Phylogenetic analysis and rapid identification of Candida dubliniensis based on analysis of ACT1 intron and exon sequences
Department of Oral Medicine and Oral Pathology, School of Dental Science, University of Dublin, Trinity College, Dublin 2, Republic of Ireland Author for correspondence: Derek J. Sullivan. Tel: + 353 1 6126275. Fax: +353 1 6711255. e-mail: djsullvn@dental.ted.ie ABSTRACT The phylogenetic position of...
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| Veröffentlicht in: | Microbiology (Society for General Microbiology) 1999-08, Vol.145 (8), p.1871-1882 |
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| creator | Donnelly, Samantha M Sullivan, Derek J Shanley, Diarmuid B Coleman, David C |
| description | Department of Oral Medicine and Oral Pathology, School of Dental Science, University of Dublin, Trinity College, Dublin 2, Republic of Ireland
Author for correspondence: Derek J. Sullivan. Tel: + 353 1 6126275. Fax: +353 1 6711255. e-mail: djsullvn@dental.ted.ie
ABSTRACT
The phylogenetic position of Candida dubliniensis has previously been established on the basis of the sequence of rRNA genes. In order to confirm the relationship between C. dubliniensis and other yeast species, particularly Candida albicans , using non-rRNA gene sequences the ACT1 gene was chosen for analysis. Three overlapping fragments that together span the entire C. dubliniensis ACT1 gene ( CdACT1 ) were amplified from a recombinant phage isolated from a genomic DNA library using PCR. These were cloned and used to determine the contiguous sequence of the gene. Analysis of the sequence data revealed the presence of a 1131 bp ORF interrupted by a single 632 bp intron at the 5' extremity of the gene. Comparison of the CdACT1 sequence with the C. albicans homologue ( CaACT1 ) revealed that although the exons are 97.9% identical the introns are only 83.4% identical. Phylogenetic trees generated using ACT1 exon and intron sequences from a range of yeast species unequivocally confirmed the phylogenetic position of C. dubliniensis as a unique taxon within the genus Candida. Analysis of the ACT1 -associated intron sequences from 10 epidemiologically unrelated C. dubliniensis isolates from disparate geographical locations showed a very low level of intraspecies sequence variation. In order to develop an accurate and rapid method to identify C. dubliniensis from primary isolation plates the significant divergence between the C. dubliniensis and C. albicans ACT1 intron sequences was exploited by designing C. dubliniensis -specific PCR primers. Using a rapid boiling method to produce template DNA directly from colonies from primary isolation plates in 10 min, these primers were used in a blind test with 122 isolates of C. dubliniensis , 53 isolates of C. albicans , 10 isolates of C. stellatoidea and representative isolates of other clinically relevant Candida and other yeast species. Only the C. dubliniensis isolates yielded the C. dubliniensis -specific 288 bp amplimer. Use of this technique on colonies suspected to be C. dubliniensis allows their correct identification as C. dubliniensis in as little as 4 h.
Keywords: ACT1 , phylogenetics, Candida dubliniensis , PCR identification
The EMBL acces |
| doi_str_mv | 10.1099/13500872-145-8-1871 |
| format | Article |
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Author for correspondence: Derek J. Sullivan. Tel: + 353 1 6126275. Fax: +353 1 6711255. e-mail: djsullvn@dental.ted.ie
ABSTRACT
The phylogenetic position of Candida dubliniensis has previously been established on the basis of the sequence of rRNA genes. In order to confirm the relationship between C. dubliniensis and other yeast species, particularly Candida albicans , using non-rRNA gene sequences the ACT1 gene was chosen for analysis. Three overlapping fragments that together span the entire C. dubliniensis ACT1 gene ( CdACT1 ) were amplified from a recombinant phage isolated from a genomic DNA library using PCR. These were cloned and used to determine the contiguous sequence of the gene. Analysis of the sequence data revealed the presence of a 1131 bp ORF interrupted by a single 632 bp intron at the 5' extremity of the gene. Comparison of the CdACT1 sequence with the C. albicans homologue ( CaACT1 ) revealed that although the exons are 97.9% identical the introns are only 83.4% identical. Phylogenetic trees generated using ACT1 exon and intron sequences from a range of yeast species unequivocally confirmed the phylogenetic position of C. dubliniensis as a unique taxon within the genus Candida. Analysis of the ACT1 -associated intron sequences from 10 epidemiologically unrelated C. dubliniensis isolates from disparate geographical locations showed a very low level of intraspecies sequence variation. In order to develop an accurate and rapid method to identify C. dubliniensis from primary isolation plates the significant divergence between the C. dubliniensis and C. albicans ACT1 intron sequences was exploited by designing C. dubliniensis -specific PCR primers. Using a rapid boiling method to produce template DNA directly from colonies from primary isolation plates in 10 min, these primers were used in a blind test with 122 isolates of C. dubliniensis , 53 isolates of C. albicans , 10 isolates of C. stellatoidea and representative isolates of other clinically relevant Candida and other yeast species. Only the C. dubliniensis isolates yielded the C. dubliniensis -specific 288 bp amplimer. Use of this technique on colonies suspected to be C. dubliniensis allows their correct identification as C. dubliniensis in as little as 4 h.
