10:00 – 10:30 Registration
10:30 – 10:45 Introduction by the Chair: Dr. David Whitehouse, UK
10:45 – 11:15 Improving standards in real time PCR
Dr Rob Powell, Southampton University, UK
It is easy to generate real time PCR data plots. However the correct interpretation depends on the fundamental characteristics of the assay that produced the data. Priming specificity and efficiency are critical components. The fundamental criteria for good primer and experimental design will be presented and illustrated with data from Southampton university and from the literature
11:15 – 11:45 The PCR in medical bacteriology diagnostics and research
Dr Craig Winstanley, University of Liverpool, UK
The talk will include an overview of the application of PCR-based techniques of relevance in medical microbiology. In addition, there will be a focus on the application of PCR-based technologies to the development and use of diagnostic PCR tests for specific cystic fibrosis pathogens.
11:45 – 11:50 Speakers photo
11:50 - 12:00 Introduction to the Biopark
12:00 – 13:00 Lunch and Poster Viewing
13:00 – 13:30 qPCR – the SYBR Green solution
Dr David Sugden, Kings College London
As a physiologist interested in making use of qPCR to measure expression of many different genes in a variety of different projects, I want assays that are easy to set-up, simple to use and reliable. This talk will describe our experience using the SYBR Green detection strategy for qPCR and will highlight the way we’ve approached assay design and set-up, preparation of standards and quantitative analysis of gene expression. The specificity, sensitivity, reliability of SYBR Green assays will be illustrated.
13:30 – 14:00 RT-qPCR Assay Validation, Primer Selection, Reverse Transcription and mRNA Integrity
Professor Stephen A. Bustin, Institute of Cell and Molecular Science, Queen Mary's School of Medicine and Dentistry, University of London
Preassay validation is a critical section of any experiment designed to quantitate cellular mRNA. Choice and localisation of RT primer, optimisation of PCR primers, choice of RT and assessment of mRNA quality are all essential if mRNA quantification is to have any real relevance. This presentation will provide a detailed analysis of the priming and reverse transcription steps and demonstrate the difficulties associated with measuring of RNA integrity.
14:00 – 14:30 Afternoon Tea/Coffee and Last Poster Viewing
14:30 – 15:00 Detection of foot-and-mouth disease virus by RT-PCR: insider tricks
Scott Reid, Institute of Animal Health, UK
Rapid and accurate diagnosis is essential for effective control of foot-and-mouth disease (FMD). I will describe the practical steps taken to use real-time RT-PCR (rRT-PCR) as a front-line laboratory diagnostic tool during the 2007 outbreaks of FMD in the United Kingdom. The practical considerations adopted for the processing of the samples, the deployment of rRT-PCR during the outbreaks and the advantages of the assay for laboratory diagnosis of FMD to support control decisions during the outbreaks will be discussed.
15:00 – 15:30 Building quality control in a qPCR multi-user research facility
Dr Frederique Ponchel, Senior Academic Research Fellow, Leeds University
This talk will describe the issues in running a multi-user facility in a research institute with multiple type of real time PCR applications and how to implement certain rules for assay validation, quality control and protection.
15:30 – 16:00 Chairman’s summing up
16:30 Soiree at *The Best Western Homestead Court Hotel for all the participants
About the Chair:
Dr David Whitehouse is an experienced academic and commercial scientist. He has more than 20 years research experience in the university sector, mostly with the MRC Human Biochemical Genetics Unit in UCL focusing on protein detection, human molecular genetics and genomics and the development of rapid diagnostic tests using monoclonal antibodies. In 2000 he transferred to the commercial sector where he specialized in the development of optical and electrophoretic devices for microbial detection and new approaches to DNA based diagnostics. He is an experienced freelance manager of intellectual property including patent applications in the biotechnological and neurosciences fields. He writes, lectures, and devises presentations and learning modules in biotechnology and healthcare for the commercial and higher education sectors.
About the Speakers
Professor Stephen Bustin obtained his PhD from Trinity College, University of Dublin in molecular genetics in 1983. He carried out post-doctoral research at the Animal Virus Research Institute in Pirbright before his interest in biotechnology led him to join Corporate Research, Amersham International, as a senior research scientist, eventually becoming research manager, gene expression. His main achievements were producing a synthethic HRP gene, generating humanised antibodies, cloning a HIV-1 strain and inventing a lyophilized reagent system for PCR. He joined the London Hospital Medical College as a senior research fellow, aiming to apply his research in a more direct, practical setting. Following promotion to Senior Lecturer (1995) and Reader in Molecular Medicine (2002) he was awarded a personal chair by the University of London in 2004
Dr Scott Reid Obtained a BSc. Biochemistry from Edinburgh University followed by an MSc in Forensic Science from the University of Strathclyde. Worked for seven years as a research scientist in industry before taking up a lectureship in microbiology at the College of Health Sciences, Abha, Saudi Arabia. From there, joined the World Reference Laboratory for foot-and-mouth disease (FMD) at the Institute for Animal Health (IAH), Pirbright to develop improved RT-PCR procedures for rapid and sensitive detection of FMD virus and other vesicular disease-causing viruses. Recently completed a PhD thesis on the basis of published work carried out at the IAH.
