Table Of Content..
U. REISCRL C. WITTWER F. COCKERILL (Eds.)
Rapid Cycle Real-Time PCR - Methods and Applications
Microbiology and Food Analysis
..
Springer
Berlin
Heidelberg
New York
Barcelona
Hong Kong
London
Milan
Paris
Tokyo
lID
U. Reischl C. Wittwer F. Cockerill (Eds.)
Rapi eye e
Real-Time peR -
Methods and App ieat-ons
Microbiology and Food Analysis
With 76 Figures and 127 Tables
, Springer
..
Dr. Uno REISCHL
Universitatsklinikum Regensburg
Institut fUr Medizinische Mikrobiologie und Hygiene
Franz-Josef-Strau6-Allee 11
93053 Regensburg, Germany
Prof. Dr. CARL WITTWER
University of Utah Medical School
Department of Pathology
Salt Lake City, UT 84132, USA
Prof. Dr. FRANKLIN COCKERILL
Division of Clinical Microbiology
Mayo Clinic
Rochester, Minnesota 55905, USA
ISBN-13: 978-3-642-48353-0
Library of Congress applied for
Die Deutsche Bibliothek - CIP-Einheitsaufnahme
Rapid cycle real-time PCR : methods and applications; microbiology and food analysis 1 Udo Reischl ...
(ed.). -Berlin; Heidelberg; New York; Barcelona; Hong Kong; London; Mailand ; Paris; Singapore;
Tokyo: Springer, 2001
ISBN-13: 978-3-642-48353-0 e-lSBN-13: 978-3-642-48351-6
DOl: 10.1007/978-3-642-48351-6
This work is subject to copyright. All rights are reserved, whether the whole or part of the material is
concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcas
ting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this
publication or parts thereof is permitted only under the provisions of the German copyright Law of
September 9, 1965, in its current version, and permission for use must always be obtained from
Springer-Verlag. Violations are liable for prosecution under the German Copyright Law.
Springer-Verlag Berlin Heidelberg New York
a member of BertelsmannSpringer Science+ Business Media GmbH
http://www.springer.de/medizin
© Springer-Verlag Berlin Heidelberg 2002
Softcover reprint of the hardcover 1 st edition 2002
The use of general descriptive names, registered names, trademarks, etc. in this publication does not
imply, even in the absence of a specific statement, that such names are exempt from the relevant pro
tective laws and regulations and therefore free for general use.
Product liability: The publisher cannot guarantee the accuracy of any information about dosage and
application thereof contained in this book. In every individual case the user must check such infor
mation by consulting the relevant literature.
Cover Design: design & production, 69121 Heidelberg, Germany
Production: ProEdit GmbH, 69126 Heidelberg, Germany
Typesetting: TBS, 69207 Sandhausen, Germany
Printed on acid free paper SPIN 10832920 18/3130 Re - 5432 1 0
•
Table of Contents
Introduction for Microbiology Volume of Rapid Cycle Real-Time PCR
FRANKLIN COCKERILL
Applications and Challenges of Real-Time PCR
for the Clinical Microbiology Laboratory .. ............ ................. 3
FRANKLIN R. COCKERILL, III*, JAMES R. UHL
Part I
Bacteriology
Rapid Detection and Simultaneous Differentiation of Bordetella pertussis
and Bordetella parapertussis in Clinical Specimens by LightCycier PCR ........ 31
UDO REISCHL, KATRIN KOSTERS, BIRGIT LEPPMEIER,
HANS-JORG LINDE, NORBERT LEHN
Rapid Detection and Simultaneous Differentiation
of Legionella spp. and L. pneumophila in Potable Water Samples
and Respiratory Specimens by LightCycier PCR .......................... 45
NELE WELLINGHAUSEN, OLFERT LANDT, UDO REISCHL
Homogeneous Assays for the Rapid PCR detection
of Burkholderia pseudomallei 16s rRNA gene
on a Real-Time Fluorometric Capillary Thermocycler .. .................... 59
ERIC P-H YAP, SOON-MENG ANG, SHIRLEY G-K SEAH,
SENG-MENG PHANG
Rapid Detection ofToxigenic Corynebacterium diphtheriae
by LightCycier PCR ................................................. 71
UDO REISCHL, ELENA SAMOILOVICH, VALENTINA KOLODKINA,
