Improving Clinical & Diagnostic PCR

Tuesday, 09 May 2006

Improving Clinical & Diagnostic PCR
Tuesday, 09 May 2006 09:00 - 17:00

Birkbeck College
Basement Lecture Theatre
43 Gordon Square
United Kingdom


09:00 �½ 09:20              Registration � Tea, coffee and biscuits

09:20 � 09:30              Introduction by the Chair: Dr Ralph Rapley - Department of Biosciences,

                                      University of Hertfordshire

09:30 - 10:00                Real-Time Reverse Transcription PCR and molecular staging in Colorectal Cancer -
Professor Stephen A. Bustin, Institute of Cell and Molecular Science, Barts and the London, Queen Mary's School of Medicine and Dentistry, University of London

The detection of lymph node (LN) metastasis constitutes the most important prognostic factor in colorectal cancer and as the primary indicator of systemic disease spread, LN status determines the choice of postoperative adjuvant chemotherapy. However, its limitations are emphasised by the considerable prognostic heterogeneity of patients within a given tumour stage: not all patients with LN-negative cancers are cured and not all patients with LN-positive tumours die from their disease. This has resulted in a search for more accurate staging protocols and has seen the introduction of the concept of �molecular staging�, the incorporation of molecular parameters into clinical tumour staging. One such parameter is the qRT-PCR assay�s potential for generating quantitative results that are not only more informative than qualitative data, but contribute to assay standardisation and quality management.

10:00 - 10:30               Int22h-related inversions causing haemophilia A: a new more discriminant PCR test for their detection - Dr Richard Bagnall - Department of Medical and Molecular Genetics, King's College School of Medicine, Guy's Hospital , London

Intrachromosomal homologous recombination between int22h duplicons cause rearrangements that include inversions accounting for 45% of severe haemophilia A. RT-PCR and long-range PCR reactions designed to detect and distinguish inversions, deletions and duplications due to recombination between int22h sequences will be discussed.

10:30-11:00                Molecular diagnosis of tropical infectious diseases
Dr Jim Huggett, Centre for Infectious Diseases & International Health, University College London

Improving diagnosis represents one of simplest and fastest strategies by which biotechnology can impact on the infectious diseases afflicting the developing world today. Yet this factor is overlooked and barely mentioned by some of the strategies which aim to tackle the problems caused by these diseases. This talk will discuss molecular diagnosis of infectious diseases in the developing world, some of the research that has been performed and outline some considerations when designing a diagnostic assay.

11:00 - 11:30             Morning coffee/tea break in the exhibition hall


11:30 - 12:00             Multiplex Real-time PCR assays for human infectious disease detection

Dr Vicki Chalker - Respiratory and Systemic Infection Laboratory, Health Protection Agency Centre for Infections, London


A range of multiplex real-time PCRs are in use at the HPA to aid clinical diagnostics of humans with suspected infectious diseases. With a focus on those tests for respiratory and sexually transmitted bacteria the assays and issues encountered during development, validation and introduction of assays into clinical service will be discussed.

12.00 - 12.30            Molecular detection and characterisation of human enteroviruses directly from clinical samples using RT-PCR and DNA sequencing - Dr Iturriza-Gomara M - Enteric Virus Unit, Virus Reference Division, Centre for Infections, Health Protection Agency, London

Enteroviruses are common human pathogens associated with a wide spectrum of symptoms ranging from asymptomatic infection, to acute flaccid paralysis and neonatal multi-organ failure. Molecular methods that provide rapid diagnosis and increased sensitivity have been developed for the diagnosis of EV infection using oligonucleotide primers complementary to conserved sequences located in the 5� untranslated region, but data generated from these regions is not sufficiently discriminatory for typing due to the lack of correlation between their nucleic acid sequence and serotype specificity. Sequences derived from the gene encoding the capsid VP1 correlate with serotype, and therefore provide the opportunity for the development of molecular typing methods consistent with present immunological methods. Molecular methods significantly increase the sensitivity of detection  and of characterisation of enteroviruses when compared to classical methods.

