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Real-Time PCR Assays Targeting a Unique Chromosomal Sequence of Yersinia pestis
Yersinia pestis, the causative agent of the zoonotic infection plague, is a major concern as a potential bioweapon. Current real-time PCR assays used for Y. pestis detection are based on plasmid targets, some of which may generate false-positive results. Using the yp48 gene of Y. pestis, we designed...
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| Published in: | Clinical chemistry (Baltimore, Md.) Md.), 2005-10, Vol.51 (10), p.1778-1785 |
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| cited_by | cdi_FETCH-LOGICAL-c471t-a210b61b595f549653714459a8f53dcd6d1ec7894df4f81b998aa81ad33661313 |
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| cites | cdi_FETCH-LOGICAL-c471t-a210b61b595f549653714459a8f53dcd6d1ec7894df4f81b998aa81ad33661313 |
| container_end_page | 1785 |
| container_issue | 10 |
| container_start_page | 1778 |
| container_title | Clinical chemistry (Baltimore, Md.) |
| container_volume | 51 |
| creator | Chase, Catherine J Ulrich, Melanie P Wasieloski, Leonard P., Jr Kondig, John P Garrison, Jeffrey Lindler, Luther E Kulesh, David A |
| description | Yersinia pestis, the causative agent of the zoonotic infection plague, is a major concern as a potential bioweapon. Current real-time PCR assays used for Y. pestis detection are based on plasmid targets, some of which may generate false-positive results.
Using the yp48 gene of Y. pestis, we designed and tested 2 real-time TaqMan minor groove binder (MGB) assays that allowed us to use chromosomal genes as both confirmatory and differential targets for Y. pestis. We also designed several additional assays using both Simple-Probe and MGB Eclipse probe technologies for the selective differentiation of Yersinia pseudotuberculosis from Y. pestis. These assays were designed around a 25-bp insertion site recently identified within the yp48 gene of Y. pseudotuberculosis.
The Y. pestis-specific assay distinguished this bacterium from other Yersinia species but had unacceptable low-level detection of Y. pseudotuberculosis, a closely related species. Simple-Probe and MGB Eclipse probes specific for the 25-bp insertion detected only Y. pseudotuberculosis DNA. Probes that spanned the deletion site detected both Y. pestis and Y. pseudotuberculosis DNA, and the 2 species were clearly differentiated by a post-PCR melting temperature (Tm) analysis. The Simple-Probe assay produced an almost 7 degrees C Tm difference and the MGB Eclipse probe a slightly more than 4 degrees C difference.
Our method clearly discriminates Y. pestis DNA from all other Yersinia species tested and from the closely related Y. pseudotuberculosis. These chromosomal assays are important both to verify the presence of Y. pestis based on a chromosomal target and to easily distinguish it from Y. pseudotuberculosis. |
| doi_str_mv | 10.1373/clinchem.2005.051839 |
| format | article |
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The Y. pestis-specific assay distinguished this bacterium from other Yersinia species but had unacceptable low-level detection of Y. pseudotuberculosis, a closely related species. Simple-Probe and MGB Eclipse probes specific for the 25-bp insertion detected only Y. pseudotuberculosis DNA. Probes that spanned the deletion site detected both Y. pestis and Y. pseudotuberculosis DNA, and the 2 species were clearly differentiated by a post-PCR melting temperature (Tm) analysis. The Simple-Probe assay produced an almost 7 degrees C Tm difference and the MGB Eclipse probe a slightly more than 4 degrees C difference.
