detection of category A Biothreat pathogens in the field _______________________________________________ 25 A Introduction ___________________________________________________ 27 A.1 Biological Threat Agents ____________________________________________ 27 A.2 Integrated Molecular Diagnostic platform _______________________________ 29 A.3 Lab-on-chip platform for nucleic acid testing _____________________________ 30 A.4 Centrifugal LabDisc platform for Nucleic acid testing_______________________ 32 A.5 Aim of this study __________________________________________________ 34 B Materials and Methods ___________________________________________ 36 B.1 Cell culture _______________________________________________________ 36 B.2 Classic virology methods ____________________________________________ 37 B.3 Sample pre-treatment ______________________________________________ 39 B.4 Purification of nucleic acids __________________________________________ 41 B.5 Nucleic acid amplification and detection ________________________________ 42 B.6 Quantitative Taqman real-time PCR ___________________________________ 44 B.7 Recombinase polymerase amplification for nucleic acid amplification __________ 46 B.8 Centrifugal LabDisc platform _________________________________________ 48 B.9 Cloning of DNA fragments and preparation of standard plasmid for PCR _______ 50 B.10 Transformation with the calcium chloride method _________________________ 51 B.11 DNA sequencing __________________________________________________ 51 C Results _______________________________________________________ 53 C.1 Development of methods for the Lab-on-chip platform _____________________ 53 C.2 Development and optimizing NAT methods for the Centrifugal LabDisc platform _ 56 D Discussion ____________________________________________________ 73 D.1 Development of methods for the Lab-on-chip platform _____________________ 73 D.2 Development of molecular methods for the Centrifugal LabDisc platform _______ 74 D.3 Establishment and Optimization of nucleic acid detection ___________________ 79 D.4 “Sample to Answer” testing of RPA LabDisc for the detection of biothreat agents_ 80 Chapter 2. Real-time PCR platform ____________________________________ 83 Development of one-step real-time reverse transcription PCR for the rapid detection of flaviviruses _____________________________________________ 83 A Introduction ___________________________________________________ 85 A.1 Flaviviruses ______________________________________________________ 85 A.2 Diagnosis of flaviviruses _____________________________________________ 86 A.3 Aim of this study ___________________________________________________ 88 B Materials and Methods __________________________________________ 89 B.1 Viruses and isolation of viral RNA _____________________________________ 89 B.2 Taqman primers and probe __________________________________________ 89 B.3 Pan-Flavivirus real-time RT-PCR assay _________________________________ 90 B.4 Specific real-time RT-PCR for different flaviviruses ________________________ 91 C Results _______________________________________________________ 93 C.1 Design and optimization of Pan-Flavi assay _____________________________ 93 C.2 Evaluation of specificity of the Pan-Flavi assay ___________________________ 95 C.3 Evaluation of sensitivity of the Pan-Flavi assay ___________________________ 95 C.4 Comparison of the Pan-Flavi assay with virus species-specific real-time RT-PCR 95 D Discussion ____________________________________________________ 98 Chapter 3. RapidSTRIPE Detection System ____________________________ 101 RapidSTRIPE H1N1 test for diagnosis of the pandemic swine-origin Influenza A (H1N1) ___________________________________________________________ 101 A Introduction __________________________________________________ 103 A.1 Swine Origin Influenza A (H1N1) virus _________________________________ 103 A.2 Diagnosis of S-OIV A (H1N1) ________________________________________ 103 A.3 Aim of this study __________________________________________________ 104 B Materials and Methods _________________________________________ 105 B.1 Virus strains and clinical specimens ___________________________________ 105 B.2 RNA extraction and cDNA synthesis __________________________________ 105 B.3 Reverse transcription quantitative real-time PCR (RT-qPCR) _______________ 106 B.4 Oligonucleotides for lateral flow strip test ______________________________ 106 B.5 Lateral flow strip (LFS) assay based on nucleic acid amplification ___________ 107 C Results ______________________________________________________ 109 C.1 Sensitivity of the LFS assay and comparison with RT-qPCR _______________ 109 C.2 Specificity of the LFS assay _________________________________________ 110 C.3 Diagnostic performance of the rapidSTRIPE H1N1 Assay KF system ________ 111 C.4 Instrument, cost and work load ______________________________________ 112 D Discussion ___________________________________________________ 114 Chapter 4. Recombinase Polymerase amplification assay for low-resource settings 117 Recombinase Polymerase amplification assay for the detection of Yellow fever virus in low-resource settings _______________________________________ 117 A Introduction __________________________________________________ 119 A.1 Yellow fever virus _________________________________________________ 119 A.2 Vaccination and adverse events after vaccination ________________________ 120 A.3 Diagnosis of YFV _________________________________________________ 120 A.4 Aim of this study _________________________________________________ 121 B Materials and Methods __________________________________________ 122 B.1 Viruses and isolation of viral RNA ____________________________________ 122 B.2 Primer and probe for RPA __________________________________________ 122 B.3 Real-time PCR and plasmid standard _________________________________ 122 B.4 Lateral-flow stripe RPA assay _______________________________________ 123 B.5 Real-time RPA assay ______________________________________________ 124 C Results ______________________________________________________ 125 C.1 Development of LFS-RPA assay for YFV ______________________________ 125 C.2 Performance of the LFS-RPA assay and comparison with RT-qPCR _________ 126 C.3 Specificity of the LFS assay _________________________________________ 127 C.4 Testing clinical samples ____________________________________________ 129 C.5 Performance of real-time RPA assay for YFV ___________________________ 129 D Discussion ___________________________________________________ 131 General conclusion and Outlook _______________________________________ 135 References _________________________________________________________ 137 Supplementary data __________________________________________________ 147 Lab equipment and materials ________________________________________ 147 List of chemicals and enzymes ______________________________________ 148 Media for cell culture _______________________________________________ 149 Cell lines _________________________________________________________ 150