caa a22 4500 60549942X CHVBK 20210128100551.0 cr unu---uuuuu 210128e20151001xx s 000 0 eng 10.1007/s00253-015-6875-8 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6875-8 Sponge-associated actinobacterial diversity: validation of the methods of actinobacterial DNA extraction and optimization of 16S rRNA gene amplification [Elektronische Daten] [Qi Yang, Christopher Franco, Wei Zhang] Experiments were designed to validate the two common DNA extraction protocols (CTAB-based method and DNeasy Blood & Tissue Kit) used to effectively recover actinobacterial DNA from sponge samples in order to study the sponge-associated actinobacterial diversity. This was done by artificially spiking sponge samples with actinobacteria (spores, mycelia and a combination of the two). Our results demonstrated that both DNA extraction methods were effective in obtaining DNA from the sponge samples as well as the sponge samples spiked with different amounts of actinobacteria. However, it was noted that in the presence of the sponge, the bacterial 16S rRNA gene could not be amplified unless the combined DNA template was diluted. To test the hypothesis that the extracted sponge DNA contained inhibitors, dilutions of the DNA extracts were tested for six sponge species representing five orders. The results suggested that the inhibitors were co-extracted with the sponge DNA, and a high dilution of this DNA was required for the successful PCR amplification for most of the samples. The optimized PCR conditions, including primer selection, PCR reaction system and program optimization, further improved the PCR performance. However, no single PCR condition was found to be suitable for the diverse sponge samples using various primer sets. These results highlight for the first time that the DNA extraction methods used are effective in obtaining actinobacterial DNA and that the presence of inhibitors in the sponge DNA requires high dilution coupled with fine tuning of the PCR conditions to achieve success in the study of sponge-associated actinobacterial diversity. Springer-Verlag Berlin Heidelberg, 2015 Sponge (Porifera) nationallicence DNA extraction efficiency nationallicence Inhibitor nationallicence PCR optimization nationallicence Validation nationallicence Actinobacteria nationallicence Yang Qi Centre for Marine Bioproducts Development, 5042, Adelaide, SA, Australia aut Franco Christopher Centre for Marine Bioproducts Development, 5042, Adelaide, SA, Australia aut Zhang Wei Centre for Marine Bioproducts Development, 5042, Adelaide, SA, Australia aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/20(2015-10-01), 8731-8740 0175-7598 99:20<8731 2015 99 253 https://doi.org/10.1007/s00253-015-6875-8 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6875-8 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Yang Qi Centre for Marine Bioproducts Development, 5042, Adelaide, SA, Australia aut NATIONALLICENCE 700 1- Franco Christopher Centre for Marine Bioproducts Development, 5042, Adelaide, SA, Australia aut NATIONALLICENCE 700 1- Zhang Wei Centre for Marine Bioproducts Development, 5042, Adelaide, SA, Australia aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/20(2015-10-01), 8731-8740 0175-7598 99:20<8731 2015 99 253