WAVLD Symposium Handbook_V4.indd - csiro

World Association of Veterinary Laboratory Diagnosticians – 13 th International Symposium, Melbourne, Australia, 11-14 November 2007
LATE-PCR WITH PRIMESAFE - MAXIMUM DIAGNOSTIC INFORMATION FROM A CLOSED-TUBE
L.J. Wangh, C. Hartshorn, K.E. Pierce, J.A. Sanchez, J.E. Rice, and A.H. Reis, Jr.
Department of Biology, Brandeis University, Waltham MA, USA, 02454-9110
(email: Wangh@brandeis.edu)
Introduction: In veterinary and human medicine there is a growing need for diagnostic assays that can
rapidly detect and analyze numerous species and strains of infectious organisms and viruses. RNA viruses
are particularly challenging because they evolve rapidly. In the case of veterinary medicine, tests have to be
done at pen-side or in the field and the findings can have great economic impact, including slaughter of
many animals. Currently the use of RT-PCR is limited in scope and few, if any assays provide results from
multiplex data in the field. We have overcome these limitations by constructing assays based on LATE-PCR
and (RT)-LATE-PCR. We are collaborating with Smiths Detection to implement these assays on an
automated “pen side” sample preparation and PCR system (see C.Volpe et. al. this volume).
Materials & Methods: Linear-After-The-Exponential (LATE)-PCR, invented in our laboratory, is an advanced
form of asymmetric PCR in which each amplicon is generated using a Limiting Primer whose initial,
concentration-adjusted melting temperature (Tm L ) is at least as high as that of the Excess Primer (Tm X ).
Under these conditions amplification begins with efficient exponential amplification of double-stranded DNA
and then abruptly switches to linear amplification of one strand, as the limiting primer is depleted from the
reaction. Each single-stranded amplicon can be quantitatively detected in real-time or at end-point using
fluorescent probes of different color. Moreover, in LATE-PCR, product detection is carried out separately
from primer annealing in the thermal cycle, thereby making it possible to use both sequence-specific and
mis-match tolerant fluorescent probes that hybridize to their target strands over a broad temperature range
below the annealing temperature of the reaction. In addition, each single-strand can be sequenced by a
convenient Dilute-‘N’-Go procedure, even when multiple sequences are generated in the same reaction.
PrimeSafe TM , also invented in our laboratory, is a PCR additive that suppresses mis-priming throughout all
thermal cycles, thereby simplifying construction of multiplexed LATE-PCR assays. (RT)-LATE-PCR assays
use random hexamers to generate cDNA molecules (two-step protocol), or use the LATE-PCR primers
themselves (one-step protocol). In either case, cDNA synthesis and amplification can be carried out in the
same tube.
Results: Using LATE-PCR we are constructing assays that are quantitative for the number of target
molecules present at the start over seven orders of magnitude and down to single DNA or cDNA molecules.
In this conference we present new results using multiplexed LATE-PCR assays for Foot and Mouth Disease
(see Pierce et al. ) or Avian Flu (see Hartshorn et al.) are described in this volume. These assays are being
implemented on Smiths Detections “pen side” device . Conserved sequences characteristic of particular
pathogens can be reliably detected with sequence-specific probes that form probe-target hybrids at
characteristic temperatures. Alternatively, variable sequences that differ slightly from one pathogenic strain
to another can be distinguished using mis-match tolerant probes that allow for probe-target hybridization
over a range of low temperatures. In either case, the complete nucleotide-by-nucleotide sequence of the
amplicon within which the probe sequence lies can be determined conveniently and cost effectively using the
Dilute-‘N’-Go dideoxy sequencing protocol. Thus, when fully implemented, LATE-PCR assays installed in a
Portable Veterinary PCR Laboratory currently under development by Smiths Detection, Inc. will generate
sophisticated information in the field, as well as the amplicons needed for thorough sequence analysis in the
laboratory.
Discussion & Conclusions: LATE-PCR and its related novel technologies promise a new generation of
highly informative diagnostic assays for use in the laboratory or in the field. Smiths Detections “pen side”
system is the only portable instrument specifically designed to take full advantage of these improvements.
References:
All of our publications on LATE-PCR are available at: http://www.brandeis.edu/projects/wanghlab/
Wed 14 November
Wed 14 November
World Association of Veterinary Laboratory Diagnosticians – 13 th International Symposium, Melbourne, Australia, 11-14 November 2007
A COMPLETE WORKFLOW FOR NUCLEIC ACID BASED
HIGH THROUGHPUT PATHOGEN DETECTION
XW Fang*, A Burrell, R C Willis, Q Hoang, W Xu, M Bounpheng, W Ge and J El-Attrache
Ambion, Inc. An Applied Biosystems Business. 2130 Woodward St., Austin, TX 78744
Introduction
Nucleic acid-based technology is increasingly used for pathogen detection and classification, as well as for
differentiation of infected animals from vaccinated animals. Nucleic acid isolation is the crucial step for this
technology. The variety of sample matrix and pathogen types poses great challenges in developing a
method for universal sample preparation easily amendable for all applications. An additional challenge is to
automate the sample preparation to minimize human exposure to the infectious pathogens and to reduce the
variation of the process. Moreover, qRT-PCR buffer optimization and careful primer/probe design are critical
to assure analytic sensitivity and detection sensitivity and specificity.
Materials & methods
Pathogen DNA/RNA is isolated using MagMAXTM kits on KingFisher Magnetic Particle Processors. RNA
quality is evaluated for purity (by A260/A280 ratio and RT-PCR inhibition), intactness (with gelelectrophoresis).
Pathogen detection is performed on ABI 7500 Fast and 7900HT real-time instruments
using AgPath-IDTM Reagent Kits.
Results
High quality DNA/RNA can be isolated with MagMAX Kits from a variety of sample matrices, such as swabs,
feces, growth media, serum, plasma, cultured cells, tissue and blood. The purified RNA/DNA is ready for
quantification by real-time PCR and sequencing. Samples can be process in microfuge tubes and 96-well
plates. The process can be easily automated.
KingFisher Magnetic Particle Processors automate magnetic bead-based processes with the coordinated
move of the permanent magnetic rods and disposable tip combs. This unique approach makes washing and
elution more efficient, accelerates processing, and also makes walk-away automation feasible. The
processors can do up to 15, 24 or 96 samples per run depending on the model of the instrument. A typical
nucleic acid isolation on a KingFisher machine takes