Comparison of different approaches to quantify Staphylococcus aureus cells by real-time quantitative PCR and application of this technique for examination of cheese
Two different real-time quantitative PCR (RTQ-PCR) approaches were applied for PCR-based quantification of Staphylococcus aureus cells by targeting the thermonuclease (nuc) gene. Purified DNA extracts from pure cultures of S. aureus were quantified in a LightCycler system using SYBR Green I. Quantif...
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| Veröffentlicht in: | Applied and environmental microbiology 2001-07, Vol.67 (7), p.3122-3126 |
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| container_title | Applied and environmental microbiology |
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| creator | HEIN, Ingeborg LEHNER, Angelika RIECK, Petra KLEIN, Kurt BRANDL, Ernst WAGNER, Martin |
| description | Two different real-time quantitative PCR (RTQ-PCR) approaches were applied for PCR-based quantification of Staphylococcus aureus cells by targeting the thermonuclease (nuc) gene. Purified DNA extracts from pure cultures of S. aureus were quantified in a LightCycler system using SYBR Green I. Quantification proved to be less sensitive (60 nuc gene copies/microl) than using a fluorigenic TaqMan probe (6 nuc gene copies/microl). Comparison of the LightCycler system and the well-established ABI Prism 7700 SDS with TaqMan probes revealed no statistically significant differences with respect to sensitivity and reproducibility. Application of the RTQ-PCR assay to quantify S. aureus cells in artificially contaminated cheeses of different types achieved sensitivities from 1.5 x 10(2) to 6.4 x 10(2) copies of the nuc gene/2 g, depending on the cheese matrix. The coefficients of correlation between log CFU and nuc gene copy numbers ranged from 0.979 to 0.998, thus enabling calculation of the number of CFU of S. aureus in cheese by performing RTQ-PCR. |
| doi_str_mv | 10.1128/AEM.67.7.3122-3126.2001 |
| format | Article |
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Purified DNA extracts from pure cultures of S. aureus were quantified in a LightCycler system using SYBR Green I. Quantification proved to be less sensitive (60 nuc gene copies/microl) than using a fluorigenic TaqMan probe (6 nuc gene copies/microl). Comparison of the LightCycler system and the well-established ABI Prism 7700 SDS with TaqMan probes revealed no statistically significant differences with respect to sensitivity and reproducibility. Application of the RTQ-PCR assay to quantify S. aureus cells in artificially contaminated cheeses of different types achieved sensitivities from 1.5 x 10(2) to 6.4 x 10(2) copies of the nuc gene/2 g, depending on the cheese matrix. The coefficients of correlation between log CFU and nuc gene copy numbers ranged from 0.979 to 0.998, thus enabling calculation of the number of CFU of S. aureus in cheese by performing RTQ-PCR.</description><identifier>ISSN: 0099-2240</identifier><identifier>EISSN: 1098-5336</identifier><identifier>DOI: 10.1128/AEM.67.7.3122-3126.2001</identifier><identifier>PMID: 11425731</identifier><identifier>CODEN: AEMIDF</identifier><language>eng</language><publisher>Washington, DC: American Society for Microbiology</publisher><subject>Bacteria ; Benzothiazoles ; Biological and medical sciences ; Cells ; Cheese ; Cheese - microbiology ; Colony Count, Microbial ; Diamines ; DNA, Bacterial - analysis ; Fluorescent Dyes ; Fluorometry ; Food contamination & poisoning ; Food industries ; Food Microbiology ; Food science ; Fundamental and applied biological sciences. Psychology ; Gene Dosage ; Hot Temperature ; Micrococcal Nuclease - genetics ; Milk and cheese industries. Ice creams ; Organic Chemicals ; Quinolines ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction ; Sensitivity and Specificity ; Staphylococcus aureus ; Staphylococcus aureus - genetics ; Staphylococcus aureus - isolation & purification ; Taq Polymerase - metabolism ; thermonuclease (nuc) gene</subject><ispartof>Applied and environmental microbiology, 2001-07, Vol.67 (7), p.3122-3126</ispartof><rights>2002 INIST-CNRS</rights><rights>Copyright American Society for Microbiology Jul 2001</rights><rights>Copyright © 2001, American Society for Microbiology 