Quantitative PCR for Detection and Enumeration of Genetic Markers of Bovine Fecal Pollution
Accurate assessment of health risks associated with bovine (cattle) fecal pollution requires a reliable host-specific genetic marker and a rapid quantification method. We report the development of quantitative PCR assays for the detection of two recently described bovine feces-specific genetic marke...
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| Veröffentlicht in: | Applied and Environmental Microbiology 2008-02, Vol.74 (3), p.745-752 |
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| container_title | Applied and Environmental Microbiology |
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| creator | Shanks, Orin C Atikovic, Emina Blackwood, A. Denene Lu, Jingrang Noble, Rachel T Domingo, Jorge Santo Seifring, Shawn Sivaganesan, Mano Haugland, Richard A |
| description | Accurate assessment of health risks associated with bovine (cattle) fecal pollution requires a reliable host-specific genetic marker and a rapid quantification method. We report the development of quantitative PCR assays for the detection of two recently described bovine feces-specific genetic markers and a method for the enumeration of these markers using a Markov chain Monte Carlo approach. Both assays exhibited a range of quantification from 25 to 2 x 10⁶ copies of target DNA, with a coefficient of variation of |
| doi_str_mv | 10.1128/AEM.01843-07 |
| format | Article |
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Denene ; Lu, Jingrang ; Noble, Rachel T ; Domingo, Jorge Santo ; Seifring, Shawn ; Sivaganesan, Mano ; Haugland, Richard A</creator><creatorcontrib>Shanks, Orin C ; Atikovic, Emina ; Blackwood, A. Denene ; Lu, Jingrang ; Noble, Rachel T ; Domingo, Jorge Santo ; Seifring, Shawn ; Sivaganesan, Mano ; Haugland, Richard A</creatorcontrib><description>Accurate assessment of health risks associated with bovine (cattle) fecal pollution requires a reliable host-specific genetic marker and a rapid quantification method. We report the development of quantitative PCR assays for the detection of two recently described bovine feces-specific genetic markers and a method for the enumeration of these markers using a Markov chain Monte Carlo approach. Both assays exhibited a range of quantification from 25 to 2 x 10⁶ copies of target DNA, with a coefficient of variation of <2.1%. One of these assays can be multiplexed with an internal amplification control to simultaneously detect the bovine-specific genetic target and presence of amplification inhibitors. The assays detected only cattle fecal specimens when tested against 204 fecal DNA extracts from 16 different animal species and also demonstrated a broad distribution among individual bovine samples (98 to 100%) collected from five geographically distinct locations. The abundance of each bovine-specific genetic marker was measured in 48 individual samples and compared to quantitative PCR-enumerated quantities of rRNA gene sequences representing total Bacteroidetes, Bacteroides thetaiotaomicron, and enterococci in the same specimens. Acceptable assay performance combined with the prevalence of DNA targets across different cattle populations provides experimental evidence that these quantitative assays will be useful in monitoring bovine fecal pollution in ambient waters.</description><identifier>ISSN: 0099-2240</identifier><identifier>EISSN: 1098-5336</identifier><identifier>DOI: 10.1128/AEM.01843-07</identifier><identifier>PMID: 18065617</identifier><identifier>CODEN: AEMIDF</identifier><language>eng</language><publisher>Washington, DC: American Society for Microbiology</publisher><subject>Animals ; Bacteria ; Bacteroides thetaiotaomicron ; Biological and medical sciences ; Cattle ; DNA Primers ; DNA, Bacterial - analysis ; DNA, Bacterial - isolation & purification ; DNA, Ribosomal - analysis ; Feces ; Feces - microbiology ; Fundamental and applied biological sciences. Psychology ; Genes, rRNA ; Genetic Markers - genetics ; Genetics ; Markov Chains ; Methods ; Microbiology ; Monte Carlo Method ; Monte Carlo simulation ; Plasmids - genetics ; Pollution ; Polymerase Chain Reaction - methods ; Species Specificity ; Water Pollution - analysis</subject><ispartof>Applied and Environmental Microbiology, 2008-02, Vol.74 (3), p.745-752</ispartof><rights>2008 INIST-CNRS</rights><rights>Copyright American Society for Microbiology Feb 2008</rights><rights>Copyright © 2008, American Society for