Abstract Book of EAVLD2012 - eavld congress 2012

S4 - P - 04
AUTOMATION OF A CAPTURE PROBE MAGNETIC BEAD DNA EXTRACTION METHOD FOR 3 GRAM
FAECAL SAMPLES FROM RED FOX INTENDED FOR PCR-DETECTION OF ECHINOCOCCUS
MULTILOCULARIS
Isaksson, M 1 , Hagström, Å 1 , Lukacs, M 2 , Holmberg, A 3 , Juremalm, M 1
1
National Veterinary Institute, Department of Virology, Immunobiology and Parasitology, Uppsala, Sweden
2
Nordiag ASA, Research and development, Oslo, Norway
3
Nordiag AB, Research and development, Stockholm, Sweden
Automation, capture probe, DNA extraction, faecal samples, Echinococcus multilocularis
Introduction
As part of the Echinococcus multilocularis (EM) surveillance in
Sweden, 4000 red fox faecal samples will be collected and
analyzed by PCR. This necessitates an automated DNA
extraction method. Due to the heterogeneous presence of EMeggs
in faeces, 3 grams of sample material is needed for the
DNA extraction of this analysis. Earlier findings show that a
capture probe magnetic bead extraction method (1) increases
sensitivity of the assay compared to the standard egg-flotation
and DNA extraction previously used at the National Veterinary
Institute in Sweden (not published). In this study, we show how
automation of the manual capture probe magnetic bead DNA
extraction method capable of handling 3 grams of faeces per
sample can increase the sample throughput capacity compared
to manual extraction.
Materials & methods
The robot used in this study is a Nordiag Bullet (Nordiag ASA). It
is a flexible robot for microplate format and includes a gripper for
plate transfers, a splittable 1ml eight channel pipette head,
magnetic separation station, bar code capability and external
waste. To be able to automate the large sample volume of the
three grams of faeces in lysis buffer, 10ml 24-well deepwell
plates were used together with two high power magnet stations.
Results
Essentially the automation consists of the five wash steps of the
magnetic beads and the magnetic pelleting between washes,
eliminating the need to manually pipette the wash steps of the
samples. The robot can be loaded with up to 48 individual
samples consisting of 3 grams of homogenized fox faeces in
buffer with biotinylated hybridization probes hybridized to the
target and attached to streptavidin coated magnetic beads. When
the robot is finished, the target DNA is melted off the probes and
the probe/magnetic bead pellet is discarded. The manual capture
probe magnetic bead DNA extraction method allows a laboratory
technician to finish 12-20 samples in one day. The automation
allows the same technician to finish 48 samples in one day, with
time to do other things while the robot is working. 96 samples in
one long day are also possible, but in this case with more or less
100% hands on time, preparing the next batch of samples while
the robot is processing the first batch.
Tab 1. Throughput of samples per week per technician, time for
weighing and division of samples not included.
Samples per
week per
technician
Manual extraction
Automated extraction
100 336
Fig 1. The Nordiag Bullet robot used in the study
Discussion & conclusions
The preparation of faecal samples before extraction, manual or
automated, is difficult to automate. The samples have to be
divided and put in the correct format tube or well before the
extraction method itself can be initiated. Automating the washing
steps of this method means that the sample throughput is
increased enough for the hybridization capture probe method to
be implemented as a routine, large scale DNA extraction method
for the PCR detection of EM eggs in red fox faecal samples.
Current work is aimed at modifying the automated capture probe
DNA extraction method to be usable also for RNA targets in a
standardized format, mixing RNA and DNA targets in the same
robot run, or even in the same sample well. This would make the
automated capture probe extraction method even more
interesting for a number of difficult sample materials such as
organ material, soil, sewage water etc.
Acknowledgements
This project is funded by the Swedish Civil Contingencies
Agency.
References
1. Opsteegh, M, Langlelaar, M, Sprong, H, den Hartog, L, De Craeye, S,
Bokken, G, Ajzenberg, D, Kijlstra, A, van der Giessen, J. 2010. Direct
detection and genotyping of Toxoplasma gondii in meat samples using
magnetic capture and PCR. International Journal of Food Microbiology,
139(3), 193-201.