2001</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c553t-c4b381cf25f7f8bdc09dacdc968344b394f50ba38f8d4ad980b90055c50c567b3</citedby><cites>FETCH-LOGICAL-c553t-c4b381cf25f7f8bdc09dacdc968344b394f50ba38f8d4ad980b90055c50c567b3</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/PMC92990/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC92990/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,723,776,780,881,3175,27901,27902,53766,53768</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=14068841$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/11425731$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>HEIN, Ingeborg</creatorcontrib><creatorcontrib>LEHNER, Angelika</creatorcontrib><creatorcontrib>RIECK, Petra</creatorcontrib><creatorcontrib>KLEIN, Kurt</creatorcontrib><creatorcontrib>BRANDL, Ernst</creatorcontrib><creatorcontrib>WAGNER, Martin</creatorcontrib><title>Comparison of different approaches to quantify Staphylococcus aureus cells by real-time quantitative PCR and application of this technique for examination of cheese</title><title>Applied and environmental microbiology</title><addtitle>Appl Environ Microbiol</addtitle><description>Two different real-time quantitative PCR (RTQ-PCR) approaches were applied for PCR-based quantification of Staphylococcus aureus cells by targeting the thermonuclease (nuc) gene. Purified DNA extracts from pure cultures of S. aureus were quantified in a LightCycler system using SYBR Green I. Quantification proved to be less sensitive (60 nuc gene copies/microl) than using a fluorigenic TaqMan probe (6 nuc gene copies/microl). Comparison of the LightCycler system and the well-established ABI Prism 7700 SDS with TaqMan probes revealed no statistically significant differences with respect to sensitivity and reproducibility. Application of the RTQ-PCR assay to quantify S. aureus cells in artificially contaminated cheeses of different types achieved sensitivities from 1.5 x 10(2) to 6.4 x 10(2) copies of the nuc gene/2 g, depending on the cheese matrix. The coefficients of correlation between log CFU and nuc gene copy numbers ranged from 0.979 to 0.998, thus enabling calculation of the number of CFU of S. aureus in cheese by performing RTQ-PCR.</description><subject>Bacteria</subject><subject>Benzothiazoles</subject><subject>Biological and medical sciences</subject><subject>Cells</subject><subject>Cheese</subject><subject>Cheese - microbiology</subject><subject>Colony Count, Microbial</subject><subject>Diamines</subject><subject>DNA, Bacterial - analysis</subject><subject>Fluorescent Dyes</subject><subject>Fluorometry</subject><subject>Food contamination & poisoning</subject><subject>Food industries</subject><subject>Food Microbiology</subject><subject>Food science</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Gene Dosage</subject><subject>Hot Temperature</subject><subject>Micrococcal Nuclease - genetics</subject><subject>Milk and cheese industries. Ice creams</subject><subject>Organic Chemicals</subject><subject>Quinolines</subject><subject>Reproducibility of Results</subject><subject>Reverse Transcriptase Polymerase Chain Reaction</subject><subject>Sensitivity and Specificity</subject><subject>Staphylococcus aureus</subject><subject>Staphylococcus aureus - genetics</subject><subject>Staphylococcus aureus - isolation & purification</subject><subject>Taq Polymerase - metabolism</subject><subject>thermonuclease (nuc) gene</subject><issn>0099-2240</issn><issn>1098-5336</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2001</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFks9u1DAQxiMEokvhFcBCKrcs4yR2bIlLtSotUhGIP2dr4tisqyRO7aRi34cHxdFGLXDhMiN5ft-MZ_Rl2SsKW0oL8fb84uOW19t6W9KiyFPg2wKAPso2FKTIWVnyx9kGQMq8KCo4yZ7FeAMAFXDxNDuhtCpYXdJN9mvn-xGDi34g3pLWWWuCGSaC4xg86r2JZPLkdsZhcvZAvk447g-d117rORKcg0lJm66LpDmQYLDLJ9ebVTHh5O4M-bz7QnBol6ad0-ntOG3au9Td6P3gbmdDrA_E_MTeDfdEmm-ieZ49sdhF82LNp9n39xffdlf59afLD7vz61wzVk65rppSUG0LZmsrmlaDbFG3WnJRVqkmK8ugwVJY0VbYSgGNBGBMM9CM1015mr079h3npjetTncI2KkxuB7DQXl06u_K4Pbqh79TspASkvzNKg8-7RMn1bu4nAYH4-eoapC8gor-F6S1BCHkAr7-B7zxcxjSDVQBTPKS8mVsfYR08DEGY-8_TEEtblHJLYrXqlaLW5bA1eKWpHz5574PutUeCThbAYwaOxtw0C4-cIudRFroN4bKzW8</recordid><startdate>20010701</startdate><enddate>20010701</enddate><creator>HEIN, Ingeborg</creator><creator>LEHNER, Angelika</creator><creator>RIECK, Petra</creator><creator>KLEIN, Kurt</creator><creator>BRANDL, Ernst</creator><creator>WAGNER, Martin</creator><general>American Society for Microbiology</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>7QO</scope><scope>7SN</scope><scope>7SS</scope><scope>7ST</scope><scope>7T7</scope><scope>7TM</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>SOI</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20010701</creationdate><title>Comparison of different approaches to quantify Staphylococcus aureus cells by real-time quantitative PCR and application of this technique for examination of cheese</title><author>HEIN, Ingeborg ; LEHNER, Angelika ; RIECK, Petra ; KLEIN, Kurt ; BRANDL, Ernst ; WAGNER, Martin</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c553t-c4b381cf25f7f8bdc09dacdc968344b394f50ba38f8d4ad980b90055c50c567b3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2001</creationdate><topic>Bacteria</topic><topic>Benzothiazoles</topic><topic>Biological and medical sciences</topic><topic>Cells</topic><topic>Cheese</topic><topic>Cheese - microbiology</topic><topic>Colony Count, Microbial</topic><topic>Diamines</topic><topic>DNA, Bacterial - analysis</topic><topic>Fluorescent Dyes</topic><topic>Fluorometry</topic><topic>Food contamination & poisoning</topic><topic>Food industries</topic><topic>Food Microbiology</topic><topic>Food science</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Gene Dosage</topic><topic>Hot Temperature</topic><topic>Micrococcal Nuclease - genetics</topic><topic>Milk and cheese industries. Ice creams</topic><topic>Organic Chemicals</topic><topic>Quinolines</topic><topic>Reproducibility of Results</topic><topic>Reverse Transcriptase Polymerase Chain Reaction</topic><topic>Sensitivity and Specificity</topic><topic>Staphylococcus aureus</topic><topic>Staphylococcus aureus - genetics</topic><topic>Staphylococcus aureus - isolation & purification</topic><topic>Taq Polymerase - metabolism</topic><topic>thermonuclease (nuc) gene</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>HEIN, Ingeborg</creatorcontrib><creatorcontrib>LEHNER, Angelika</creatorcontrib><creatorcontrib>RIECK, Petra</creatorcontrib><creatorcontrib>KLEIN, Kurt</creatorcontrib><creatorcontrib>BRANDL, Ernst</creatorcontrib><creatorcontrib>WAGNER, Martin</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>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Ecology Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Environment Abstracts</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>Environment Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Applied and environmental microbiology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>HEIN, Ingeborg</au><au>LEHNER, Angelika</au><au>RIECK, Petra</au><au>KLEIN, Kurt</au><au>BRANDL, Ernst</au><au>WAGNER, Martin</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Comparison of different approaches to quantify Staphylococcus aureus cells by real-time quantitative PCR and application of this technique for examination of cheese</atitle><jtitle>Applied and environmental microbiology</jtitle><addtitle>Appl Environ Microbiol</addtitle><date>2001-07-01</date><risdate>2001</risdate><volume>67</volume><issue>7</issue><spage>3122</spage><epage>3126</epage><pages>3122-3126</pages><issn>0099-2240</issn><eissn>1098-5336</eissn><coden>AEMIDF</coden><abstract>Two different real-time quantitative PCR (RTQ-PCR) approaches were applied for PCR-based quantification of Staphylococcus aureus cells by targeting the thermonuclease (nuc) gene. Purified DNA extracts from pure cultures of S. aureus were quantified in a LightCycler system using SYBR Green I. Quantification proved to be less sensitive (60 nuc gene copies/microl) than using a fluorigenic TaqMan probe (6 nuc gene copies/microl). Comparison of the LightCycler system and the well-established ABI Prism 7700 SDS with TaqMan probes revealed no statistically significant differences with respect to sensitivity and reproducibility. Application of the RTQ-PCR assay to quantify S. aureus cells in artificially contaminated cheeses of different types achieved sensitivities from 1.5 x 10(2) to 6.4 x 10(2) copies of the nuc gene/2 g, depending on the cheese matrix. The coefficients of correlation between log CFU and nuc gene copy numbers ranged from 0.979 to 0.998, thus enabling calculation of the number of CFU of S. aureus in cheese by performing RTQ-PCR.</abstract><cop>Washington, DC</cop><pub>American Society for Microbiology</pub><pmid>11425731</pmid><doi>10.1128/AEM.67.7.3122-3126.2001</doi><tpages>5</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Bacteria Benzothiazoles Biological and medical sciences Cells Cheese Cheese - microbiology Colony Count, Microbial Diamines DNA, Bacterial - analysis Fluorescent Dyes Fluorometry Food contamination & poisoning Food industries Food Microbiology Food science Fundamental and applied biological sciences. Psychology Gene Dosage Hot Temperature Micrococcal Nuclease - genetics Milk and cheese industries. Ice creams Organic Chemicals Quinolines Reproducibility of Results Reverse Transcriptase Polymerase Chain Reaction Sensitivity and Specificity Staphylococcus aureus Staphylococcus aureus - genetics Staphylococcus aureus - isolation & purification Taq Polymerase - metabolism thermonuclease (nuc) gene |
| title | Comparison of different approaches to quantify Staphylococcus aureus cells by real-time quantitative PCR and application of this technique for examination of cheese |
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