NORBERT LEHN, HANS-JORG LINDE
Rapid Detection of Resistance Associated Mutations
in Mycobacterium tuberculosis by LightCycier PCR .................... ... 83
MARIA J. TORRES, ANTONIO CRIADO, JOSE C. PALOMARES,
JAVIER AZNAR
• Table of Contents
Duplex LightCycier PCR Assay for the Rapid Detection of Methicillin-Resistant
Staphylococcus aureus and Simultaneous Species Confirmation ............. 93
UDO REISCHL, HANS-JORG LINDE, BIRGIT LEPPMEIER,
NORBERT LEHN
Rapid Detection of Group BS treptococci Using the LightCycier Instrument 107
DANBING KE, CHRISTIAN MENARD, FRAN<;:OIS J. PICARD,
MICHEL G. BERGERON
Rapid Detection and Quantification of Chlamydia trachomatis
in Clinical Specimens by LightCycier PCR ............................... ll5
HEIDI WOOD, UDO REISCHL, ROSANNA W. PEELING
Quantification of Toxoplasma gondiiin Amniotic Fluid
by Rapid Cycle Real-Time PCR ....................................... 133
FRAN<;:OIS DELHOMMEAU, FRAN<;:OIS FORESTIER
Genospecies-Specific Melting Temperature of the recA PCR Product
for the Detection of Borrelia burgdorferi sensu lato and Differentiation
of Borrelia garinii from Borrelia afzelii and Borrelia burgdorferi sensu stricto 139
JOHANNA MAKINEN, QIUSHUI HE, MATTI K. VILJANEN
Rapid and Specific Detection of Coxiella burnetiiby LightCycier PCR 149
MARKUS STEMMLER, HERMANN MEYER
Molecular Identification of Campylobarter coli and Campylobarter jejuni
Using Automated DNA Extraction and Real-Time PCR with Species-Specific
TaqMan Probes ................ .................................. 155
LYNN A. COOPER, LUKE T. DAUM, ROBERT ROUDABUSH,
KENTON 1. LOHMAN
Rapid and Specific Detection of Plesiomonas shigelloides Directly
from Stool by LightCycier PCR ....................................... 161
JIN PHANG LOH, ERIC PENG HUAT YAP
Quantification of Thermostable Direct Hemolysin-Producing
Vibrio parahaemolyticusfrom Foods Assumed to Cause Food Poisoning
Using the LightCycier Instrument .................................... 171
YOSHITO IWADE, AKINORI YAMAUCHI, AKIRA SUGIYAMA,
OSAMU NAKAYAMA, NORICHIKA H. KUMAZAWA
Table of Contents go
Part [J
Detection of Fungal Pathogens
Quantification and Speciation of Fungal DNA in Clinical Specimens
Using the LightCycier Instrument ................................... . 179
JUERGEN LOEFFLER, HOLGER HEBART, NORBERT HENKE,
KATHRIN SCHMIDT, HERMANN EINSELE
Part III
Virology
Development, Implementation, and Optimization of LightCycier PCR Assays
for Detection of Herpes Simplex Virus and Varicella-Zoster Virus
from Clinical Specimens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 189
THOMAS F. SMITH, MARK J. Espy, ARLO D. WOLD
Qualitative Detection of Herpes Simplex Virus DNA
in the Routine Diagnostic Laboratory ................................. 201
HARALD H. KESSLER, GERHARD MUHLBAUER,
EVELYN STELZL, EGON MARTH
Use of Real-Time PCR to Monitor Human Herpesvirus 6 (HHV-6) Reactivation ... 211
JUNKO H. OHYASHIKI, KAZUMA OHYASHIKI, KOHTARO YAMAMOTO
Simultaneous Identification of Five HCV Genotypes in a Single Reaction 217
OLFERT LANDT, ULRICH LASS, HEINZ-HuBERT FEUCHT
Rapid Detection of West Nile Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 227
OLFERT LANDT, JASMIN DEHNHARDT, ANDREAS NITSCHE,
GARY MILBURN, SHAWN D. CARVER
Use of Rapid Cycle Real-Time PCR for the Detection of Rabies Virus ........... 235
LORRAINE McELHINNEY, JASON SAWYER, CHRISTOPHER J. FINNEGAN,
JEMMA SMITH, ANTHONY R. FOOKS
Development of One-Step RT -PCR for the Detection of an RNA Virus, Dengue,
on the Capillary Real-Time Thermocycler .......... ................... .. 241
BOON-HuAN TAN, E'EIN SEE, ELIZABETH LIM, ERIC PENG-HUAT YAP
mn
Part IV
Detection of Genetically Modified Organisms (GMO)
Variation Analysis of Seven LightCycier Based Real-Time PCR Systems
to Detect Genetically Modified Products (RRS, Btl 76, Btl 1, Mon81 0, T25,
