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Multiplex RT-PCR in the Diagnosis of Human Picornaviruses and Human Respiratory Viruses

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Multiplex RT-PCR in the Diagnosis of Human Picornaviruses and Human Respiratory Viruses Multiplex RT-PCR in the Diagnosis of Human Picornaviruses and Human Respiratory Viruses Pia Jokela Department of Virology Haartman Institute Faculty of Medicine University of Helsinki and Department of Virology and Immunology Section of Clinical Microbiology HUSLAB Helsinki University Central Hospital and Division of General Microbiology Department of Biosciences Faculty of Biological and Environmental Sciences University of Helsinki ACADEMIC DISSERTATION To be presented for public examination, with the permission of the Faculty of Biological and Environmental Sciences of the University of Helsinki, in the Small Lecture Hall, Haartman Institute, Haartmaninkatu 3, Helsinki, on May 25th, at 12 o’clock noon. Helsinki 2012 Supervisors: Docent Maija Lappalainen Department of Virology and Immunology Section of Clinical Microbiology HUSLAB Helsinki University Central Hospital Helsinki, Finland Docent Heli Piiparinen Department of Virology Haartman Institute University of Helsinki Helsinki, Finland Reviewers: Docent Leena Maunula Department of Food Hygiene and Environmental Health University of Helsinki Helsinki, Finland Docent Merja Roivainen Intestinal Viruses Unit National Institute for Healts and Welfare Helsinki, Finland Opponent: Docent Janne-Juhana Aittoniemi Fimlab Laboratories Tampere, Finland ISBN 978-952-10-7963-4 (paperback) ISBN 978-952-10-7964-1 (PDF) http://ethesis.helsinki.fi) Unigrafia Oy University Printing House Helsinki 2012 To the Boys, Mikko, Aaro and Reino TABLE OF CONTENTS LIST OF ORIGINAL PUBLICATIONS ............................................................................ 6 ABBREVIATIONS ............................................................................................................. 7 SUMMARY......................................................................................................................... 8 TIIVISTELMÄ (SUMMARY IN FINNISH) .................................................................... 10 REVIEW OF THE LITERATURE ................................................................................... 12 1 HUMAN PICORNAVIRUSES ................................................................................ 12 1.1 Human enteroviruses ....................................................................................... 12 1.2 Human rhinoviruses ......................................................................................... 13 1.3 Human parechoviruses .................................................................................... 15 1.4 Hepatitis A virus .............................................................................................. 16 1.5 Aichi virus ....................................................................................................... 16 2 HUMAN RESPIRATORY VIRUSES ..................................................................... 17 2.1 Influenza viruses .............................................................................................. 18 2.2 Adenoviruses ................................................................................................... 20 2.3 Respiratory syncytial virus .............................................................................. 20 2.4 Parainfluenza viruses ....................................................................................... 21 2.5 Human metapneumovirus ................................................................................ 22 2.6 Human coronaviruses ...................................................................................... 23 2.7 Human bocavirus ............................................................................................. 24 3 DIAGNOSTIC APPROACHES FOR HUMAN PICORNAVIRUSES AND HUMAN RESPIRATORY VIRUSES ..................................................................................... 24 3.1 Virus culture .................................................................................................... 25 3.2 Antigen detection ............................................................................................. 27 3.3 Serology ........................................................................................................... 28 3.4 Nucleic acid detection ..................................................................................... 30 3.4.1 Conventional PCR ..................................................................................... 30 3.4.2 Real-time PCR ........................................................................................... 32 3.4.3 Multiplex PCR ........................................................................................... 33 AIMS OF THE STUDY .................................................................................................... 36 MATERIALS AND METHODS ...................................................................................... 37 1 CLINICAL SAMPLES (I –III).................................................................................. 37 2 QUALITY ASSURANCE SAMPLES (II, III) ........................................................ 37 3 NUCLEIC ACID EXTRACTION (I –III)................................................................. 39 4 PRODUCTION OF VIRAL RNAs (I–III) ............................................................... 41 4.1 RNA transcripts of the picornaviruses (I, III) .................................................. 41 4.2 RNA transcripts of RSV and hMPV (II) ......................................................... 42 5 MULTIPLEX RT-PCR (I–III).................................................................................. 43 5.1 Multiplex RT-PCR and liquid hybridization for human picornaviruses (I) .... 43 5.1.1 RT reactions (I) .......................................................................................... 43 5.1.2 PCR (I) ....................................................................................................... 45 5.1.3 Liquid hybridization (I) ............................................................................. 45 5.2 Real-time duplex RT-PCR for detection of HEVs and HRVs (III) ................. 47 5.3 Real-time duplex RT-PCR for detection of RSV and hMPV (II) ................... 48 6 DIRECT FLUORESCENT ASSAY (II, III) ............................................................ 48 7 RVP FAST ASSAY (III) .......................................................................................... 48 8 STATISTICS (III) .................................................................................................... 49 RESULTS AND DISCUSSION ........................................................................................ 50 1 MULTIPLEX RT-PCR ASSAYS FOR DETECTION OF HUMAN PICORNAVIRUSES (I, III) ..................................................................................... 50 1.1 Performance of the multiplex RT-PCR and liquid hybridization assay (I) ..... 50 1.1.1 Analytical sensitivity and precision of the multiplex RT-PCR and liquid hybridization assay (I) ............................................................................... 51 1.1.2 Clinical sensitivity and specificity of the assay (I) .................................... 53 1.2 Performance of the real-time duplex HEV/HRV RT-PCR (III) ...................... 54 1.2.1 Analytical sensitivity of the real-time duplex HEV/HRV RT-PCR (III) .. 54 1.2.2 Analytical specificity of the real-time duplex HEV/HRV RT-PCR (III) .. 55 2 REAL-TIME MULTIPLEX RT-PCR ASSAY FOR DETECTION OF RSV AND hMPV AND EVALUATION OF ANTIGEN DETECTION FOR hMPV (II) ........ 56 2.1 Performance of the real-time duplex RSV/hMPV RT-PCR assay (II) ............ 56 2.1.1 Analytical sensitivity and precision of the real-time duplex RSV/hMPV assay (II) ................................................................................................................... 57 2.1.2 Analytical specificity of the real-time duplex RSV/hMPV assay (II) ............. 57 2.2 Antigen detection of hMPV (II) ...................................................................... 58 3 PERFORMANCE OF THE RVP FAST ASSAY (III) ............................................ 58 3.1 Analytical specificity of the RVP Fast assay (III) ........................................... 59 3.2 Experiences with the RVP Fast assay in analysis of clinical samples (III) ..... 59 4 DETECTION OF HUMAN RESPIRATORY VIRUSES IN CLINICAL SAMPLES (II, III) ....................................................................................................................... 61 4.1 Detection of HEVs and HRVs (III) ................................................................. 62 4.2 Detection of RSV and hMPV (II, III) .............................................................. 63 4.3 Detection of influenza viruses (III) ................................................................. 68 4.4 Detection of AdV (III) ..................................................................................... 70 4.5 Detection of PIVs (III) ..................................................................................... 71 4.6 Detection of hCoVs and hBoV (III) ................................................................ 71 4.7 Mixed infections (III) ...................................................................................... 72 CONCLUDING REMARKS ............................................................................................ 73 ACKNOWLEDGEMENTS............................................................................................... 76 REFERENCES
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