Sample Processing - Waterborne Pathogens: Detection Methods and Applications - page 95

et al. successfully used the PowerSoil DNA extraction kit (MO BIO Labo-
ratories, Inc., Carlsbad, Calif.) with a slightly modified protocol to extract
DNA after ultrafiltration of drinking water and raw water (ground and sur-
face) spiked with various bacteria, bacterial endospores, and Cryptosporidium
oocysts. 81 We have tested other kits also intended for extraction of DNA
from soil for extraction of water concentrates after ultrafiltration of surface
water and surface raw water. Although some PCR inhibition was observed
in all DNA preparations, the bacteria used for spiking were detected in 10
or 100× diluted DNA using a standard qPCR-kit and in undiluted DNA
using a kit designed for environmental samples (unpublished). Dineen et al.
evaluated a number of commercial DNA extraction kits for isolation of
bacterial endospore DNA from different types of soil, a study that may con-
stitute a good basis for choice of kits also for DNA extraction from water
concentrates. 82
Hill et al. 21 used membrane filtration for secondary concentration after
ultrafiltration of spiked tap water samples and extracted DNA by bead beat-
ing of filters in PBS after which a noncommercial guanidine thiocyanate-
based lysis buffer was added and the lysate purified on a silica spin column.
They concluded that tap water quality can affect the analytical performance
in qPCR assays, although no specific water quality parameter could be
identified as responsible for the reduced performance. In this study, vegeta-
tive bacteria and endospores were targeted, but later the same technique was
used also for C. parvum oocysts. 8
In conclusion, more work is needed to develop a DNA extraction
method that can be used for inhibitor-free template DNA from bacteria
and protozoa from different types of water.
4.6. ANALYTICAL CONTROLS
Whenever possible, analytical controls should be included in the
sample processing. For water analysis, this may be more difficult than for
e.g. food. For instance, in an outbreak situation, no noncontaminated
water sample from the same source will be available to serve as a negative
sample process control, or for spiking with the target organisms, e.g. for
detection of a bacterial pathogen by cultivation. Further, process controls
of the same volume as the water sample should be included, which may
not be possible to include when large water volumes are analyzed. Next,
we give two examples of analytical controls that can be used in water
sample processing.