Lectin,lnvertase) ......... ... ................................... .. 251
ISABELLE DAHINDEN, ANDREAS ZIMMERMANN, MARIANNE LINIGER,
URS PAULI
lEt
Abbreviations
Bp Base pair
EDTA Ethylenediamine Tetraacetic Acid
F Fluorescein
LCRed640 LightCycler Red 640 Fluorescent Dye
LCRed705 LightCycler Red 705 Fluorescent Dye
Nt Nucleotide( s)
P Phosphate
PBS Phosphate Buffered Saline
SDS Sodium Dodecylsulfate
TE 1xTE Buffer Contains 0.01 M Tris, 0.001 M EDTA, pH 8.0
TE' 1xTE' Buffer Contains 0.01 M Tris, 0.0001 M EDTA, pH 8.0
TEN (=STE) 1xTEN Buffer Contains 0.01 M Tris (pH 8.0), 150 mM NaCI,
0.05% Tween 20
Tm(°C) Melting Temperature
n-n nearest neighbor
FRET Fluorescence Resonance Energy Transfer
~H Enthalpy
~S Entropy
~G Free Energy
TPMT Thiopurine Methyltransferase
XO Xanthine Oxidase
AA Amino Acid
ORF Open Reading Frame
LightCycler is a trademark of a member of the Roche Group
MagNA Pure LC is a trademark of a member of the Roche Group
.,
Introduction for Microbiology Volume
of Rapid Cycle Real-Time PCR
Several commercial testing platforms have become available or soon will be avail
able which combine rapid cycle polymerase chain reaction with fluorescent probe
detection of amplified target nucleic acid in the same closed vessel. This technol
ogy is referred to as rapid cycle real-time PCR.
One testing platform which exploits this technology, the LightCycler instru
ment (Roche Diagnostics Corporation), has become particularly popular. The
LightCycler system was the first system available that permitted the operator to
evaluate target amplification after each PCR cycle. Recently an automated nucle
ic acid extraction system, the MagNA Pure LC (Roche Diagnostics Corporation),
has been developed to complement the LightCycler system. The combination of
the MagNA Pure LC and LightCycler system now permits complete automation
of extraction, purification, concentration, amplification and detection of target
nucleic acid from patient samples.
Although the LightCycler system was initially intended for research purposes,
it became quickly obvious to many microbiologists, that the system had immense
potential for diagnostic microbiology testing. The LightCycler-MagNA Pure LC
system could not only replace cumbersome and work intense procedures
required for conventional, "home-brewed" PCR assays, but as well could replace
standard antigen or culture-based assays. As an example, the accepted diagnostic
approach for detecting Group A Streptococcus (Streptococcus pyogenes) from
throat swabs is to screen for group A streptococcal antigens using a commercial
direct immunoassay. If the result for this "rapid antigen" assay is negative, a cul
ture is performed. This two-step procedure follows guidelines provided by the
Infectious Diseases Society of America and other professional organizations and
is required because current rapid antigen assays lack sensitivity. We have devel
oped a LightCycler assay which is -10% more sensitive and approximately half
the cost ($1-$2 vs $2-$3) and requires much less time to perform « 1 hr vs up to
48 h) than the combined antigen-culture assay. In addition to accuracy, speed and
cost issues, the LightCycler system and other rapid cycle real-time PCR platforms
have other advantages over conventional PCR methods. These include decreased
risk for contamination (specimen or amplicon) and adaptability for a wide range
of testing. The containment of the PCR reaction and amplicon detection in the
same closed vessel, as is possible with the LightCycler system, lessens the possi
bility of amplicon contamination including amplicon "carryover" from previous
assay runs. As well, the possibility of specimen to specimen contamination is
essentially eliminated. The versatility for testing is exemplified by applications