12:30 - 13:00             Diagnosis of foot-and-mouth disease by RT-PCR at the IAH Pirbright: a review - Dr Scott Reid - Institute for Animal Health, Pirbright Laboratory


A brief review of the development and evaluation of improved RT-PCR methodologies for routine diagnosis of foot-and-mouth disease (FMD) at the World Reference Laboratory (WRL) for FMD in Pirbright is presented. The talk will provide an introduction to the disease and the causative virus followed by discussion of the RT-PCR methods evaluated at the WRL over the last 7-8 years. These include conventional, immuno-capture, PCR-ELISA and real-time formats. Present RT-PCR research topics will be discussed along with assay validation, future objectives and the impact made by the 2001 epidemic on the development of RT-PCR methodologies for diagnosis of FMD.

13:00 - 14:00              Lunch and meet the companies


Afternoon Session



14:00 - 14:30              PCR applications in medical microbiology - Dr Craig Winstanley, Department of Medical Microbiology and Genitourinary Medicine, University of Liverpool 

An overview of the main PCR-based techniques of relevance in medical microbiology.  Brief description of PCR-based subtractive hybridisation and its application to designing diagnostic PCR tests for specific cystic fibrosis pathogens.

14:30 - 15:00            Implementation of conventional and real-time PCR for routine diagnosis of Helicobacter pylori infection and antibiotic resistance - Dr Stephanie Chisholm - Health Protection Agency Centre for Infections Campylobacter and Helicobacter Reference Unit London.

The Campylobacter and Helicobacter Reference Unit provides specialist services for Helicobacter pylori culture and antibiotic susceptibility testing and now offers additional tests including a multiplex PCR detection assay for H. pylori and/or �H. heilmannii�, and two real-time PCR assays to detect SNPs associated with clarithromycin and tetracycline resistance. Since 2003, PCR detection was 99% sensitive in the H. pylori culture-positive biopsies and detected H. pylori in a further 16% of all patients where culture had failed.  Susceptibilities to clarithromycin and to tetracycline were determined in 96% of these. Molecular testing is an invaluable adjunct to culture methods for diagnosis and drug resistance determination of H. pylori infection.

15:00 - 15:30               Accurate gene expression profiling - facing the issues of normalisation and efficiency - Dr Tania Nolan, Sigma-Genosys

The real-time reverse transcription (RT) polymerase chain reaction (PCR) (qRT-PCR) is often described as a �gold� standard for measuring differences in gene transcript quantities. However there are significant problems caused by variability of RNA template, assay design, data normalisation and inconsistent data analysis . As a first step towards standardisation, we illustrate some of the problems associated with the RT step of RT-qPCR and how this effects final quantification and data interpretation and describe a qRT-PCR protocol that illustrates the essential technical steps required to generate quantitative data that are reliable and reproducible.


15:30 - 16:00              Afternoon Tea/Coffee and cakes with the companies



16:00 - 16:30              Taqman vs Molecular Beacons.  Towards an improved probe configuration for Real-Time PCR detection - Dr Rob Powell - Southampton University School of Medicine


Taqman probes are highly fluorogenic.  However the linear probe structure does not permit efficient quenching in the dark state.  Alternatively Molecular Beacon Probes are well quenched but are not hydrolysed during amplification and therefore are not as fluorogenic.  We have optimised a novel probe design that is a hybrid of these two probe configurations.   Data is presented to show that these probes have improved signal to noise ratio�s and give rise to earlier CT value detection that Taqman or Beacon probes.

16:30 - 17:00            Development of a universal primer set for flaviviruses -
Dr S. Maher, School of Molecular and Microbial Sciences, University of Queensland, Australian Biosecurity CRC, St Lucia, QLD, Australia  4067

17:00                         Close


Payment Instructions

    • Payment can be made by Visa or Mastercard online by secure server or by post
    • Cheques should be made payable to Euroscicon and mailed (together with a print out of the invoice which will be available at the end of the registration process) to

    Sally Wheatland
    PO Box 49717
    N20 8WH.

    Payment must be received prior to the meeting

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