Our method clearly discriminates Y. pestis DNA from all other Yersinia species tested and from the closely related Y. pseudotuberculosis. These chromosomal assays are important both to verify the presence of Y. pestis based on a chromosomal target and to easily distinguish it from Y. pseudotuberculosis.</description><identifier>ISSN: 0009-9147</identifier><identifier>EISSN: 1530-8561</identifier><identifier>DOI: 10.1373/clinchem.2005.051839</identifier><identifier>PMID: 16099940</identifier><identifier>CODEN: CLCHAU</identifier><language>eng</language><publisher>Washington, DC: Am Assoc Clin Chem</publisher><subject>Analytical, structural and metabolic biochemistry ; Base Sequence ; Biological and medical sciences ; Chromosomes - genetics ; DNA - genetics ; Fundamental and applied biological sciences. Psychology ; Gene Targeting - methods ; Genes ; Genetic testing ; Infections ; Investigative techniques, diagnostic techniques (general aspects) ; Medical sciences ; Molecular Sequence Data ; Plasmids ; Reverse Transcriptase Polymerase Chain Reaction - methods ; Sensitivity and Specificity ; Sequence Analysis, DNA - methods ; Transition Temperature ; Virulence ; Yersinia pestis ; Yersinia pestis - classification ; Yersinia pestis - genetics ; Yersinia pseudotuberculosis ; Yersinia pseudotuberculosis - classification ; Yersinia pseudotuberculosis - genetics</subject><ispartof>Clinical chemistry (Baltimore, Md.), 2005-10, Vol.51 (10), p.1778-1785</ispartof><rights>2005 INIST-CNRS</rights><rights>Copyright American Association for Clinical Chemistry Oct 2005</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c471t-a210b61b595f549653714459a8f53dcd6d1ec7894df4f81b998aa81ad33661313</citedby><cites>FETCH-LOGICAL-c471t-a210b61b595f549653714459a8f53dcd6d1ec7894df4f81b998aa81ad33661313</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>315,786,790,27957,27958</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=17144460$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/16099940$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Chase, Catherine J</creatorcontrib><creatorcontrib>Ulrich, Melanie P</creatorcontrib><creatorcontrib>Wasieloski, Leonard P., Jr</creatorcontrib><creatorcontrib>Kondig, John P</creatorcontrib><creatorcontrib>Garrison, Jeffrey</creatorcontrib><creatorcontrib>Lindler, Luther E</creatorcontrib><creatorcontrib>Kulesh, David A</creatorcontrib><title>Real-Time PCR Assays Targeting a Unique Chromosomal Sequence of Yersinia pestis</title><title>Clinical chemistry (Baltimore, Md.)</title><addtitle>Clin Chem</addtitle><description>Yersinia pestis, the causative agent of the zoonotic infection plague, is a major concern as a potential bioweapon. Current real-time PCR assays used for Y. pestis detection are based on plasmid targets, some of which may generate false-positive results.
Using the yp48 gene of Y. pestis, we designed and tested 2 real-time TaqMan minor groove binder (MGB) assays that allowed us to use chromosomal genes as both confirmatory and differential targets for Y. pestis. We also designed several additional assays using both Simple-Probe and MGB Eclipse probe technologies for the selective differentiation of Yersinia pseudotuberculosis from Y. pestis. These assays were designed around a 25-bp insertion site recently identified within the yp48 gene of Y. pseudotuberculosis.
The Y. pestis-specific assay distinguished this bacterium from other Yersinia species but had unacceptable low-level detection of Y. pseudotuberculosis, a closely related species. Simple-Probe and MGB Eclipse probes specific for the 25-bp insertion detected only Y. pseudotuberculosis DNA. Probes that spanned the deletion site detected both Y. pestis and Y. pseudotuberculosis DNA, and the 2 species were clearly differentiated by a post-PCR melting temperature (Tm) analysis. The Simple-Probe assay produced an almost 7 degrees C Tm difference and the MGB Eclipse probe a slightly more than 4 degrees C difference.
