High-Resolution Melting Analysis of the spa Repeat Region of Staphylococcus aureus
The staphylococcal protein A (spa) locus of Staphylococcus aureus contains a complex repeat structure and is commonly used for single-locus sequence-based genotyping. The real-time PCR platform supports genotyping methods that are single step and closed tube and potentially can be carried out simult...
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| Veröffentlicht in: | Clinical chemistry (Baltimore, Md.) Md.), 2008-02, Vol.54 (2), p.432-436 |
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| creator | Stephens, Alex J Inman-Bamber, John Giffard, Philip M Huygens, Flavia |
| description | The staphylococcal protein A (spa) locus of Staphylococcus aureus contains a complex repeat structure and is commonly used for single-locus sequence-based genotyping. The real-time PCR platform supports genotyping methods that are single step and closed tube and potentially can be carried out simultaneously with diagnosis. We describe here a method for genotyping S. aureus using high-resolution melting (HRM) analysis of the spa polymorphic region X.
The conventional PCR spa assay was modified and optimized for the Rotor-Gene 6000 instrument (Corbett Life Science). HRM analysis on the Corbett Rotor-Gene 6000 instrument was used to test 22 known spa sequences obtained from 44 diverse methicillin-resistant S. aureus (MRSA) isolates. Criteria for calling pairs of melting curves "same" or "different" were developed empirically by converting the data to difference graph format with one curve defined as the control. HRM curve comparison between runs was done to determine the portability of the method. The assay performance was assessed by genotyping uncharacterized isolates, carrying out blind trials, and comparing HRM profiles from different runs.
HRM analysis of 44 diverse MRSA isolates generated 20 profiles from 22 spa sequence types. The 2 unresolved HRM spa types differed by only 1 bp. Two blind trials demonstrated complete reproducibility with respect to calling the different spa types. Interrun comparisons of HRM curves were successfully developed, indicating the robustness of the method.
Analysis of the spa locus by HRM resolves spa sequence variants. This single- and closed-tube single-step method for S. aureus genotyping can be easily combined with the interrogation of other genetic markers. |
| doi_str_mv | 10.1373/clinchem.2007.093658 |
| format | Article |
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The conventional PCR spa assay was modified and optimized for the Rotor-Gene 6000 instrument (Corbett Life Science). HRM analysis on the Corbett Rotor-Gene 6000 instrument was used to test 22 known spa sequences obtained from 44 diverse methicillin-resistant S. aureus (MRSA) isolates. Criteria for calling pairs of melting curves "same" or "different" were developed empirically by converting the data to difference graph format with one curve defined as the control. HRM curve comparison between runs was done to determine the portability of the method. The assay performance was assessed by genotyping uncharacterized isolates, carrying out blind trials, and comparing HRM profiles from different runs.
HRM analysis of 44 diverse MRSA isolates generated 20 profiles from 22 spa sequence types. The 2 unresolved HRM spa types differed by only 1 bp. Two blind trials demonstrated complete reproducibility with respect to calling the different spa types. Interrun comparisons of HRM curves were successfully developed, indicating the robustness of the method.
