Development of a real-time PCR assay for monitoring anaerobic fungal and cellulolytic bacterial populations within the rumen
Abstract Traditional methods for enumerating and identifying microbial populations within the rumen can be time consuming and cumbersome. Methods that involve culturing and microscopy can also be inconclusive, particularly when studying anaerobic rumen fungi. A real-time PCR SYBR Green assay, using...
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| Veröffentlicht in: | FEMS microbiology ecology 2006-12, Vol.58 (3), p.572-582 |
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| creator | Denman, Stuart E. McSweeney, Christopher S. |
| description | Abstract
Traditional methods for enumerating and identifying microbial populations within the rumen can be time consuming and cumbersome. Methods that involve culturing and microscopy can also be inconclusive, particularly when studying anaerobic rumen fungi. A real-time PCR SYBR Green assay, using PCR primers to target total rumen fungi and the cellulolytic bacteria Ruminococcus flavefaciens and Fibrobacter succinogenes, is described, including design and validation. The DNA and crude protein contents with respect to the fungal biomass of both polycentric and monocentric fungal isolates were investigated across the fungal growth stages to aid in standard curve generation. The primer sets used were found to be target specific with no detectable cross-reactivity. Subsequently, the real-time PCR assay was employed in a study to detect these populations within cattle rumen. The anaerobic fungal target was observed to increase 3.6-fold from 0 to 12 h after feeding. The results also indicated a 5.4-fold increase in F. succinogenes target between 0 and 12 h after feeding, whereas R. flavefaciens was observed to maintain more or less consistent levels. This is the first report of a real-time PCR assay to estimate the rumen anaerobic fungal population. |
| doi_str_mv | 10.1111/j.1574-6941.2006.00190.x |
| format | Article |
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Traditional methods for enumerating and identifying microbial populations within the rumen can be time consuming and cumbersome. Methods that involve culturing and microscopy can also be inconclusive, particularly when studying anaerobic rumen fungi. A real-time PCR SYBR Green assay, using PCR primers to target total rumen fungi and the cellulolytic bacteria Ruminococcus flavefaciens and Fibrobacter succinogenes, is described, including design and validation. The DNA and crude protein contents with respect to the fungal biomass of both polycentric and monocentric fungal isolates were investigated across the fungal growth stages to aid in standard curve generation. The primer sets used were found to be target specific with no detectable cross-reactivity. Subsequently, the real-time PCR assay was employed in a study to detect these populations within cattle rumen. The anaerobic fungal target was observed to increase 3.6-fold from 0 to 12 h after feeding. The results also indicated a 5.4-fold increase in F. succinogenes target between 0 and 12 h after feeding, whereas R. flavefaciens was observed to maintain more or less consistent levels. This is the first report of a real-time PCR assay to estimate the rumen anaerobic fungal population.</description><identifier>ISSN: 0168-6496</identifier><identifier>EISSN: 1574-6941</identifier><identifier>DOI: 10.1111/j.1574-6941.2006.00190.x</identifier><identifier>PMID: 17117998</identifier><language>eng</language><publisher>Oxford, UK: Blackwell Publishing Ltd</publisher><subject>anaerobic fungi ; Anaerobiosis ; Animals ; Assaying ; Bacteria ; Bacteria - genetics ; Bacteria - isolation & purification ; Bacteriology ; Biological and medical sciences ; Calibration ; Cattle ; Cellulolytic bacteria ; Cellulose - metabolism ; Colony Count, Microbial ; Cross-reactivity ; Ecology ; Feeding ; Fibrobacter succinogenes ; Fundamental and applied biological sciences. Psychology ; Fungi ; Fungi - genetics ; Fungi - isolation & purification ; Identification methods ; Microbiology ; Microorganisms ; Miscellaneous ; Mycology ; Polymerase chain reaction ; Polymerase Chain Reaction - methods ; Population studies ; Populations ; Real time ; real‐time PCR ; Rumen ; Rumen - microbiology ; Ruminococcus flavefaciens</subject><ispartof>FEMS microbiology ecology, 2006-12, Vol.58 (3), p.572-582</ispartof><rights>2006 Federation of European Microbiological Societies 2006</rights><rights>2007 INIST-CNRS</rights><rights>2006 Federation of European Microbiological Societies</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c5350-23aca57cb6033ea8fbfb1fc48b7f8772ebc5ff7ab124433c25dddaeaea7ecf13</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1111%2Fj.1574-6941.2006.00190.x$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1111%2Fj.1574-6941.2006.00190.x$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>315,781,785,1418,27929,27930,45579,45580</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=18285633$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/17117998$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Denman, Stuart E.</creatorcontrib><creatorcontrib>McSweeney, Christopher S.</creatorcontrib><title>Development of a real-time PCR assay for monitoring anaerobic fungal and cellulolytic bacterial populations within the rumen</title><title>FEMS microbiology ecology</title><addtitle>FEMS Microbiol Ecol</addtitle><description>Abstract