Keywords: ACT1 , phylogenetics, Candida dubliniensis , PCR identification
The EMBL accession numbers for the nucleotide sequences reported in this paper are AJ236897 ( Candida dubliniensis CD36), AJ237918 ( Candida tropicalis NCPF 3111) and AJ237919 ( Candida stellatoidea ATCC 11006).
Abbreviations: HIV, human immunodeficiency virus.</description><identifier>ISSN: 1350-0872</identifier><identifier>EISSN: 1465-2080</identifier><identifier>DOI: 10.1099/13500872-145-8-1871</identifier><identifier>PMID: 10463153</identifier><language>eng</language><publisher>Reading: Soc General Microbiol</publisher><subject>Actins - genetics ; Base Sequence ; Biological and medical sciences ; Candida - classification ; Candida - genetics ; Candida dubliniensis ; DNA Primers ; DNA, Fungal - genetics ; Exons - genetics ; Fundamental and applied biological sciences. Psychology ; Fungal Proteins - genetics ; Genes, Fungal - genetics ; Growth, nutrition, metabolism, transports, enzymes. Molecular biology ; Humans ; Introns - genetics ; Microbiology ; Molecular Sequence Data ; Mycology ; Phylogeny ; Polymerase Chain Reaction - methods ; Species Specificity</subject><ispartof>Microbiology (Society for General Microbiology), 1999-08, Vol.145 (8), p.1871-1882</ispartof><rights>1999 INIST-CNRS</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c440t-b781f76f12da8290393885b30e8263d5ddc535bc2ce577ade0b5a207ad709d003</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,27901,27902</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=1912152$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/10463153$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Donnelly, Samantha M</creatorcontrib><creatorcontrib>Sullivan, Derek J</creatorcontrib><creatorcontrib>Shanley, Diarmuid B</creatorcontrib><creatorcontrib>Coleman, David C</creatorcontrib><title>Phylogenetic analysis and rapid identification of Candida dubliniensis based on analysis of ACT1 intron and exon sequences</title><title>Microbiology (Society for General Microbiology)</title><addtitle>Microbiology</addtitle><description>Department of Oral Medicine and Oral Pathology, School of Dental Science, University of Dublin, Trinity College, Dublin 2, Republic of Ireland
Author for correspondence: Derek J. Sullivan. Tel: + 353 1 6126275. Fax: +353 1 6711255. e-mail: djsullvn@dental.ted.ie
ABSTRACT
The phylogenetic position of Candida dubliniensis has previously been established on the basis of the sequence of rRNA genes. In order to confirm the relationship between C. dubliniensis and other yeast species, particularly Candida albicans , using non-rRNA gene sequences the ACT1 gene was chosen for analysis. Three overlapping fragments that together span the entire C. dubliniensis ACT1 gene ( CdACT1 ) were amplified from a recombinant phage isolated from a genomic DNA library using PCR. These were cloned and used to determine the contiguous sequence of the gene. Analysis of the sequence data revealed the presence of a 1131 bp ORF interrupted by a single 632 bp intron at the 5' extremity of the gene. Comparison of the CdACT1 sequence with the C. albicans homologue ( CaACT1 ) revealed that although the exons are 97.9% identical the introns are only 83.4% identical. Phylogenetic trees generated using ACT1 exon and intron sequences from a range of yeast species unequivocally confirmed the phylogenetic position of C. dubliniensis as a unique taxon within the genus Candida. Analysis of the ACT1 -associated intron sequences from 10 epidemiologically unrelated C. dubliniensis isolates from disparate geographical locations showed a very low level of intraspecies sequence variation. In order to develop an accurate and rapid method to identify C. dubliniensis from primary isolation plates the significant divergence between the C. dubliniensis and C. albicans ACT1 intron sequences was exploited by designing C. dubliniensis -specific PCR primers. Using a rapid boiling method to produce template DNA directly from colonies from primary isolation plates in 10 min, these primers were used in a blind test with 122 isolates of C. dubliniensis , 53 isolates of C. albicans , 10 isolates of C. stellatoidea and representative isolates of other clinically relevant Candida and other yeast species. Only the C. dubliniensis isolates yielded the C. dubliniensis -specific 288 bp amplimer. Use of this technique on colonies suspected to be C. dubliniensis allows their correct identification as C. dubliniensis in as little as 4 h.
