Specificity and sensitivity of eubacterial primers utilized for molecular profiling of bacteria within complex microbial ecosystems

Huws, Sharon; Edwards, Joan E.; Kim, Eun J.; Scollan, Nigel D.

doi:10.1016/j.mimet.2007.06.013

Cited by 114 publications

(88 citation statements)

References 24 publications

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“…The primers used in this study were previously validated (Tajima et al, 2001;Huws et al, 2010). Furthermore, the primers were checked using the probe match tool in the RDP (Huws et al, 2007;Cole et al, 2014). One interesting point is that the sum of the selected bacterial populations corresponded to half of the total bacterial, and this observation was independent of the diet used.…”

Section: Discussionmentioning

confidence: 99%

“…Primers for A. lipolytica (Tajima et al, 2001), F. succinogenes (Tajima et al, 2001), B. proteoclasticus (Huws et al, 2010) and P. bryantii (Tajima et al, 2001) were those reported in previous research. Once obtained, the primers were tested for specificity using the probe match function at the Ribosomal Database Project (RDP; https://rdp.cme.msu.edu/probematch/search.jsp), as described by Huws et al (2007). The oligonucleotides from each target bacteria were synthetized from Integrated DNA Technologies (Coralville, IA, USA).…”

Section: Dna Extractionmentioning

confidence: 99%

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Quantitative analysis of ruminal bacterial populations involved in lipid metabolism in dairy cows fed different vegetable oils

Vargas‐Bello‐Pérez

Cancino‐Padilla

Romero

et al. 2016

Animal

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Vegetable oils are used to increase energy density of dairy cow diets, although they can provoke changes in rumen bacteria populations and have repercussions on the biohydrogenation process. The aim of this study was to evaluate the effect of two sources of dietary lipids: soybean oil (SO, an unsaturated source) and hydrogenated palm oil (HPO, a saturated source) on bacterial populations and the fatty acid profile of ruminal digesta. Three non-lactating Holstein cows fitted with ruminal cannulae were used in a 3 × 3 Latin square design with three periods consisting of 21 days. Dietary treatments consisted of a basal diet (Control, no fat supplement) and the basal diet supplemented with SO (2.7% of dry matter (DM)) or HPO (2.7% of DM). Ruminal digesta pH, NH 3 -N and volatile fatty acids were not affected by dietary treatments. Compared with control and HPO, total bacteria measured as copies of 16S ribosomal DNA/ml by quantitative PCR was decreased ( P < 0.05) by SO. Fibrobacter succinogenes, Butyrivibrio proteoclasticus and Anaerovibrio lipolytica loads were not affected by dietary treatments. In contrast, compared with control, load of Prevotella bryantii was increased ( P < 0.05) with HPO diet. Compared with control and SO, HPO decreased ( P < 0.05) C18:2 cis n-6 in ruminal digesta. Contents of C15:0 iso, C18:11 trans-11 and C18:2 cis-9, trans-11 were increased ( P < 0.05) in ruminal digesta by SO compared with control and HPO. In conclusion, supplementation of SO or HPO do not affect ruminal fermentation parameters, whereas HPO can increase load of ruminal P. bryantii. Also, results observed in our targeted bacteria may have depended on the saturation degree of dietary oils.

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Section: Discussionmentioning

confidence: 99%

Section: Dna Extractionmentioning

confidence: 99%

Quantitative analysis of ruminal bacterial populations involved in lipid metabolism in dairy cows fed different vegetable oils

Vargas‐Bello‐Pérez

Cancino‐Padilla

Romero

et al. 2016

Animal

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“…In addition, the number of bands or peaks detected by these techniques is several orders of magnitude lower than the estimated diversity in the rumen (Hess et al, 2011;Kim et al, 2011). As for all PCR-based techniques, the selection of primers and inherent problems during amplification can distort the diversity profiles obtained (Kanagawa, 2003;Huws et al, 2007). In spite of these shortcomings, fingerprinting techniques will continue to be used as they provide a quick snapshot of the microbiota.…”

