Diagnostics
Automation in Molecular Diagnostics – Towards an Open Future with the BD MAX™
Martin Altwegg Head, Molecular Biology Department, Bioanalytica
Abstract
Polymerase chain reaction (PCR) has very rapidly made its way from the research environment to the routine diagnostic laboratory. This would not have been possible without significant improvements in nucleic acid extraction and the availability of realtime amplification formats. Today, the main focus is on automation with a view to increasing efficiency when working with large volumes of specimens. However, automation is also important in bringing molecular tests closer to the patient, providing significantly more rapid results that may be crucial for both therapeutic and epidemiological measures. BD MAX™ is the first fully automated system that allows the running of laboratory-developed tests in various formats in addition to in vitro diagnostic (IVD) kits. It is flexible, reliable and easy to use for one to 24 specimens with the capability of different cycling protocols within a single run.
Keywords
Realtime PCR, full automation, emergency testing, open system, laboratory-developed tests, Chlamydia trachomatis, process control, hybridisation, SybrGreen
Disclosure: This work was supported by BD. The author has received fees from BD for oral presentations on various occasions, but is not an employee of BD. Received: 27 June 2011 Accepted: 8 July 2011 Citation: European Infectious Disease, 2011;5(2):135–8 Correspondence: Martin Altwegg, Head, Molecular Biology Department, Bioanalytica, Maihofstrasse 95a, CH-6000 Luzern 6, Switzerland. E:
m.altwegg@
bioanalytica.ch
Support: The publication of this article was funded by BD.
The Limits of ‘Conventional’ Microbiology More than a century ago, Robert Koch used a solid medium for culturing bacteria for the first time: the surface of a boiled potato. This allowed him to isolate single colonies, a prerequisite for the characterisation of strains/species and for establishing a link between particular bacteria and given diseases. Since then, culture techniques have improved dramatically through the development of very sophisticated selective and differential media. However, at the same time it became increasingly evident that not only a majority of environmental but also clinically important bacteria could not be cultured in vitro (e.g., Mycobacterium leprae, Tropheryma whipplei). With viruses, the situation is even more pronounced. These limitations called for alternative approaches to the detection of infectious agents.
Significant improvements were achieved by using immunological methods for the direct detection of antigens and of antibodies produced by infected individuals. However, immunological tests also have their disadvantages mainly with regard to sensitivity: antigen detection assays require a high concentration of the target molecule whereas serology is often negative in the acute stage of a disease and thus allows a definite diagnosis only retrospectively.
The Evolution of Molecular Methods Increased sensitivity was achieved by introducing DNA hybridisation and finally by the availability of amplification methods, especially the polymerase chain reaction (PCR), that – at least theoretically – are capable of detecting single target molecules per assay.
© TOUCH BRIEFINGS 2011
In its early days, PCR was confined to research laboratories because of the very tedious procedures involving many manual steps.1
Isolation
of nucleic acids was based on phenol–chloroform extraction and alcohol precipitation, whereas amplification required the addition of polymerase in each cycle (including opening the reaction tubes) and manual movement of tubes between three water baths with different temperatures. Detection of amplicons was essentially achieved by agarose or polyacrylamide gel electrophoresis based exclusively on size. Accordingly, contamination was an inherent problem of PCR.
Amplification methods made their way into diagnostic laboratories through parallel improvements in different areas:
•
Nucleic acid extraction was facilitated by the availability of solid matrices capable of binding both DNA and RNA under appropriate conditions after cell lysis using chaotropic salts.2
Initially a manual
procedure, this step has now become fully automated and commercial kits optimised for different types of specimens are available from various suppliers.
•
Amplification became significantly less cumbersome very rapidly through the development of thermocyclers and the availability of heat-stable polymerases from thermophilic organisms (e.g. Thermus aquaticus) allowing many amplification cycles in a closed tube.3
•
Several strategies were developed to prevent or at least reduce the problem of false positive results due to contamination (amplicon carryover). These included strict separation of different working areas (master mix preparation, extraction/PCR
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