Diagnostics Figure 1a: BD MAX™ Instrument
process was now possible without any post-amplification steps associated with opening tubes and possible generation of aerosols. In addition, it provided a sound basis for further automation of molecular tests.
Fully Automated Molecular Tests
The BD MAX™ instrument, with its 2x12 extraction positions (front) and the two microfluidic amplification cartridges (rear). Each amplification chamber can be addressed individually, making it a convenient tool for optimisation of assays and/or the parallel analysis of various targets that require different cycling protocols.
Figure 1b: BD MAX™ Extraction Cartridge
DNA Unitised Reagent Strips with all assay reagents added
Extraction kits Master mix kits
Full automation, i.e. complete integration of nucleic acid preparation, amplification and detection, has started with large-volume tests such as the detection of hepatitis viruses or Chlamydia trachomatis/Neisseria gonorrhoeae, resulting in true walk-away systems and thus minimising hands-on time and the necessity of well-trained staff. However, this has created some pressure on the concentration of diagnostic activities in order to reach the (high) number of tests required for optimal efficiency of the whole process. On the other hand, there is a steadily increasing demand for short turnaround times in the case of acute infections or when epidemiological measures are necessary to prevent further spread of infectious agents (e.g. M. tuberculosis, methicillin-resistant Staphylococcus aureus [MRSA], influenza or noroviruses).
Reaction tube
Extraction tube PCR tube Conical tube
Pipette tips 175 μl Pipette tips 1 ml Waste
DNA extraction solutions
The extraction cartridge contains all the reagents necessary for DNA extraction and the set-up of the amplification reaction. DNA is eluted from the magnetic beads in 10–15 µl of buffer at high pH, which is then neutralised resulting in a total volume of 20–25 µl DNA (in the conical tube 3), of which 10 µl is then used to dissolve the PCR reagent followed by filling the microfluidic amplification cartridges. The total amplification volume is 4 µl. The remainder of the extracted DNA can subsequently be used for other applications. PCR = polymerase chain reaction.
setup and amplification), chemical destruction of amplified material, cross-linking of DNA with isopsoralen and ultraviolet (UV) irradiation, and the very clever selective destruction of DNA synthesised in vitro with deoxyuridine triphosphate (dUTP) instead of deoxythymidine triphosphate (dTTP) by incubation with uracil-N-glycosylase.4
•
As amplicon detection based exclusively on size was repeatedly shown not to be sufficiently specific, hybridisation methods were developed for a sequence-based confirmation of the amplicons (e.g. by Southern or dot blots, reverse hybridisation with capture probes bound in microtitre plates or on nitrocellulose strips, etc.). A major step towards routine diagnostic use of amplification methods was the development of realtime PCR (rtPCR) using radiolabelled probes5 various formats,1
and later probes with fluorescent dyes in
e.g., fluorescence-based energy transfer (FRET), 5’ exonuclease activity of polymerases (TaqMan), molecular beacons or scorpions. This not only significantly increased the specificity of the assays by an additional hybridisation step but also was an important step forwards regarding the problem of amplicon carryover because the entire amplification/detection
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Emergency testing (near-patient or even point-of-care testing) is usually associated with single specimens or at most short series of specimens. In addition, qualified staff experienced in running molecular tests is often not available, especially after regular working hours and during the night. This calls for fully automated, simple and rapid tests that require as few manual steps as possible. Such a concept has been successfully realised by Cepheid with the development of the GeneXpert™ system, for which already an impressive menu of tests is available. Hardware configurations from single to large arrays of individually addressable units can be tailored to a laboratory’s or a hospital’s needs. Major drawbacks include the relatively high price for reagents and the fact that the system is not suitable for laboratory-developed (‘homebrew’) tests, should there be a need, because no commercial reagents are available (as is the case for a norovirus assay, which is among the most commonly requested emergency tests in our laboratory).
BD MAX™ – An Open Concept
A different approach was chosen by a small company called Handylab with the development of the Jaguar system, which was renamed BD MAX™ after Handylab was acquired by BD in 2009. The BD MAX (see Figure 1a) is a fully integrated device for the purification, amplification and detection of nucleic acids. It can be used in different modes.
• Type 1 or in vitro diagnostic (IVD) mode – All reagents are supplied in a ready-to-use kit (see Figure 1b). Manual steps are limited to pipetting the clinical specimen into the sample preparation tube, adding the two snap-in tubes containing the lyophilised extraction reagents (including a process control) and the lyophilised PCR reagents (primers and probes for target and process control) to the extraction cartridge and loading the instrument. All reagents can be stored at room temperature.
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Open mode – DNA extraction and amplification reagents (Taq polymerase with/without process control) are provided lyophilised. Liquid primers and probe(s) according user-defined protocols designed to detect DNA targets are manually added to the conical tube 3 (see Figure 1b). Extraction reagents for several types of specimens are available.
•
Research mode – Liquid master mix with polymerase, primers and probes is added to tube 3 (see Figure 1b) for running user-defined
EUROPEAN INFECTIOUS DISEASE
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