40 cycles in 40 minutes
--Rapid Eco 48 Thermal Cycler critical to qPCR expert Professor Stephen Bustin's assay development
Despite substantial advances in the accessibility and ease-of-use of PCR for diagnostics, generating an assay that delivers reliable, reproducible and meaningful results is still a challenging task.
Here,eminent PCR expert Professor Stephen Bustin examines how the Eco 48 real-time quantitative PCR (qPCR) Thermal Cycler from PCRmax allows the rapid development of robust bacterial and fungal assays.
Introducing the Assay Expert.
Stephen Bustin is Professor of Molecular Medicine, Anglia Ruskin University and lead-author of the "Minimum Information for Publication of Quantitative real-time PCR Experiments" (MIQE) guidelines. His team relies on the speed and precision of the Eco 48 for a range of assay applications, from quality assurance through to intra-operative diagnostics.
PCRmax: What are the major challenges for users of qPCR in diagnostics?
Prof Bustin: Over the past 15 years qPCR has become a commonplace technique throughout clinical diagnostics. As qPCR instrumentation has advanced, however, users have become more distanced from their raw data, making it harder to assess the true efficiency and reliability of the technique. Robust quality assessment (QA) of an assay is therefore critical before sample analysis. Since assessment and validation are intensive processes, the practical challenge of qPCR is to ensure the procedure is fast and cost effective while complying with the MIQE framework.
PCRmax: Can you give us an example of your current research and how you have overcome some of the challenges you just highlighted?
Prof Bustin: We are constantly developing new assays for the quantification of potential cellular mRNA biomarkers as well as the diagnosis of fungal and bacterial pathogens; this sometimes involves the conversion of less reliable conventional endpoint PCR assays to the qPCR format. I believe that, where possible, such legacy assays should no longer be used as they are poor at detecting the large expression range differences characteristic of cellular mRNA, while the need to run a gel means there is always the potential for contamination.
The Eco 48 package is an ideal system for assay development and optimisation because it performs runs so quickly without any impact on data quality. The machine has four channels for multiplex development which enables us to run process 40 cycles in around 16 minutes, with performance aligned to the MIQE guidelines. Moreover the operating software is easy to use, reducing the time and resources required for developmental work.
PCRmax: How important has qPCR instrumentation, and particularly the Eco 48, been to your research?
Prof Bustin: The importance of speed in assay development has made the Eco 48 a key tool throughout our research. For example our team has recently worked on a project aimed at developing a qPCR-based test for the intra-operative assessment of lymph nodes in patients with breast cancer. Here, the sentinel lymph node is removed from the patient whilst in theatre and rushed to the lab for RNA extraction. A reverse transcription (RT)-qPCR reaction is then performed to screen for epithelial cell mRNA markers indicative of metastasis.
The ability to perform inter-operative diagnosis is extremely valuable because it cuts time in the operating theatre and can avoid the need for an additional operation, greatly reducing patient trauma as well as the cost of treatment. With the Eco 48 Thermal Cycler the time taken to perform the entire assay from extraction to analysis has been reduced to just 29 minutes, a clear benefit of employing this instrument over alternative techniques.
The innovations behind the research
The Eco 48 delivers this industry leading speed and precision through a variety of advanced features. Innovative block technology achieves complete heating uniformity and rapid cycles in under 40 minutes, with the sensitivity to deliver ±0.1oC uniformity across the whole block instantly after every temperature change for precise amplification. Combined with a high-performance optical system and easy-to-use software, the Eco 48 improves analytical productivity for practically all qPCR applications.
PCRmax is a Bibby Scientific company specialising in providing real time instrumentation, consumables and reagents for the life science laboratory.