Analysis of the spa locus by HRM resolves spa sequence variants. This single- and closed-tube single-step method for S. aureus genotyping can be easily combined with the interrogation of other genetic markers.</description><identifier>ISSN: 0009-9147</identifier><identifier>EISSN: 1530-8561</identifier><identifier>DOI: 10.1373/clinchem.2007.093658</identifier><identifier>PMID: 18223135</identifier><identifier>CODEN: CLCHAU</identifier><language>eng</language><publisher>Washington, DC: Am Assoc Clin Chem</publisher><subject>Analytical, structural and metabolic biochemistry ; Biological and medical sciences ; Data analysis ; Fluorescence ; Fundamental and applied biological sciences. Psychology ; Genetic markers ; Genotype & phenotype ; Humans ; Investigative techniques, diagnostic techniques (general aspects) ; Life sciences ; Medical sciences ; Melting ; Methicillin Resistance ; Methods ; Polymerase Chain Reaction - methods ; Staphylococcal Protein A - genetics ; Staphylococcal Protein A - isolation & purification ; Staphylococcus aureus ; Staphylococcus aureus - genetics ; Staphylococcus infections ; Studies ; Transition Temperature</subject><ispartof>Clinical chemistry (Baltimore, Md.), 2008-02, Vol.54 (2), p.432-436</ispartof><rights>2008 INIST-CNRS</rights><rights>Copyright American Association for Clinical Chemistry Feb 2008</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c469t-3ab229e93085867c9755c4ccf623e2fc8646a539b9da57f7421ca799d07ee47a3</citedby><cites>FETCH-LOGICAL-c469t-3ab229e93085867c9755c4ccf623e2fc8646a539b9da57f7421ca799d07ee47a3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,27903,27904</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=20053349$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/18223135$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Stephens, Alex J</creatorcontrib><creatorcontrib>Inman-Bamber, John</creatorcontrib><creatorcontrib>Giffard, Philip M</creatorcontrib><creatorcontrib>Huygens, Flavia</creatorcontrib><title>High-Resolution Melting Analysis of the spa Repeat Region of Staphylococcus aureus</title><title>Clinical chemistry (Baltimore, Md.)</title><addtitle>Clin Chem</addtitle><description>The staphylococcal protein A (spa) locus of Staphylococcus aureus contains a complex repeat structure and is commonly used for single-locus sequence-based genotyping. The real-time PCR platform supports genotyping methods that are single step and closed tube and potentially can be carried out simultaneously with diagnosis. We describe here a method for genotyping S. aureus using high-resolution melting (HRM) analysis of the spa polymorphic region X.
The conventional PCR spa assay was modified and optimized for the Rotor-Gene 6000 instrument (Corbett Life Science). HRM analysis on the Corbett Rotor-Gene 6000 instrument was used to test 22 known spa sequences obtained from 44 diverse methicillin-resistant S. aureus (MRSA) isolates. Criteria for calling pairs of melting curves "same" or "different" were developed empirically by converting the data to difference graph format with one curve defined as the control. HRM curve comparison between runs was done to determine the portability of the method. The assay performance was assessed by genotyping uncharacterized isolates, carrying out blind trials, and comparing HRM profiles from different runs.
HRM analysis of 44 diverse MRSA isolates generated 20 profiles from 22 spa sequence types. The 2 unresolved HRM spa types differed by only 1 bp. Two blind trials demonstrated complete reproducibility with respect to calling the different spa types. Interrun comparisons of HRM curves were successfully developed, indicating the robustness of the method.
Analysis of the spa locus by HRM resolves spa sequence variants. This single- and closed-tube single-step method for S. aureus genotyping can be easily combined with the interrogation of other genetic markers.</description><subject>Analytical, structural and metabolic biochemistry</subject><subject>Biological and medical sciences</subject><subject>Data analysis</subject><subject>Fluorescence</subject><subject>Fundamental and applied biological sciences. 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Psychology</topic><topic>Genetic markers</topic><topic>Genotype & phenotype</topic><topic>Humans</topic><topic>Investigative techniques, diagnostic techniques (general aspects)</topic><topic>Life sciences</topic><topic>Medical sciences</topic><topic>Melting</topic><topic>Methicillin Resistance</topic><topic>Methods</topic><topic>Polymerase Chain Reaction - methods</topic><topic>Staphylococcal Protein A - genetics</topic><topic>Staphylococcal Protein A - isolation & purification</topic><topic>Staphylococcus aureus</topic><topic>Staphylococcus aureus - genetics</topic><topic>Staphylococcus infections</topic><topic>Studies</topic><topic>Transition Temperature</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Stephens, Alex J</creatorcontrib><creatorcontrib>Inman-Bamber, John</creatorcontrib><creatorcontrib>Giffard, Philip