Traditional methods for enumerating and identifying microbial populations within the rumen can be time consuming and cumbersome. Methods that involve culturing and microscopy can also be inconclusive, particularly when studying anaerobic rumen fungi. A real-time PCR SYBR Green assay, using PCR primers to target total rumen fungi and the cellulolytic bacteria Ruminococcus flavefaciens and Fibrobacter succinogenes, is described, including design and validation. The DNA and crude protein contents with respect to the fungal biomass of both polycentric and monocentric fungal isolates were investigated across the fungal growth stages to aid in standard curve generation. The primer sets used were found to be target specific with no detectable cross-reactivity. Subsequently, the real-time PCR assay was employed in a study to detect these populations within cattle rumen. The anaerobic fungal target was observed to increase 3.6-fold from 0 to 12 h after feeding. The results also indicated a 5.4-fold increase in F. succinogenes target between 0 and 12 h after feeding, whereas R. flavefaciens was observed to maintain more or less consistent levels. This is the first report of a real-time PCR assay to estimate the rumen anaerobic fungal population.</description><subject>anaerobic fungi</subject><subject>Anaerobiosis</subject><subject>Animals</subject><subject>Assaying</subject><subject>Bacteria</subject><subject>Bacteria - genetics</subject><subject>Bacteria - isolation & purification</subject><subject>Bacteriology</subject><subject>Biological and medical sciences</subject><subject>Calibration</subject><subject>Cattle</subject><subject>Cellulolytic bacteria</subject><subject>Cellulose - metabolism</subject><subject>Colony Count, Microbial</subject><subject>Cross-reactivity</subject><subject>Ecology</subject><subject>Feeding</subject><subject>Fibrobacter succinogenes</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Fungi</subject><subject>Fungi - genetics</subject><subject>Fungi - isolation & purification</subject><subject>Identification methods</subject><subject>Microbiology</subject><subject>Microorganisms</subject><subject>Miscellaneous</subject><subject>Mycology</subject><subject>Polymerase chain reaction</subject><subject>Polymerase Chain Reaction - methods</subject><subject>Population studies</subject><subject>Populations</subject><subject>Real time</subject><subject>real‐time PCR</subject><subject>Rumen</subject><subject>Rumen - microbiology</subject><subject>Ruminococcus flavefaciens</subject><issn>0168-6496</issn><issn>1574-6941</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2006</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><recordid>eNp1kVGL1DAQx4Mo3t7pV5CA6Ftr0rRJC77IeqfCiSL3HqZpcpclTWrSerfgh7_UXT1QTB4yzPz-k2H-CGFKSprPm11JG1EXvKtpWRHCS0JoR8q7R2jzp_AYbQjlbcHrjp-g05R2GWpYTZ6iEyooFV3XbtDP9_qHdmEatZ9xMBhw1OCK2Y4af91-w5AS7LEJEY_B2zlE668xeNAx9FZhs_hrcDkxYKWdW1xw-znne1CzjjaXpjAtDmYbfMK3dr6xHs83Gscl__gMPTHgkn5-fM_Q1cX51fZjcfnlw6ftu8tCNawhRcVAQSNUzwljGlrTm54aVbe9MK0Qle5VY4yAnlZ1zZiqmmEYQOcrtDKUnaHXh7ZTDN8XnWY52rSOC16HJUneUlFzVmfw5V_gLizR59FkxYjI2-v42u7FkVr6UQ9yinaEuJe_l5qBV0cAkgJnInhl0wPXVm3DGcvc2wN3a53eP9SJXE2WO7l6KVcv5Wqy_GWyvJMX559zkOXsIA_L9B9x8Y-Y3QOQAauN</recordid><startdate>200612</startdate><enddate>200612</enddate><creator>Denman, Stuart E.</creator><creator>McSweeney, Christopher S.</creator><general>Blackwell Publishing Ltd</general><general>Blackwell</general><general>Oxford University Press</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>3V.</scope><scope>7QL</scope><scope>7QP</scope><scope>7QR</scope><scope>7SN</scope><scope>7T7</scope><scope>7TK</scope><scope>7TM</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>P64</scope><scope>PATMY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope></search><sort><creationdate>200612</creationdate><title>Development of a real-time PCR assay for monitoring anaerobic fungal and cellulolytic bacterial populations within the rumen</title><author>Denman, Stuart E. ; McSweeney, Christopher S.