High-Resolution Melting Curve Analysis for Identification of Pasteurellaceae Species in Experimental Animal Facilities
Pasteurellaceae are among the most prevalent bacterial pathogens isolated from mice housed in experimental animal facilities. Reliable detection and differentiation of Pasteurellaceae are essential for high-quality health monitoring. In this study, we combined a real-time PCR assay amplifying a vari...
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| Veröffentlicht in: | PloS one 2015-11, Vol.10 (11), p.e0142560-e0142560 |
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| description | Pasteurellaceae are among the most prevalent bacterial pathogens isolated from mice housed in experimental animal facilities. Reliable detection and differentiation of Pasteurellaceae are essential for high-quality health monitoring. In this study, we combined a real-time PCR assay amplifying a variable region in the 16S rRNA sequence with high-resolution melting curve analysis (HRM) to identify and differentiate among the commonly isolated species Pasteurella pneumotropica biotypes "Jawetz" and "Heyl", Actinobacillus muris, and Haemophilus influenzaemurium. We used a set of six reference strains for assay development, with the melting profiles of these strains clearly distinguishable due to DNA sequence variations in the amplicon. For evaluation, we used real-time PCR/HRM to test 25 unknown Pasteurellaceae isolates obtained from an external diagnostic laboratory and found the results to be consistent with those of partial 16S rRNA sequencing. The real-time PCR/HRM method provides a sensitive, rapid, and closed-tube approach for Pasteurellaceae species identification for health monitoring of laboratory mice. |
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Reliable detection and differentiation of Pasteurellaceae are essential for high-quality health monitoring. In this study, we combined a real-time PCR assay amplifying a variable region in the 16S rRNA sequence with high-resolution melting curve analysis (HRM) to identify and differentiate among the commonly isolated species Pasteurella pneumotropica biotypes "Jawetz" and "Heyl", Actinobacillus muris, and Haemophilus influenzaemurium. We used a set of six reference strains for assay development, with the melting profiles of these strains clearly distinguishable due to DNA sequence variations in the amplicon. For evaluation, we used real-time PCR/HRM to test 25 unknown Pasteurellaceae isolates obtained from an external diagnostic laboratory and found the results to be consistent with those of partial 16S rRNA sequencing. The real-time PCR/HRM method provides a sensitive, rapid, and closed-tube approach for Pasteurellaceae species identification for health monitoring of laboratory mice.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0142560</identifier><identifier>PMID: 26556281</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Animal experimentation ; Animal Husbandry ; Animals ; Bacteria ; Biotypes ; Brucella ; Deoxyribonucleic acid ; Diagnostic systems ; DNA ; DNA, Bacterial - genetics ; Gene amplification ; Genes ; Genetic aspects ; High resolution ; Housing, Animal ; Identification ; Laboratory animals ; Listeria ; Listeria monocytogenes ; Melting ; Melting curve ; Methods ; Mice ; Mycoplasma ; Nucleotide sequence ; Pasteurella ; Pasteurellaceae - isolation & purification ; Polymerase chain reaction ; Real time ; Real-Time Polymerase Chain Reaction ; RNA, Ribosomal, 16S - genetics ; Rodent Diseases - microbiology ; rRNA 16S ; Salmonella ; Species ; Staphylococcus aureus ; Staphylococcus infections ; Strains (organisms) ; Variable region</subject><ispartof>PloS one, 2015-11, Vol.10 (11), p.e0142560-e0142560</ispartof><rights>COPYRIGHT 2015 Public Library of Science</rights><rights>2015 Miller et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2015 Miller et al 2015 Miller et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c692t-4d4dbf13011ac463a10f2502a5a48a7825fe1942533a6f98f16ed587379898803</citedby><cites>FETCH-LOGICAL-c692t-4d4dbf13011ac463a10f2502a5a48a7825fe1942533a6f98f16ed587379898803</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/PMC4640598/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4640598/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23845,27901,27902,53766,53768,79342,79343</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/26556281$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Miller, Manuel</creatorcontrib><creatorcontrib>Zorn, Julia</creatorcontrib><creatorcontrib>Brielmeier, Markus</creatorcontrib><title>High-Resolution Melting Curve Analysis for Identification of Pasteurellaceae Species in Experimental Animal Facilities</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Pasteurellaceae are among the most prevalent bacterial pathogens isolated from mice housed in experimental animal facilities. Reliable detection and differentiation of Pasteurellaceae are essential for high-quality health monitoring. In this study, we combined a real-time PCR assay amplifying a variable region in the 16S rRNA sequence with high-resolution melting curve analysis (HRM) to identify and differentiate among the commonly isolated species Pasteurella pneumotropica biotypes "Jawetz" and "Heyl", Actinobacillus muris, and Haemophilus influenzaemurium. We used a set of six reference strains for assay development, with the melting profiles of these strains clearly distinguishable due to DNA sequence variations in the amplicon. For evaluation, we used real-time PCR/HRM to test 25 unknown Pasteurellaceae isolates obtained from an external diagnostic laboratory and found the results to be consistent with those of partial 16S rRNA sequencing. The real-time PCR/HRM method provides a sensitive, rapid, and closed-tube approach for Pasteurellaceae species identification for health monitoring of laboratory mice.