Keywords: ACT1 , phylogenetics, Candida dubliniensis , PCR identification
The EMBL accession numbers for the nucleotide sequences reported in this paper are AJ236897 ( Candida dubliniensis CD36), AJ237918 ( Candida tropicalis NCPF 3111) and AJ237919 ( Candida stellatoidea ATCC 11006).
Abbreviations: HIV, human immunodeficiency virus.</description><subject>Actins - genetics</subject><subject>Base Sequence</subject><subject>Biological and medical sciences</subject><subject>Candida - classification</subject><subject>Candida - genetics</subject><subject>Candida dubliniensis</subject><subject>DNA Primers</subject><subject>DNA, Fungal - genetics</subject><subject>Exons - genetics</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Fungal Proteins - genetics</subject><subject>Genes, Fungal - genetics</subject><subject>Growth, nutrition, metabolism, transports, enzymes. Molecular biology</subject><subject>Humans</subject><subject>Introns - genetics</subject><subject>Microbiology</subject><subject>Molecular Sequence Data</subject><subject>Mycology</subject><subject>Phylogeny</subject><subject>Polymerase Chain Reaction - methods</subject><subject>Species Specificity</subject><issn>1350-0872</issn><issn>1465-2080</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1999</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkU-LFDEQxYMo7jr6CQTJQQQPrVVJpzs5LoP_YEEP6zmkk_ROpCc9Jj2s46e3xhl1b-aQPFK_V1XwGHuO8AbBmLcoFYDuRYOtanSDuscH7BLbTjUCNDwkTURzRC7Yk1q_AVAR8DG7QGg7iUpesp9fNodpvo05Lslzl910qKmSCLy4XQo8hZiXNCbvljRnPo98TcUUHA_7YUo5xXw0DK7GwAn424LIq_UN8pSX8vs_8PiDRI3f9zH7WJ-yR6Obanx2flfs6_t3N-uPzfXnD5_WV9eNb1tYmqHXOPbdiCI4LQxII7VWg4SoRSeDCsErqQYvfFR970KEQTkBpHowAUCu2KtT312ZaXRd7DZVH6fJ5Tjvq-2MMWAA_wtiL-nQvWLyBPoy11riaHclbV05WAR7zMb-ycZSNlbbYzbkenFuvx-2MdzznMIg4OUZcNW7aSwu-1T_cQYFKkHY6xO2Sbebu1Sipfi2iXYZ0kwr-3szfwHcH6Vk</recordid><startdate>19990801</startdate><enddate>19990801</enddate><creator>Donnelly, Samantha M</creator><creator>Sullivan, Derek J</creator><creator>Shanley, Diarmuid B</creator><creator>Coleman, David C</creator><general>Soc General Microbiol</general><general>Society for General 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>M7N</scope><scope>7X8</scope></search><sort><creationdate>19990801</creationdate><title>Phylogenetic analysis and rapid identification of Candida dubliniensis based on analysis of ACT1 intron and exon sequences</title><author>Donnelly, Samantha M ; Sullivan, Derek J ; Shanley, Diarmuid B ; Coleman, David C</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c440t-b781f76f12da8290393885b30e8263d5ddc535bc2ce577ade0b5a207ad709d003</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1999</creationdate><topic>Actins - genetics</topic><topic>Base Sequence</topic><topic>Biological and medical sciences</topic><topic>Candida - classification</topic><topic>Candida - genetics</topic><topic>Candida dubliniensis</topic><topic>DNA Primers</topic><topic>DNA, Fungal - genetics</topic><topic>Exons - genetics</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Fungal Proteins - genetics</topic><topic>Genes, Fungal - genetics</topic><topic>Growth, nutrition, metabolism, transports, enzymes. Molecular biology</topic><topic>Humans</topic><topic>Introns - genetics</topic><topic>Microbiology</topic><topic>Molecular Sequence Data</topic><topic>Mycology</topic><topic>Phylogeny</topic><topic>Polymerase Chain Reaction - methods</topic><topic>Species Specificity</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Donnelly, Samantha M</creatorcontrib><creatorcontrib>Sullivan, Derek J</creatorcontrib><creatorcontrib>Shanley, Diarmuid B</creatorcontrib><creatorcontrib>Coleman, David C</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>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>MEDLINE - Academic</collection><jtitle>Microbiology (Society for General Microbiology)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Donnelly, Samantha M</au><au>Sullivan, Derek J</au><au>Shanley, Diarmuid B</au><au>Coleman, David C</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Phylogenetic analysis and rapid identification of Candida dubliniensis based on analysis of ACT1 intron and exon sequences</atitle><jtitle>Microbiology (Society for General Microbiology)</jtitle><addtitle>Microbiology</addtitle><date>1999-08-01</date><risdate>1999</risdate><volume>145</volume><issue>8</issue><spage>1871</spage><epage>1882</epage><pages>1871-1882</pages><issn>1350-0872</issn><eissn>1465-2080</eissn><abstract>Department of Oral Medicine and Oral Pathology, School of Dental Science, University of Dublin, Trinity College, Dublin 2, Republic of Ireland