Our method clearly discriminates Y. pestis DNA from all other Yersinia species tested and from the closely related Y. pseudotuberculosis. These chromosomal assays are important both to verify the presence of Y. pestis based on a chromosomal target and to easily distinguish it from Y. pseudotuberculosis.</description><subject>Analytical, structural and metabolic biochemistry</subject><subject>Base Sequence</subject><subject>Biological and medical sciences</subject><subject>Chromosomes - genetics</subject><subject>DNA - genetics</subject><subject>Fundamental and applied biological sciences. 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Academic</collection><jtitle>Clinical chemistry (Baltimore, Md.)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Chase, Catherine J</au><au>Ulrich, Melanie P</au><au>Wasieloski, Leonard P., Jr</au><au>Kondig, John P</au><au>Garrison, Jeffrey</au><au>Lindler, Luther E</au><au>Kulesh, David A</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Real-Time PCR Assays Targeting a Unique Chromosomal Sequence of Yersinia pestis</atitle><jtitle>Clinical chemistry (Baltimore, Md.)</jtitle><addtitle>Clin Chem</addtitle><date>2005-10-01</date><risdate>2005</risdate><volume>51</volume><issue>10</issue><spage>1778</spage><epage>1785</epage><pages>1778-1785</pages><issn>0009-9147</issn><eissn>1530-8561</eissn><coden>CLCHAU</coden><notes>ObjectType-Article-1</notes><notes>SourceType-Scholarly Journals-1</notes><notes>ObjectType-Feature-2</notes><notes>content type line 23</notes><abstract>Yersinia pestis, the causative agent of the zoonotic infection plague, is a major concern as a potential bioweapon. Current real-time PCR assays used for Y. pestis detection are based on plasmid targets, some of which may generate false-positive results.
Using the yp48 gene of Y. pestis, we designed and tested 2 real-time TaqMan minor groove binder (MGB) assays that allowed us to use chromosomal genes as both confirmatory and differential targets for Y. pestis. We also designed several additional assays using both Simple-Probe and MGB Eclipse probe technologies for the selective differentiation of Yersinia pseudotuberculosis from Y. pestis. These assays were designed around a 25-bp insertion site recently identified within the yp48 gene of Y. pseudotuberculosis.
The Y. pestis-specific assay distinguished this bacterium from other Yersinia species but had unacceptable low-level detection of Y. pseudotuberculosis, a closely related species. Simple-Probe and MGB Eclipse probes specific for the 25-bp insertion detected only Y. pseudotuberculosis DNA. Probes that spanned the deletion site detected both Y. pestis and Y. pseudotuberculosis DNA, and the 2 species were clearly differentiated by a post-PCR melting temperature (Tm) analysis. The Simple-Probe assay produced an almost 7 degrees C Tm difference and the MGB Eclipse probe a slightly more than 4 degrees C difference.
Our method clearly discriminates Y. pestis DNA from all other Yersinia species tested and from the closely related Y. pseudotuberculosis. These chromosomal assays are important both to verify the presence of Y. pestis based on a chromosomal target and to easily distinguish it from Y. pseudotuberculosis.</abstract><cop>Washington, DC</cop><pub>Am Assoc Clin Chem</pub><pmid>16099940</pmid><doi>10.1373/clinchem.2005.051839</doi><tpages>8</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | Clinical chemistry (Baltimore, Md.), 2005-10, Vol.51 (10), p.1778-1785 |
| issn | 0009-9147 1530-8561 |
| language | eng |
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| source | Oxford Academic Journals (OUP) |
| subjects | Analytical, structural and metabolic biochemistry Base Sequence Biological and medical sciences Chromosomes - genetics DNA - genetics Fundamental and applied biological sciences. Psychology Gene Targeting - methods Genes Genetic testing Infections Investigative techniques, diagnostic techniques (general aspects) Medical sciences Molecular Sequence Data Plasmids Reverse Transcriptase Polymerase Chain Reaction - methods Sensitivity and Specificity Sequence Analysis, DNA - methods Transition Temperature Virulence Yersinia pestis Yersinia pestis - classification Yersinia pestis - genetics Yersinia pseudotuberculosis Yersinia pseudotuberculosis - classification Yersinia pseudotuberculosis - genetics |
| title | Real-Time PCR Assays Targeting a Unique Chromosomal Sequence of Yersinia pestis |
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