Section: Rumen Microbial Diversitymentioning

confidence: 99%

Rumen microbial (meta)genomics and its application to ruminant production

Morgavi

Kelly

Janssen

et al. 2013

Animal

193

151

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Meat and milk produced by ruminants are important agricultural products and are major sources of protein for humans. Ruminant production is of considerable economic value and underpins food security in many regions of the world. However, the sector faces major challenges because of diminishing natural resources and ensuing increases in production costs, and also because of the increased awareness of the environmental impact of farming ruminants. The digestion of feed and the production of enteric methane are key functions that could be manipulated by having a thorough understanding of the rumen microbiome. Advances in DNA sequencing technologies and bioinformatics are transforming our understanding of complex microbial ecosystems, including the gastrointestinal tract of mammals. The application of these techniques to the rumen ecosystem has allowed the study of the microbial diversity under different dietary and production conditions. Furthermore, the sequencing of genomes from several cultured rumen bacterial and archaeal species is providing detailed information about their physiology. More recently, metagenomics, mainly aimed at understanding the enzymatic machinery involved in the degradation of plant structural polysaccharides, is starting to produce new insights by allowing access to the total community and sidestepping the limitations imposed by cultivation. These advances highlight the promise of these approaches for characterising the rumen microbial community structure and linking this with the functions of the rumen microbiota. Initial results using high-throughput culture-independent technologies have also shown that the rumen microbiome is far more complex and diverse than the human caecum. Therefore, cataloguing its genes will require a considerable sequencing and bioinformatic effort. Nevertheless, the construction of a rumen microbial gene catalogue through metagenomics and genomic sequencing of key populations is an attainable goal. A rumen microbial gene catalogue is necessary to understand the function of the microbiome and its interaction with the host animal and feeds, and it will provide a basis for integrative microbiome–host models and inform strategies promoting less-polluting, more robust and efficient ruminants.

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“…While the use of universal primers has undoubtedly favored the detection and analysis of sequences, some authors indicate that they are deficient in detecting a considerable number of uncultured bacterial species from environmental samples (Baker et al 2003, Huws et al 2007). Several studies have reported the coverage of known universal primers and their combinations with sequences obtained from metagenomic studies.…”

Section: Arnr 16smentioning

confidence: 99%

“…El uso de los iniciadores universales ha favorecido la detección y análisis de secuencias; sin embargo, algunos autores señalan la deficiencia que tienen para detectar un número considerable de especies bacterianas no cultivadas provenientes de muestras medioambientales (Baker et al 2003, Huws et al 2007. Existen varios trabajos que reportan la cobertura de iniciadores universales y sus combinaciones, con secuencias obtenidas de estudios metagenómicos.…”

Section: Características Del Arnr 16sunclassified

The 16S rRNA gene in the study of marine microbial communities

et al. 2015

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ABSTRACT. New sequencing technologies and analytical capabilities have stimulated the study of microbial communities from specific environments, enabling researchers to understand the complexity of those systems. The 16S rRNA gene has proved very useful in describing the diversity and characterization of marine microbial communities, particularly of uncultivated organisms. The development of new sequencing techniques has contributed to the exponential increase in the number of reported 16S rRNA sequences as barcodes for microorganisms, forcing a review of concepts and methods for the taxonomic classification of these organisms. Manipulation and analysis of large amounts of genetic information have prompted the development of specific databases, specialized algorithms, and computational tools to compare thousands of such sequences and make a taxonomic assignment. Complete 16S rRNA sequences are thus needed for accurate and reproducible taxonomy assignment in the study of marine bacterial communities.Key words: metagenomics, marine microbial communities, 16S rRNA databases. RESUMEN. Las nuevas tecnologías de secuenciación y capacidades analíticas han estimulado el estudio de las comunidades microbianas deambientes específicos, lo cual ha permitido conocer la complejidad de estos sistemas. El gen ARNr 16S ha demostrado ser de gran utilidad para describir y caracterizar las comunidades microbianas marinas, especialmente los organismos no cultivados. Las nuevas técnicas de secuenciación han contribuido al incremento exponencial de registro de secuencias, aunque parciales, del ARNr 16S como elemento de código de barras para microorganismos. Consecuentemente, ha sido necesaria una revisión de conceptos y métodos de clasificación taxonómica para estos organismos. El manejo y análisis de una gran cantidad de información génica han impulsado el desarrollo de bases de datos específicas, algoritmos y herramientas computacionales especializadas para comparar miles de secuencias semejantes y hacer la asignación taxonómica. Por lo tanto, las secuencias completas del ARNr 16S son necesarias para una asignación taxonómica certera y reproducible en los estudios de comunidades microbianas marinas.Palabras clave: metagenómica, comunidades microbianas marinas, bases de datos ARNr 16S.

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Specificity and sensitivity of eubacterial primers utilized for molecular profiling of bacteria within complex microbial ecosystems

Cited by 114 publications

References 24 publications

Quantitative analysis of ruminal bacterial populations involved in lipid metabolism in dairy cows fed different vegetable oils

Quantitative analysis of ruminal bacterial populations involved in lipid metabolism in dairy cows fed different vegetable oils

Rumen microbial (meta)genomics and its application to ruminant production

The 16S rRNA gene in the study of marine microbial communities

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