Microbes on building materials--evaluation of DNA extraction protocols as common basis for molecular analysis.
The study of microbial life in building materials is an emerging topic concerning biodeterioration of materials as well as health risks in houses and at working places. Biodegradation and potential health implications associated with microbial growth in our residues claim for more precise methods for quantification and identification. To date, cultivation experiments are commonly used to gain insight into the microbial diversity. Nowadays, molecular techniques for the identification of microorganisms provide efficient methods that can be applied in this field. The efficiency of DNA extraction is decisive in order to perform a reliable and reproducible quantification of the microorganisms by qPCR or to characterize the structure of the microbial community. In this study we tested thirteen DNA extraction methods and evaluated their efficiency for identifying (1) the quantity of DNA, (2) the quality and purity of DNA and (3) the ability of the DNA to be amplified in a PCR reaction using three universal primer sets for the ITS region of fungi as well as one primer pair targeting the 16S rRNA of bacteria with three typical building materials - common plaster, red brick and gypsum cardboard. DNA concentration measurements showed strong variations among the tested methods and materials. Measurement of the DNA yield showed up to three orders of magnitude variation from the same samples, whereas A260/A280 ratios often prognosticated biases in the PCR amplifications. Visualization of the crude DNA extracts and the comparison of DGGE fingerprints showed additional drawbacks of some methods. The FastDNA Spin kit for soil showed to be the best DNA extraction method and could provide positive results for all tests with the three building materials. Therefore, we suggest this method as a gold standard for quantification of indoor fungi and bacteria in building materials.
VIBT-BOKU, University of Natural Resources and Life Sciences, Department of Biotechnology, Muthgasse 11, A-1190 Vienna, Austria. joerg.ettenauer@boku.ac.at
Jörg D
JD
Ettenauer
Guadalupe
G
Piñar
Ksenija
K
Lopandic
Bernhard
B
Spangl
Günther
G
Ellersdorfer
Christian
C
Voitl
Katja
K
Sterflinger
D000704:Analysis of Variance; D003252:Construction Materials / Q000382:microbiology* / Q000592:standards*; D016172:DNA Fingerprinting; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D004271:DNA, Fungal / Q000032:analysis* / Q000235:genetics; D058645:Denaturing Gradient Gel Electrophoresis; D004783:Environmental Microbiology; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D012329:RNA, Bacterial / Q000235:genetics; D012331:RNA, Fungal / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics
D004269:DNA, Bacterial; D004271:DNA, Fungal; D012329:RNA, Bacterial; D012331:RNA, Fungal; D012336:RNA, Ribosomal, 16S