Microbiology</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c628t-543ce05bb47356557d45e065963524616aec3e4378eb88e5eaf3bbf1b9a4b59d3</citedby><cites>FETCH-LOGICAL-c628t-543ce05bb47356557d45e065963524616aec3e4378eb88e5eaf3bbf1b9a4b59d3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2227726/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2227726/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,724,777,781,882,3175,3176,27905,27906,53772,53774</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=20245777$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/18065617$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Shanks, Orin C</creatorcontrib><creatorcontrib>Atikovic, Emina</creatorcontrib><creatorcontrib>Blackwood, A. Denene</creatorcontrib><creatorcontrib>Lu, Jingrang</creatorcontrib><creatorcontrib>Noble, Rachel T</creatorcontrib><creatorcontrib>Domingo, Jorge Santo</creatorcontrib><creatorcontrib>Seifring, Shawn</creatorcontrib><creatorcontrib>Sivaganesan, Mano</creatorcontrib><creatorcontrib>Haugland, Richard A</creatorcontrib><title>Quantitative PCR for Detection and Enumeration of Genetic Markers of Bovine Fecal Pollution</title><title>Applied and Environmental Microbiology</title><addtitle>Appl Environ Microbiol</addtitle><description>Accurate assessment of health risks associated with bovine (cattle) fecal pollution requires a reliable host-specific genetic marker and a rapid quantification method. We report the development of quantitative PCR assays for the detection of two recently described bovine feces-specific genetic markers and a method for the enumeration of these markers using a Markov chain Monte Carlo approach. Both assays exhibited a range of quantification from 25 to 2 x 10⁶ copies of target DNA, with a coefficient of variation of <2.1%. One of these assays can be multiplexed with an internal amplification control to simultaneously detect the bovine-specific genetic target and presence of amplification inhibitors. The assays detected only cattle fecal specimens when tested against 204 fecal DNA extracts from 16 different animal species and also demonstrated a broad distribution among individual bovine samples (98 to 100%) collected from five geographically distinct locations. The abundance of each bovine-specific genetic marker was measured in 48 individual samples and compared to quantitative PCR-enumerated quantities of rRNA gene sequences representing total Bacteroidetes, Bacteroides thetaiotaomicron, and enterococci in the same specimens. Acceptable assay performance combined with the prevalence of DNA targets across different cattle populations provides experimental evidence that these quantitative assays will be useful in monitoring bovine fecal pollution in ambient waters.</description><subject>Animals</subject><subject>Bacteria</subject><subject>Bacteroides thetaiotaomicron</subject><subject>Biological and medical sciences</subject><subject>Cattle</subject><subject>DNA Primers</subject><subject>DNA, Bacterial - analysis</subject><subject>DNA, Bacterial - isolation & purification</subject><subject>DNA, Ribosomal - analysis</subject><subject>Feces</subject><subject>Feces - microbiology</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Genes, rRNA</subject><subject>Genetic Markers - genetics</subject><subject>Genetics</subject><subject>Markov Chains</subject><subject>Methods</subject><subject>Microbiology</subject><subject>Monte Carlo Method</subject><subject>Monte Carlo simulation</subject><subject>Plasmids - genetics</subject><subject>Pollution</subject><subject>Polymerase Chain Reaction - methods</subject><subject>Species Specificity</subject><subject>Water Pollution - analysis</subject><issn>0099-2240</issn><issn>1098-5336</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2008</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpd0ctv1DAQB-AIgehSuHGGCAlOpIzfzgWpLNuC1Iry6ImD5WQnu24Tu9jJIv57vN1VeciSLduffhp7iuIpgSNCqH5zvDg_AqI5q0DdK2YEal0JxuT9YgZQ1xWlHA6KRyldAQAHqR8WB0SDFJKoWfH982T96EY7ug2WF_MvZRdi-R5HbEcXfGn9slz4acBob_ehK0_R4-ja8tzGa4xpe_QubJzH8gRb25cXoe-nLX5cPOhsn_DJfj0sLk8W3-YfqrNPpx_nx2dVK6keK8FZiyCahismpBBqyQXm-mrJBOWSSIstQ86UxkZrFGg71jQdaWrLG1Ev2WHxdpd7MzUDLlv0Y7S9uYlusPGXCdaZf2-8W5tV2BhKqVJU5oBX-4AYfkyYRjO41GLfW49hSobUUnEFW_jiP3gVpujz4wwFUWsuhM7o9Q61MaQUsburhIDZtszklpnblhlQmT_7u_o_eN-jDF7ugU35f7tofevSnaNAuVBq68qdW7vV-qeLaGwajMXBKG5YnkQmz3eks8HYVcwxl18pEAagBeN5_Aavp7I3</recordid><startdate>20080201</startdate><enddate>20080201</enddate><creator>Shanks, Orin C</creator><creator>Atikovic, Emina</creator><creator>Blackwood, A. 