M</creatorcontrib><creatorcontrib>Huygens, Flavia</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>ProQuest Central (Corporate)</collection><collection>University Readers</collection><collection>Biotechnology Research Abstracts</collection><collection>Nursing & Allied Health Database</collection><collection>Nucleic Acids Abstracts</collection><collection>Toxicology Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Science Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Public Health Database</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Technology Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>Materials Science & Engineering Collection</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest One Sustainability</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>ProQuest Central</collection><collection>Technology Collection</collection><collection>Natural Science Collection</collection><collection>Earth, Atmospheric & Aquatic Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Materials Science Collection</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>Materials Science Database</collection><collection>Nursing & Allied Health Database (Alumni Edition)</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Science Database</collection><collection>Nursing & Allied Health Premium</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Earth, Atmospheric & Aquatic Science Database</collection><collection>Materials Science Collection</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central Basic</collection><collection>Genetics Abstracts</collection><collection>SIRS Editorial</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>MEDLINE - Academic</collection><jtitle>Clinical chemistry (Baltimore, Md.)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Stephens, Alex J</au><au>Inman-Bamber, John</au><au>Giffard, Philip M</au><au>Huygens, Flavia</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>High-Resolution Melting Analysis of the spa Repeat Region of Staphylococcus aureus</atitle><jtitle>Clinical chemistry (Baltimore, Md.)</jtitle><addtitle>Clin Chem</addtitle><date>2008-02-01</date><risdate>2008</risdate><volume>54</volume><issue>2</issue><spage>432</spage><epage>436</epage><pages>432-436</pages><issn>0009-9147</issn><eissn>1530-8561</eissn><coden>CLCHAU</coden><abstract>The staphylococcal protein A (spa) locus of Staphylococcus aureus contains a complex repeat structure and is commonly used for single-locus sequence-based genotyping. The real-time PCR platform supports genotyping methods that are single step and closed tube and potentially can be carried out simultaneously with diagnosis. We describe here a method for genotyping S. aureus using high-resolution melting (HRM) analysis of the spa polymorphic region X.
The conventional PCR spa assay was modified and optimized for the Rotor-Gene 6000 instrument (Corbett Life Science). HRM analysis on the Corbett Rotor-Gene 6000 instrument was used to test 22 known spa sequences obtained from 44 diverse methicillin-resistant S. aureus (MRSA) isolates. Criteria for calling pairs of melting curves "same" or "different" were developed empirically by converting the data to difference graph format with one curve defined as the control. HRM curve comparison between runs was done to determine the portability of the method. The assay performance was assessed by genotyping uncharacterized isolates, carrying out blind trials, and comparing HRM profiles from different runs.
HRM analysis of 44 diverse MRSA isolates generated 20 profiles from 22 spa sequence types. The 2 unresolved HRM spa types differed by only 1 bp. Two blind trials demonstrated complete reproducibility with respect to calling the different spa types. Interrun comparisons of HRM curves were successfully developed, indicating the robustness of the method.
Analysis of the spa locus by HRM resolves spa sequence variants. This single- and closed-tube single-step method for S. aureus genotyping can be easily combined with the interrogation of other genetic markers.</abstract><cop>Washington, DC</cop><pub>Am Assoc Clin Chem</pub><pmid>18223135</pmid><doi>10.1373/clinchem.2007.093658</doi><tpages>5</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Analytical, structural and metabolic biochemistry Biological and medical sciences Data analysis Fluorescence Fundamental and applied biological sciences. Psychology Genetic markers Genotype & phenotype Humans Investigative techniques, diagnostic techniques (general aspects) Life sciences Medical sciences Melting Methicillin Resistance Methods Polymerase Chain Reaction - methods Staphylococcal Protein A - genetics Staphylococcal Protein A - isolation & purification Staphylococcus aureus Staphylococcus aureus - genetics Staphylococcus infections Studies Transition Temperature |
| title | High-Resolution Melting Analysis of the spa Repeat Region of Staphylococcus aureus |
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