Sample Processing - Waterborne Pathogens: Detection Methods and Applications - page 94

4.5. NUCLEI ACID EXTRACTION FOR MOLECULAR
DETECTION
Based on the type of water concentrate, a variety of methods to
extract RNA and DNA from viruses, bacteria, and parasites, while concur-
rently reducing the level of PCR inhibitors, has been reported. 4,13,23,24,75,76
The efficiency of these methods all depends on the characteristics of the
prior water concentration.
Total nucleic acid can be released from concentrated water samples
solely by heating at 99 °C for 5 min as shown in several studies. 7,76 Chemi-
cal nucleic acid extractions may include the use of Proteinase K 77 or the
chaotropic guanidinium thiocyanate, 24,78 in conjunction with a lysis buffer
to degrade proteins and thereby disrupt the viral capsids and cell walls of
bacteria and parasites to release e.g. nucleic acids. These chemical principles
have been adapted into commercial kits for nucleic acid purification—as
has bead beating as a means for releasing nucleic acids from hard-to-lyse
structures such as bacterial endospores and (oo)cysts. The released nucleic
acid is bound specifically to silica, magnetic or paramagnetic beads, or mem-
branes. Following a few effective washing steps, the nucleic acids are eluted
from the binding matrix with an elution buffer or molecular-grade water.
Additionally, spin columns to remove inhibitors such as HiBind RNA
minicolumn may be applied. 24 Most of these and similar kits can be used in
manual, semiautomated, and fully automated formats (e.g. Magtration Sys-
tem (Precision System Science), miniMAG magnetic system (bioMérieux)),
and are thus suitable for small and large laboratories with varied throughput
requirements.
DNA for detection of bacteria and protozoa can be extracted from mate-
rial pelleted by centrifugation or collected on membrane filters. However,
this pellet is likely to contain a lot of material beside bacteria and protozoa,
especially if surface waters are being analyzed. This includes for example
algae, debris etc. that will release DNA during the extraction process. Thus,
the target DNA will only constitute a small part of the total DNA. Further,
it also will contain PCR inhibitors, e.g. humic substances and phenolic
that will hamper the subsequent PCR-based detection. 79 Various kits and
methods for DNA preparation, use of post-lysis additives such as polyvinyl-
pyrrolidone (PVP) and Chelex-100 or PCR facilitators such as PVP, BSA,
and dimethyl sulfoxide to the PCR reaction, or combinations thereof, have
been investigated, 12,80 but no universal method has been presented. Francy