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Denene ; Lu, Jingrang ; Noble, Rachel T ; Domingo, Jorge Santo ; Seifring, Shawn ; Sivaganesan, Mano ; Haugland, Richard A</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c628t-543ce05bb47356557d45e065963524616aec3e4378eb88e5eaf3bbf1b9a4b59d3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2008</creationdate><topic>Animals</topic><topic>Bacteria</topic><topic>Bacteroides thetaiotaomicron</topic><topic>Biological and medical sciences</topic><topic>Cattle</topic><topic>DNA Primers</topic><topic>DNA, Bacterial - analysis</topic><topic>DNA, Bacterial - isolation & purification</topic><topic>DNA, Ribosomal - analysis</topic><topic>Feces</topic><topic>Feces - microbiology</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Genes, rRNA</topic><topic>Genetic Markers - genetics</topic><topic>Genetics</topic><topic>Markov Chains</topic><topic>Methods</topic><topic>Microbiology</topic><topic>Monte Carlo Method</topic><topic>Monte Carlo simulation</topic><topic>Plasmids - genetics</topic><topic>Pollution</topic><topic>Polymerase Chain Reaction - methods</topic><topic>Species Specificity</topic><topic>Water Pollution - analysis</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Shanks, Orin C</creatorcontrib><creatorcontrib>Atikovic, Emina</creatorcontrib><creatorcontrib>Blackwood, A. 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Denene</au><au>Lu, Jingrang</au><au>Noble, Rachel T</au><au>Domingo, Jorge Santo</au><au>Seifring, Shawn</au><au>Sivaganesan, Mano</au><au>Haugland, Richard A</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Quantitative PCR for Detection and Enumeration of Genetic Markers of Bovine Fecal Pollution</atitle><jtitle>Applied and Environmental Microbiology</jtitle><addtitle>Appl Environ Microbiol</addtitle><date>2008-02-01</date><risdate>2008</risdate><volume>74</volume><issue>3</issue><spage>745</spage><epage>752</epage><pages>745-752</pages><issn>0099-2240</issn><eissn>1098-5336</eissn><coden>AEMIDF</coden><abstract>Accurate assessment of health risks associated with bovine (cattle) fecal pollution requires a reliable host-specific genetic marker and a rapid quantification method. We report the development of quantitative PCR assays for the detection of two recently described bovine feces-specific genetic markers and a method for the enumeration of these markers using a Markov chain Monte Carlo approach. Both assays exhibited a range of quantification from 25 to 2 x 10⁶ copies of target DNA, with a coefficient of variation of <2.1%. One of these assays can be multiplexed with an internal amplification control to simultaneously detect the bovine-specific genetic target and presence of amplification inhibitors. The assays detected only cattle fecal specimens when tested against 204 fecal DNA extracts from 16 different animal species and also demonstrated a broad distribution among individual bovine samples (98 to 100%) collected from five geographically distinct locations. The abundance of each bovine-specific genetic marker was measured in 48 individual samples and compared to quantitative PCR-enumerated quantities of rRNA gene sequences representing total Bacteroidetes, Bacteroides thetaiotaomicron, and enterococci in the same specimens. Acceptable assay performance combined with the prevalence of DNA targets across different cattle populations provides experimental evidence that these quantitative assays will be useful in monitoring bovine fecal pollution in ambient waters.</abstract><cop>Washington, DC</cop><pub>American Society for Microbiology</pub><pmid>18065617</pmid><doi>10.1128/AEM.01843-07</doi><tpages>8</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | Applied and Environmental Microbiology, 2008-02, Vol.74 (3), p.745-752 |
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| language | eng |
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| source | American Society for Microbiology; MEDLINE; PubMed Central; Alma/SFX Local Collection |
| subjects | Animals Bacteria Bacteroides thetaiotaomicron Biological and medical sciences Cattle DNA Primers DNA, Bacterial - analysis DNA, Bacterial - isolation & purification DNA, Ribosomal - analysis Feces Feces - microbiology Fundamental and applied biological sciences. Psychology Genes, rRNA Genetic Markers - genetics Genetics Markov Chains Methods Microbiology Monte Carlo Method Monte Carlo simulation Plasmids - genetics Pollution Polymerase Chain Reaction - methods Species Specificity Water Pollution - analysis |
| title | Quantitative PCR for Detection and Enumeration of Genetic Markers of Bovine Fecal Pollution |
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