A two-step protocol for the extraction and purification of total DNA from soil samples was developed. Crude DNA extracts (100 pl from 5 g of soil) were contaminated with humic acids at concentrations of 0.7 to 3.3 ,ug/,ul, depending on the type of soil extracted. The coextracted humic acid fraction of a clay silt was similar to a commercially available standard humic acid mixture, as determined by electrophoretic mobility in agarose gels, UV fluorescence, and inhibition assays with DNA-transforming enzymes. Restriction endonucleases were inhibited at humic acid concentrations of 0.5 to 17.2 ,ug/ml for the commercial product and 0.8 to 51.7 1g/ml for the coextracted humic acids. DNase I was less susceptible (MIC of standard humic acids, 912 ,Ig/ml), and RNase could not be inhibited at all (MIC, >7.6 mg/ml). High inhibitory susceptibilities for humic acids were observed with Taq polymerase. For three Taq polymerases from different commercial sources, MICs were 0.08 to 0.64 ILg of the standard humic acids per ml and 0.24 to 0.48 ,ug of the coextracted humic acids per ml. The addition ofT4 gene 32 protein increased the MIC for one Taq polymerase to 5.12 ,ug/ml. Humic acids decreased nonradioactive detection in DNA-DNA slot blot hybridizations at amounts of 0.1 ,ug and inhibited transformation of competent Escherichia coli HB101 with a broad-host-range plasmid, pUNI, at concentrations of 100 ;ig/ml. Purification of crude DNA with ion-exchange chromatography resulted in removal of 97% of the initially coextracted humic acids. Recovery rates of soil-seeded microorganisms, carrying a mammal-derived DNA sequence as a marker gene, were 72 to 79%o for Corynebacterium glutamicum ATCC 13032 pUNI, 81 to 85% for E. coli DH5-a pUN1, and 86 to 92% for Hansenula polymorpha LR9-Apr8, compared with that for DNA extracted from the respective pure cultures. Soil-extracted DNA was pure enough to detect 105 C. glutamicum pUN1 cells per g of soil by transformation ofE. coli HB101, 104 cells ofH. polymorpha per g of soil by slot blot DNA-DNA hybridizations, and 10 cells of H. polymorpha per g of soil by polymerase chain reaction amplification of the mammalian marker gene.

The gut of insects may harbour one of the largest reservoirs of a yet unexplored microbial diversity. To understand how specific insects select for their own bacterial communities, the structural diversity and variability of bacteria found in the gut of different bee species was analysed. For three successive years, adults and larvae of Apis mellifera ssp. carnica (honey bee), and Bombus terrestris (bumble bee), as well as larvae of Osmia bicornis (red mason bee) were collected at a flowering oilseed rape field. Total DNA was extracted from gut material and the bacterial diversity was analysed, independent of cultivation, by genetic profiling with single-strand conformation polymorphism (SSCP) of polymerase chain reaction (PCR)-amplified partial 16S rRNA genes. The SSCP profiles were specific for all bee species and for larvae and adults. Qualitative and quantitative differences were found in the bacterial community structure of larvae and adults of A. mellifera, but differences in B. terrestris were mainly quantitative. Sequencing of the PCR products revealed a dominance of Alpha-, Beta-, and Gammaproteobacteria, Bacteroidetes, and Firmicutes in all bee species. Single-strand conformation polymorphism profiles suggested a higher abundance and diversity of lactobacilli in adults of A. mellifera than in larvae. Further phylogenetic analyses indicated common bacterial phylotypes for all three bee species, e.g. those related to Simonsiella, Serratia, and Lactobacillus. Clades related to Delftia acidovorans, Pseudomonas aeruginosa or Lactobacillus intestinalis only contained sequences from larvae. Several of the bee-specific clusters also contained identical or highly similar sequences from bacteria detected in other A. mellifera subspecies from South Africa, suggesting the existence of cosmopolitan gut bacteria in bees.

Single-strand-conformation polymorphism (SSCP) of DNA, a method widely used in mutation analysis, was adapted to the analysis and differentiation of cultivated pure-culture soil microorganisms and noncultivated rhizosphere microbial communities. A fragment (approximately 400 bp) of the bacterial 16S rRNA gene (V-4 and V-5 regions) was amplified by PCR with universal primers, with one primer phosphorylated at the 5′ end. The phosphorylated strands of the PCR products were selectively digested with lambda exonuclease, and the remaining strands were separated by electrophoresis with an MDE polyacrylamide gel, a matrix specifically optimized for SSCP purposes. By this means, reannealing and heteroduplex formation of DNA strands during electrophoresis could be excluded, and the number of bands per organism was reduced. PCR products from 10 of 11 different bacterial type strains tested could be differentiated from each other. With template mixtures consisting of pure-culture DNAs from 5 and 10 bacterial strains, most of the single strains could be detected from such model communities after PCR and SSCP analyses. Purified bands amplified from pure cultures and model communities extracted from gels could be reamplified by PCR, but by this process, additional products were also generated, as detected by further SSCP analysis. Profiles generated with DNAs of rhizosphere bacterial communities, directly extracted from two different plant species grown in the same field site, could be clearly distinguished. This study demonstrates the potential of the selected PCR–single-stranded DNA approach for microbial community analysis.

This document provides supplementary guidance on specific topics for the allergenicity risk assessment of genetically modified plants. In particular, it supplements general recommendations outlined in previous EFSA GMO Panel guidelines and Implementing Regulation (EU) No 503/2013. The topics addressed are non-IgE-mediated adverse immune reactions to foods, in vitro protein digestibility tests and endogenous allergenicity. New scientific and regulatory developments regarding these three topics are described in this document. Considerations on the practical implementation of those developments in the risk assessment of genetically modified plants are discussed and recommended, where appropriate. (C) 2017 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority

A cultivation-independent technique for genetic profiling of PCR-amplified small-subunit rRNA genes (SSU rDNA) was chosen to characterize the diversity and succession of microbial communities during composting of an organic agricultural substrate. PCR amplifications were performed with DNA directly extracted from compost samples and with primers targeting either (i) the V4-V5 region of eubacterial 16S rRNA genes, (ii) the V3 region in the 16S rRNA genes of actinomycetes, or (iii) the V8-V9 region of fungal 18S rRNA genes. Homologous PCR products were converted to single-stranded DNA molecules by exonuclease digestion and were subsequently electrophoretically separated by their single-strand-conformation polymorphism (SSCP). Genetic profiles obtained by this technique showed a succession and increasing diversity of microbial populations with all primers. A total of 19 single products were isolated from the profiles by PCR reamplification and cloning. DNA sequencing of these molecular isolates showed similarities in the range of 92.3 to 100% to known gram-positive bacteria with a low or high G؉C DNA content and to the SSU rDNA of ␥-Proteobacteria. The amplified 18S rRNA gene sequences were related to the respective gene regions of Candida krusei and Candida tropicalis. Specific molecular isolates could be attributed to different composting stages. The diversity of cultivated bacteria isolated from samples taken at the end of the composting process was low. A total of 290 isolates were related to only 6 different species. Two or three of these species were also detectable in the SSCP community profiles. Our study indicates that community SSCP profiles can be highly useful for the monitoring of bacterial diversity and community successions in a biotechnologically relevant process.