By Dr. Norbert Klöpper, Product Manager, Bruker Daltonics, Bruker Daltonics Inc.
SubscribeAPSIS - The Bioidentification Solution Abstract: The reliable identification of harmful pathogen substances within 60 minutes is one of the most daunting challenging tasks in bio-detection today.
Bruker Daltonics presents an integrated solution combining two advanced techniques: Polymerase Chain Reaction (PCR) and microarray hybridisation. The combination of two specific identification technologies enables the control over variations of harmful pathogens. Using a unique design cartridge, integrating PCR and hybridisation into one reaction chamber with a microliter volume, a step ahead to miniaturised systems is achieved. Integrated process control and protocol facilities give a traceability and reliability of results.
BASIC TECHNOLOGY
PCR (Polymerase Chain Reaction) became a prominent technology for the detection of harmful microorganism based pathogens in the NBC arena in the last few years. The key driver for this technology came from the clinical diagnostic market. The need for a reliable identification in both emergency response and mobile lab applications lead to a boost in instrument development. Following this route, the use of micro array technology, widely used in universities and as prototypes in clinical diagnostics, is the next logical step in the development of identification devices in the biodetection arena.
Microarrays are specially designed arrays of single strand DNA attached to a glass surface to detect special variations within the amplified PCR product.
Figure 1: Basic principle, the labelled DNA from PCR is hybridised against the probes
The result of hybridisation experiments could be detected by means of fluorescence detection. One of the advantages of this approach is, the number of fluorescence dyes used does not limit that the information obtained. This approach allows more information from the same sample, as it is feasible with the present device technology. The use of Multiplex-PCR approaches gives the opportunity to go for a “shotgun approach”, to test for multiple species with only one sample set-up.
TECHNICAL REALISATION
The challenging task in the development of an integrated PCR-Microarray detector is to find a useful combination of these technologies. Bruker utilizes a specially designed cartridge, which combines PCR and hybridisation within the same reaction chamber. This cartridge was developed in collaboration with Clondiag Chip Technologies, Jena, Germany.
Figure 2: APSIS Detection Cartridge
In the first step the target DNA/RNA is amplified by PCR to achieve a detectable amount of DNA/RNA. The second step is the hybridisation against sample sequences on the glass substrate within the presence of the same buffer and the same volume where PCR was performed. The fluorescence pattern is analyzed by fluorescence detection giving a characteristic pattern. A dedicated software tool evaluates the pattern.
The design of the processing cartridge is optimised for minimal user interaction. An integrated EPROM gives a complete process control and enables the cartridge to be completely self-describing for protocol purposes.
Figure 3: Components of APSIS Detection Cartridge
The APSIS System has a modular design consisting of a process station, with eight parallel but independently operating processing slots for thermal and fluid processing, and a reader unit. Inside the reader unit the fluorescence readout is performed using microscope optics. This concept allows to process up to eight samples taken from the environment in parallel. The most time consuming step is the PCR with subsequent hybridisation, which is around 40 minutes. The time for the readout is only 1 minute.
Figure 4: APSIS System consisting of the Processor Unit and the Reader. On the right side the ruggedized version is shown.
DETECTION CAPABILITIES
Currently several detection cartridges are available. A cartridge for demo purposes containing usual microorganisms used in biological research can be provided containing:
Poly a binding protein
Saccharomyces cerevisiae
Salmonella spec.
Escherichia coli
Bacillus thuringiensis
B. globigii
MS2 Phage
For the detection of infectious diseases caused by brain viruses a cartridge with following species was developed:
CMV Menigoencephalitis
HSV1 Herpes simplex virus type 1
HSV2 Herpes simplex virus type 2
VZV Varicella-Zoster virus
Enterovirus echovirus/enterovirus/coxsackievirus
Together with WIS in Munster, Germany, a dedicated chip for the combined detection of anthrax/smallpox was designed and evaluated. The probes contain two probes for generic anthrax markers (rpoB_C and rpoB_T) and probes for markers based on the toxic plamids (pag and lef for pXO1 and capC for pXO2) to detect the entire pathogen principle and to avoid false positives from other members of the B. cereus group. For smallpox a generic region is amplified. There are several probes designed to see the variations in this region for identification down to strain level. The chip contains probes for Camel, Cow and Monkey Pox, Variola, Vaccinia and Ectromelia and two universal regions common for all of these. Furthermore a parallel detection of anthrax and smallpox was established.
OUTLOOK AND ACKNOWLEDGEMENTS
Bruker Daltonik will add additional threats to the chips. The next candidates are Yersinia pestis, Francisella tularensis, Brucella spec. and Clostridium botulinum.
We want to express our acknowledges to the colleagues from WIS Munster, especially Dr. Birgit Hülseweh, for the evaluation of the BWA chip and the group of Prof. Hufert at the University of Freiburg for the evaluation of the brain-virus chip.
