Dorn-In S.,Chair of Animal Hygiene |
Fahn C.,Chair of Animal Nutrition |
Holzel C.S.,Chair of Animal Hygiene |
Wenz S.,Chair of Animal Hygiene |
And 3 more authors.
FEMS Microbiology Letters | Year: 2014
Ground feeds for pigs were investigated for fungal contamination before and after pelleting (subsamples in total n = 24) by cultural and molecular biological methods. A fungal-specific primer pair ITS1/ITS5.8R was used to amplify fungal DNA; PCR products were processed for the PCR-SSCP method. In the resulting acrylamide gel, more than 85% of DNA bands of ground feeds were preserved after pelleting. Twenty-two DNA bands were sequenced; all represented fungal DNA. The level of fungal DNA in ground feed samples was equivalent to 4.77-5.69 log10 CFU g-1, calculated by qPCR using a standard curve of Aspergillus flavus. In pelleted feed, the level of fungal DNA was in average ± 0.07 log10 different from ground feed. Quantified by cultural methods, the fresh ground feeds contained up to 4.51 log10 CFU g-1 culturable fungi, while there was < 2.83 log10 CFU g-1 detected in pelleted feeds. This result shows that, while the process of pelleting reduced the amount of living fungi dramatically, it did not affect the total fungal DNA in feed. Thus, the described methodology was able to reconstruct the fungal microbiota in feeds and reflected a considerable fungal contamination of raw materials such as grains. © 2014 Federation of European Microbiological Societies.
PubMed | Chair of Food Safety and Chair of Animal Hygiene
Type: | Journal: Journal of microbiological methods | Year: 2015
Universal primers targeting the bacterial 16S-rRNA-gene allow quantification of the total bacterial load in variable sample types by qPCR. However, many universal primer pairs also amplify DNA of plants or even of archaea and other eukaryotic cells. By using these primers, the total bacterial load might be misevaluated, whenever samples contain high amounts of non-target DNA. Thus, this study aimed to provide primer pairs which are suitable for quantification and identification of bacterial DNA in samples such as feed, spices and sample material from digesters. For 42 primers, mismatches to the sequence of chloroplasts and mitochondria of plants were evaluated. Six primer pairs were further analyzed with regard to the question whether they anneal to DNA of archaea, animal tissue and fungi. Subsequently they were tested with sample matrix such as plants, feed, feces, soil and environmental samples. To this purpose, the target DNA in the samples was quantified by qPCR. The PCR products of plant and feed samples were further processed for the Single Strand Conformation Polymorphism method followed by sequence analysis. The sequencing results revealed that primer pair 335F/769R amplified only bacterial DNA in samples such as plants and animal feed, in which the DNA of plants prevailed.
Dollhofer V.,Bavarian State Research Center for Agriculture |
Callaghan T.M.,Bavarian State Research Center for Agriculture |
Dorn-In S.,Chair of Animal Hygiene |
Bauer J.,Chair of Animal Hygiene |
Lebuhn M.,Bavarian State Research Center for Agriculture
Journal of Microbiological Methods | Year: 2016
Anaerobic fungi (AF) decompose plant material with their rhizoid and multiple cellulolytic enzymes. They disintegrate the complex structure of lignocellulosic substrates, making them more accessible and suitable for further microbial degradation. There is also much interest in their use as biocatalysts for biotechnological applications. Here, three novel polymerase chain reaction (PCR)-based methods for detecting AF and their transcriptional activity in in vitro cultures and environmental samples were developed. Two real-time quantitative PCR (qPCR)-based methods targeting AF were developed: AF-SSU, was designed to quantify the 18S rRNA genes of AF. AF-Endo, measuring transcripts of an endoglucanase gene from the glycoside hydrolase family 5 (GH5), was developed to quantify their transcriptional cellulolytic activity. The third PCR based approach was designed for phylogenetical analysis. It targets the 28S rRNA gene (LSU) of AF revealing their phylogenetic affiliation. The in silico-designed primer/probe combinations were successfully tested for the specific amplification of AF from animal and biogas plant derived samples. In combination, these three methods represent useful tools for the analysis of AF transcriptional cellulolytic activity, their abundance and their phylogenetic placement. © 2016 Elsevier B.V.
PubMed | Chair of Animal Hygiene
Type: Journal Article | Journal: International journal of food microbiology | Year: 2013
Food processing of spoiled meat is prohibited by law, since it is a deception and does not comply with food safety aspects. In general, spoilage of meat is mostly caused by bacteria. However, a high contamination level of fungi could be also found in some meat or meat products with certain preserving conditions. In case that unhygienic meat is used to produce heat processed products, the microorganisms will be deactivated by heat, so that they cannot be detected by a standard cultivation method. Therefore, this study aimed to develop and apply a molecular biological method--polymerase chain reaction and single strand conformation polymorphism (PCR-SSCP)--to reconstruct the original fungal flora of heat processed meat. Twenty primer pairs were tested for their specificity for fungal DNA. Since none of them fully complied with all study criteria (such as high specificity and sensitivity for fungal DNA; suitability of the products for PCR-SSCP) in the matrix meat, we designed a new reverse primer, ITS5.8R. The primer pair ITS1/ITS5.8R amplified DNA from all tested fungal species, but not DNA from meat-producing animals or from ingredients of plant origin (spices). For the final test, 32 DNA bands in acrylamide gel from 15 meat products and 1 soy sauce were sequenced-all originating from fungal species, which were, in other studies, reported to contaminate meat e.g. Alternaria alternata, Aureobasidium pullulans, Candida rugosa, C. tropicalis, C. zeylanoides, Eurotium amstelodami and Pichia membranifaciens, and/or spices such as Botrytis aclada, Guignardia mangiferae, Itersonilia perplexans, Lasiodiplodia theobromae, Lewia infectoria, Neofusicoccum parvum and Pleospora herbarum. This confirms the suitability of PCR-SSCP to specifically detect fungal DNA in heat processed meat products, and thus provides an overview of fungal species contaminating raw material such as meat and spices.