Author for correspondence: Derek J. Sullivan. Tel: + 353 1 6126275. Fax: +353 1 6711255. e-mail: djsullvn@dental.ted.ie
ABSTRACT
The phylogenetic position of Candida dubliniensis has previously been established on the basis of the sequence of rRNA genes. In order to confirm the relationship between C. dubliniensis and other yeast species, particularly Candida albicans , using non-rRNA gene sequences the ACT1 gene was chosen for analysis. Three overlapping fragments that together span the entire C. dubliniensis ACT1 gene ( CdACT1 ) were amplified from a recombinant phage isolated from a genomic DNA library using PCR. These were cloned and used to determine the contiguous sequence of the gene. Analysis of the sequence data revealed the presence of a 1131 bp ORF interrupted by a single 632 bp intron at the 5' extremity of the gene. Comparison of the CdACT1 sequence with the C. albicans homologue ( CaACT1 ) revealed that although the exons are 97.9% identical the introns are only 83.4% identical. Phylogenetic trees generated using ACT1 exon and intron sequences from a range of yeast species unequivocally confirmed the phylogenetic position of C. dubliniensis as a unique taxon within the genus Candida. Analysis of the ACT1 -associated intron sequences from 10 epidemiologically unrelated C. dubliniensis isolates from disparate geographical locations showed a very low level of intraspecies sequence variation. In order to develop an accurate and rapid method to identify C. dubliniensis from primary isolation plates the significant divergence between the C. dubliniensis and C. albicans ACT1 intron sequences was exploited by designing C. dubliniensis -specific PCR primers. Using a rapid boiling method to produce template DNA directly from colonies from primary isolation plates in 10 min, these primers were used in a blind test with 122 isolates of C. dubliniensis , 53 isolates of C. albicans , 10 isolates of C. stellatoidea and representative isolates of other clinically relevant Candida and other yeast species. Only the C. dubliniensis isolates yielded the C. dubliniensis -specific 288 bp amplimer. Use of this technique on colonies suspected to be C. dubliniensis allows their correct identification as C. dubliniensis in as little as 4 h.
Keywords: ACT1 , phylogenetics, Candida dubliniensis , PCR identification
The EMBL accession numbers for the nucleotide sequences reported in this paper are AJ236897 ( Candida dubliniensis CD36), AJ237918 ( Candida tropicalis NCPF 3111) and AJ237919 ( Candida stellatoidea ATCC 11006).
Abbreviations: HIV, human immunodeficiency virus.</abstract><cop>Reading</cop><pub>Soc General Microbiol</pub><pmid>10463153</pmid><doi>10.1099/13500872-145-8-1871</doi><tpages>12</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Actins - genetics Base Sequence Biological and medical sciences Candida - classification Candida - genetics Candida dubliniensis DNA Primers DNA, Fungal - genetics Exons - genetics Fundamental and applied biological sciences. Psychology Fungal Proteins - genetics Genes, Fungal - genetics Growth, nutrition, metabolism, transports, enzymes. Molecular biology Humans Introns - genetics Microbiology Molecular Sequence Data Mycology Phylogeny Polymerase Chain Reaction - methods Species Specificity |
| title | Phylogenetic analysis and rapid identification of Candida dubliniensis based on analysis of ACT1 intron and exon sequences |
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