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| Titel |
One-step cell lysis suitable for quantitative bacteria detection in inhibitor-laden sands |
| VerfasserIn |
Hyun Jeong Lim, Jung-Hyun Choi, Ahjeong Son |
| Konferenz |
EGU General Assembly 2015
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| Medientyp |
Artikel
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| Sprache |
Englisch
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| Digitales Dokument |
PDF |
| Erschienen |
In: GRA - Volume 17 (2015) |
| Datensatznummer |
250105098
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| Publikation (Nr.) |
 EGU/EGU2015-4547.pdf |
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| Zusammenfassung |
| Complexity and heterogeneity of soils often hinder effective DNA extraction from the soil
matrix. In particular, conventional DNA extraction techniques require extensive purification
which makes DNA extraction time-consuming and labor-intensive. Other drawbacks include
lower recovery yield, degradation, and damage of DNA, which are also caused by intensive
purifications during DNA extraction. Therefore a rapid and simple and yet effective DNA
pretreatment method is preferred for environmental monitoring and screening. This study has
evaluated the feasibility of simple physical pretreatment for effective cell lysis of bacteria in
sands. Bead beating method was selected as an effective physical cell lysis method in this
study. We examined the capability of this physical lysis for Pseudomonas putida
seeded sands without additional chemical purification steps. The lysate from the
method was analysed by the quantitative polymerase chain reaction (qPCR) assay and
subsequently compared to that by commercial DNA extraction kit. The best lysis condition
(treatment with 0.1 mm glass beads at 3000 rpm for 3 minutes) was selected. The qPCR
results of bead beating treated samples showed the better performance than that of
conventional DNA extraction kit. Moreover, the qPCR assay was performed to
the sands laden with qPCR inhibitors (humic acids, clay, and magnesium), which
generally present in environmental samples. Further experiments with the sands
containing less than 10 μg/g of humic acids and 70% of clay showed successful
quantification results of qPCR assay. In conclusion, the bead beating method is useful for
simplified DNA extraction prior to qPCR analysis for sand samples of particular
composition. It is expected that this approach will be beneficial for environmental
in-situ analysis or immediate pre-screening. It also provides the groundwork for
future studies with real soil samples that have various physico-chemical properties. |
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