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c5350-23aca57cb6033ea8fbfb1fc48b7f8772ebc5ff7ab124433c25dddaeaea7ecf13</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2006</creationdate><topic>anaerobic fungi</topic><topic>Anaerobiosis</topic><topic>Animals</topic><topic>Assaying</topic><topic>Bacteria</topic><topic>Bacteria - genetics</topic><topic>Bacteria - isolation & purification</topic><topic>Bacteriology</topic><topic>Biological and medical sciences</topic><topic>Calibration</topic><topic>Cattle</topic><topic>Cellulolytic bacteria</topic><topic>Cellulose - metabolism</topic><topic>Colony Count, Microbial</topic><topic>Cross-reactivity</topic><topic>Ecology</topic><topic>Feeding</topic><topic>Fibrobacter succinogenes</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Fungi</topic><topic>Fungi - genetics</topic><topic>Fungi - isolation & purification</topic><topic>Identification methods</topic><topic>Microbiology</topic><topic>Microorganisms</topic><topic>Miscellaneous</topic><topic>Mycology</topic><topic>Polymerase chain reaction</topic><topic>Polymerase Chain Reaction - methods</topic><topic>Population studies</topic><topic>Populations</topic><topic>Real time</topic><topic>real‐time PCR</topic><topic>Rumen</topic><topic>Rumen - microbiology</topic><topic>Ruminococcus flavefaciens</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Denman, Stuart E.</creatorcontrib><creatorcontrib>McSweeney, Christopher S.</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>ProQuest Central (Corporate)</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Chemoreception Abstracts</collection><collection>Ecology Abstracts</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>Agricultural & Environmental Science Collection</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biological Science Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Environmental Science Database</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>Environmental Science Collection</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>FEMS microbiology ecology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Denman, Stuart E.</au><au>McSweeney, Christopher S.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Development of a real-time PCR assay for monitoring anaerobic fungal and cellulolytic bacterial populations within the rumen</atitle><jtitle>FEMS microbiology ecology</jtitle><addtitle>FEMS Microbiol Ecol</addtitle><date>2006-12</date><risdate>2006</risdate><volume>58</volume><issue>3</issue><spage>572</spage><epage>582</epage><pages>572-582</pages><issn>0168-6496</issn><eissn>1574-6941</eissn><abstract>Abstract
Traditional methods for enumerating and identifying microbial populations within the rumen can be time consuming and cumbersome. Methods that involve culturing and microscopy can also be inconclusive, particularly when studying anaerobic rumen fungi. A real-time PCR SYBR Green assay, using PCR primers to target total rumen fungi and the cellulolytic bacteria Ruminococcus flavefaciens and Fibrobacter succinogenes, is described, including design and validation. The DNA and crude protein contents with respect to the fungal biomass of both polycentric and monocentric fungal isolates were investigated across the fungal growth stages to aid in standard curve generation. The primer sets used were found to be target specific with no detectable cross-reactivity. Subsequently, the real-time PCR assay was employed in a study to detect these populations within cattle rumen. The anaerobic fungal target was observed to increase 3.6-fold from 0 to 12 h after feeding. The results also indicated a 5.4-fold increase in F. succinogenes target between 0 and 12 h after feeding, whereas R. flavefaciens was observed to maintain more or less consistent levels. This is the first report of a real-time PCR assay to estimate the rumen anaerobic fungal population.</abstract><cop>Oxford, UK</cop><pub>Blackwell Publishing Ltd</pub><pmid>17117998</pmid><doi>10.1111/j.1574-6941.2006.00190.x</doi><tpages>11</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | anaerobic fungi Anaerobiosis Animals Assaying Bacteria Bacteria - genetics Bacteria - isolation & purification Bacteriology Biological and medical sciences Calibration Cattle Cellulolytic bacteria Cellulose - metabolism Colony Count, Microbial Cross-reactivity Ecology Feeding Fibrobacter succinogenes Fundamental and applied biological sciences. Psychology Fungi Fungi - genetics Fungi - isolation & purification Identification methods Microbiology Microorganisms Miscellaneous Mycology Polymerase chain reaction Polymerase Chain Reaction - methods Population studies Populations Real time real‐time PCR Rumen Rumen - microbiology Ruminococcus flavefaciens |
| title | Development of a real-time PCR assay for monitoring anaerobic fungal and cellulolytic bacterial populations within the rumen |
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