Dr Craig Winstanley, has a BSc in Biochemistry 1983 (UMIST), PhD in Bacterial Genetics 1987 (Wales). He was a lecturer at Coventry University and the University of Bradford prior to becoming a Lecturer / Senior Lecturer in the School of Infection & Host Defence, University of Liverpool (since 1999). His major research interests include the study of genetic variation and the identification of virulence genes in Gram-negative bacterial pathogens and the development of diagnostic tests for bacterial pathogens in cystic fibrosis. He has published widely on various molecular aspects of bacterial pathogens.
Dr Rob Powell holds a PhD molecular virology and had an academic career working on Chronic fatigue syndrome and as a Wellcome trust fellow within Southampton University School of Medicine. PrimerDesign was established on his 10 years of research experience, including human virus diseases and asthma genetics. Dr Powell has been a consultant to the life science industry on matters relating to gene quantitation for the past three years.
Dr David Sugden obtained a BSc in Pharmacology from Leeds University then worked in the pharmaceutical industry for 6 years as a research scientist. He completed a PhD while working in industry then did a post-doc at Imperial College before taking a position as visiting associate at the National Institutes of Health, Bethesda, USA. He was tempted back to the UK to take up a Royal Society University Research Fellowship at King’s College London, and was appointed to a Lectureship at King’s in the Physiology Department.in 1994, and promoted to Reader in 2003.
Dr Frederique Ponchel, Senior Academic Research Fellow, Leeds University. 2005/10 RCUK Senior Academic Research Fellow – Senior Lecturer, Leeds Institute of Molecular Medicine, The University of Leeds, Leeds, England. 2000/5 ARC Career Development Research Fellow Molecular Medicine Unit, The University of Leeds, Leeds, England. 1998/99, Senior Postdoctoral Fellow Molecular Medicine Unit, The University of Leeds, Leeds, England. 1995/98, EC Postdoctoral Position, YCRC p53 group, The University of York, York, England. 1994/95, Postdoctoral Position Laboratory of Molecular Oncology, INSERM 453, Lyon, France, 1994 Ph.D. – IFSBM : Doctoral Thesis in Fundamental Oncology Obtained with the highest distinction and special congratulations of the jury. Special program similar to an EMBL-Ph.D. University : PARIS XI Le Kremlin Bicetre.
This meeting was organised by Euroscicon (www.euroscicon.com), a team of dedicated professionals working for the continuous improvement of technical knowledge transfer to all scientists. Euroscicon believe that they can make a positive difference to the quality of science by providing cutting edge information on new technological advancements to the scientific community. This is provided via our exceptional services to individual scientists, research institutions and industry. The event was hosted by 'BioPark' (www.biopark.co.uk), a research and development centre in Welwyn Garden City providing specialist facilities and support for bioscience and health technology businesses to grow, and to develop new products and technologies
Posters
Comparison of Immunofluorescence (IF) and real-time PCR for the diagnosis of viral respiratory infections at Southampton General Hospital
Emma Andrews, Senior Biomedical Scientist (Virology), Molecular Diagnostics, HPA Southampton
A national strategy initiated by the HPA Molecular Diagnostics Forum (MDF) was to update respiratory diagnostics in the Regional Microbiology Network (RMN) by replacing current IF techniques with molecular methods. The MDF provided a pool of methods from which we chose suitable molecular assays that were compatible with existing local platforms and resources.
All samples received for IF between 1st January 2007 and 1st May 2007 were also tested by real time PCR. There were 200 samples tested in total. Assays deployed included influenza A and B, RSV, adenovirus, and parainfluenza 1, 2 and 3.
5 influenza A, 10 RSV, 11 adenovirus and 12 parainfluenza positives were detected by PCR in comparison to 2 influenza A, 5 RSV, 0 adenovirus and 2 parainfluenza positives detected by IF.
In summary, it was found that only 22.5% of the samples that were positive by PCR were also positive by IF. The majority of infections were found in the paediatric wards with a few cases in the oncology wards.
Changing to molecular methods for diagnosing respiratory viruses at Southampton General Hospital has greatly improved detection rates. Earlier detection by PCR will have the potential to contribute to reduced mortality and morbidity and preventing the spread of outbreaks at the hospital.