</description><subject>Animal experimentation</subject><subject>Animal Husbandry</subject><subject>Animals</subject><subject>Bacteria</subject><subject>Biotypes</subject><subject>Brucella</subject><subject>Deoxyribonucleic acid</subject><subject>Diagnostic systems</subject><subject>DNA</subject><subject>DNA, Bacterial - genetics</subject><subject>Gene amplification</subject><subject>Genes</subject><subject>Genetic aspects</subject><subject>High resolution</subject><subject>Housing, Animal</subject><subject>Identification</subject><subject>Laboratory animals</subject><subject>Listeria</subject><subject>Listeria monocytogenes</subject><subject>Melting</subject><subject>Melting curve</subject><subject>Methods</subject><subject>Mice</subject><subject>Mycoplasma</subject><subject>Nucleotide sequence</subject><subject>Pasteurella</subject><subject>Pasteurellaceae - isolation & purification</subject><subject>Polymerase chain reaction</subject><subject>Real time</subject><subject>Real-Time Polymerase Chain Reaction</subject><subject>RNA, Ribosomal, 16S - genetics</subject><subject>Rodent Diseases - microbiology</subject><subject>rRNA 16S</subject><subject>Salmonella</subject><subject>Species</subject><subject>Staphylococcus aureus</subject><subject>Staphylococcus infections</subject><subject>Strains (organisms)</subject><subject>Variable region</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2015</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><sourceid>DOA</sourceid><recordid>eNqNk11v0zAUhiMEYmPwDxBEQkJw0eKvOPENUlVtrNLQ0AbcWq5z3Hpy42In1fbvcdt0atAukC9s2c95j89rnyx7i9EY0xJ_ufNdaJQbr30DY4QZKTh6lp1iQcmIE0SfH61Pslcx3iFU0Irzl9kJ4UXBSYVPs82lXSxHNxC961rrm_w7uNY2i3zahQ3kk5ThIdqYGx_yWQ1Na43Vakd6k_9QsYUugHNKg4L8dg3aQsxtk5_fryHYVYpQLsnYVZoulLbOtol4nb0wykV4089n2a-L85_Ty9HV9bfZdHI10lyQdsRqVs8NpghjpRmnCiNDCkRUoVilyooUBrBIpVOquBGVwRzqoippKSpRVYieZe_3umvno-wtixKXlFCMK14mYrYnaq_u5DpdWYUH6ZWVuw0fFlKF1moHkgmteS04w3XBCC1VLViZ7EVlMWdzIEnra5-tm6-g1qn4oNxAdHjS2KVc-I1knKFCVEngUy8Q_J8OYitXNuqtvQ34bndvikUaRUI__IM-XV1PLVQqwDbGp7x6KyonjDIsOBdbrfETVBo1rKxO_8vYtD8I-DwISEwL9-1CdTHK2e3N_7PXv4fsxyN2Ccq1y8PPjEOQ7UEdfIwBzKPJGMltexzckNv2kH17pLB3xw_0GHToB_oXLfUJyg</recordid><startdate>20151110</startdate><enddate>20151110</enddate><creator>Miller, Manuel</creator><creator>Zorn, Julia</creator><creator>Brielmeier, Markus</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</general><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>IOV</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope></search><sort><creationdate>20151110</creationdate><title>High-Resolution Melting Curve Analysis for Identification of Pasteurellaceae Species in Experimental Animal Facilities</title><author>Miller, Manuel ; Zorn, Julia ; Brielmeier, Markus</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c692t-4d4dbf13011ac463a10f2502a5a48a7825fe1942533a6f98f16ed587379898803</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2015</creationdate><topic>Animal experimentation</topic><topic>Animal Husbandry</topic><topic>Animals</topic><topic>Bacteria</topic><topic>Biotypes</topic><topic>Brucella</topic><topic>Deoxyribonucleic acid</topic><topic>Diagnostic systems</topic><topic>DNA</topic><topic>DNA, Bacterial - 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Miller, Manuel</au><au>Zorn, Julia</au><au>Brielmeier, Markus</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>High-Resolution Melting Curve Analysis for Identification of Pasteurellaceae Species in Experimental Animal Facilities</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2015-11-10</date><risdate>2015</risdate><volume>10</volume><issue>11</issue><spage>e0142560</spage><epage>e0142560</epage><pages>e0142560-e0142560</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>Pasteurellaceae are among the most prevalent bacterial pathogens isolated from mice housed in experimental animal facilities. Reliable detection and differentiation of Pasteurellaceae are essential for high-quality health monitoring. In this study, we combined a real-time PCR assay amplifying a variable region in the 16S rRNA sequence with high-resolution melting curve analysis (HRM) to identify and differentiate among the commonly isolated species Pasteurella pneumotropica biotypes "Jawetz" and "Heyl", Actinobacillus muris, and Haemophilus influenzaemurium. We used a set of six reference strains for assay development, with the melting profiles of these strains clearly distinguishable due to DNA sequence variations in the amplicon. For evaluation, we used real-time PCR/HRM to test 25 unknown Pasteurellaceae isolates obtained from an external diagnostic laboratory and found the results to be consistent with those of partial 16S rRNA sequencing. The real-time PCR/HRM method provides a sensitive, rapid, and closed-tube approach for Pasteurellaceae species identification for health monitoring of laboratory mice.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>26556281</pmid><doi>10.1371/journal.pone.0142560</doi><oa>free_for_read</oa></addata></record> |
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| subjects | Animal experimentation Animal Husbandry Animals Bacteria Biotypes Brucella Deoxyribonucleic acid Diagnostic systems DNA DNA, Bacterial - genetics Gene amplification Genes Genetic aspects High resolution Housing, Animal Identification Laboratory animals Listeria Listeria monocytogenes Melting Melting curve Methods Mice Mycoplasma Nucleotide sequence Pasteurella Pasteurellaceae - isolation & purification Polymerase chain reaction Real time Real-Time Polymerase Chain Reaction RNA, Ribosomal, 16S - genetics Rodent Diseases - microbiology rRNA 16S Salmonella Species Staphylococcus aureus Staphylococcus infections Strains (organisms) Variable region |
| title | High-Resolution Melting Curve Analysis for Identification of Pasteurellaceae Species in Experimental Animal Facilities |
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