Molecular identification of Candida isolates by Real‐time PCR‐high‐resolution melting analysis and investigation of the genetic diversity of Candida species
Background Candida species are considered as the cause of one of the most important opportunistic fungal diseases. Accurate identification of Candida species is important because of antifungal susceptibility patterns are different among these species, so proper identification helps in the selection...
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| description | Background
Candida species are considered as the cause of one of the most important opportunistic fungal diseases. Accurate identification of Candida species is important because of antifungal susceptibility patterns are different among these species, so proper identification helps in the selection of antifungal drugs for the prevention and treatment. Phenotypic methods for identification of Candida species, which are widely used in clinical microbiology laboratories, have some limitations. Real‐time PCR followed by the high‐resolution melting analysis (HRMA) is a novel approach for the rapid recognition of pathogenic fungi. Molecular phylogeny is essential for obtaining a better understanding of the evolution of the genus Candida and the identification of the relative degree of the Candida species. The purpose of this study was molecular identification of Candida isolates by Real‐time PCR‐high‐resolution melting analysis and investigation of the genetic diversity of Candida species.
Methods
Two hundred and thirty‐two Candida isolates including 111 Candida isolates obtained from 96 HIV/AIDS patients and 121 Candida isolates obtained from 98 non‐HIV persons were identified by real‐time PCR and high‐resolution melting curve analysis. To evaluate genetic diversity and relationships among Candida species, PCR products of nine clinical Candida isolates, as a representative of each kind of species, were randomly selected for DNA sequence analysis.
Results
In HIV/AIDS patients, six species of Candida spp. were identified as follows: C albicans (n = 64; 57.7%), C glabrata (n = 31; 27.92%), C parapsilosis (n = 9; 8.1%), C tropicalis (n = 4; 3.6%), C krusei (n = 2; 1.8%), and C kefyr (n = 1; 0.90%). In non‐HIV persons, we identified eight species of Candida including C albicans (n = 46; 38.33%) followed by C glabrata and C krusei (each one, n = 18; 15%), C tropicalis (n = 13; 10.83%), C lusitaniae (n = 12; 5.17%), C parapsilosis (n = 10; 4.31%), and C kefyr and C guillermondii (each one, n = 2; 1.66%). Also, the phylogenetic analysis showed the presence of two main clades and six separate subclades. Accordingly, about 88.9% of the isolates were located in clade I and 11.10% of the studied isolates were in clade II.
Conclusions
Real‐time PCR followed by high‐resolution melting analysis (HRMA) is known as a reliable, fast, and simple approach for detection and accurate identification of Candida species, especially in clinical samples.
Two hundred and thirty‐two Candid |
| doi_str_mv | 10.1002/jcla.23444 |
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Candida species are considered as the cause of one of the most important opportunistic fungal diseases. Accurate identification of Candida species is important because of antifungal susceptibility patterns are different among these species, so proper identification helps in the selection of antifungal drugs for the prevention and treatment. Phenotypic methods for identification of Candida species, which are widely used in clinical microbiology laboratories, have some limitations. Real‐time PCR followed by the high‐resolution melting analysis (HRMA) is a novel approach for the rapid recognition of pathogenic fungi. Molecular phylogeny is essential for obtaining a better understanding of the evolution of the genus Candida and the identification of the relative degree of the Candida species. The purpose of this study was molecular identification of Candida isolates by Real‐time PCR‐high‐resolution melting analysis and investigation of the genetic diversity of Candida species.
Methods
Two hundred and thirty‐two Candida isolates including 111 Candida isolates obtained from 96 HIV/AIDS patients and 121 Candida isolates obtained from 98 non‐HIV persons were identified by real‐time PCR and high‐resolution melting curve analysis. To evaluate genetic diversity and relationships among Candida species, PCR products of nine clinical Candida isolates, as a representative of each kind of species, were randomly selected for DNA sequence analysis.
Results
In HIV/AIDS patients, six species of Candida spp. were identified as follows: C albicans (n = 64; 57.7%), C glabrata (n = 31; 27.92%), C parapsilosis (n = 9; 8.1%), C tropicalis (n = 4; 3.6%), C krusei (n = 2; 1.8%), and C kefyr (n = 1; 0.90%). In non‐HIV persons, we identified eight species of Candida including C albicans (n = 46; 38.33%) followed by C glabrata and C krusei (each one, n = 18; 15%), C tropicalis (n = 13; 10.83%), C lusitaniae (n = 12; 5.17%), C parapsilosis (n = 10; 4.31%), and C kefyr and C guillermondii (each one, n = 2; 1.66%). Also, the phylogenetic analysis showed the presence of two main clades and six separate subclades. Accordingly, about 88.9% of the isolates were located in clade I and 11.10% of the studied isolates were in clade II.
Conclusions
Real‐time PCR followed by high‐resolution melting analysis (HRMA) is known as a reliable, fast, and simple approach for detection and accurate identification of Candida species, especially in clinical samples.
Two hundred and thirty‐two Candida isolates including 111 Candida isolates from 96 HIV/AIDS patients and 121 Candida isolates from 98 non‐HIV persons were used in this study. Clinical Candida isolates and eight Candida reference strains were grown on Sabouraud dextrose agar media for 18‐24 hours at 37°C. The DNA of Candida isolates was extracted. Real‐time PCR followed by HRMA was performed on clinical Candida isolates. The mean Tm ± 4 SD and the dMelt curve shape of clinical Candida isolates were compared to the Candida reference strains panel. To evaluate genetic diversity and relationships among Candida species, PCR products of nine clinical Candida isolates, as a representative of any species, were randomly selected for DNA sequence analysis.</description><identifier>ISSN: 0887-8013</identifier><identifier>EISSN: 1098-2825</identifier><identifier>DOI: 10.1002/jcla.23444</identifier><identifier>PMID: 32656934</identifier><language>eng</language><publisher>New York: John Wiley & Sons, Inc</publisher><subject>Acquired immune deficiency syndrome ; AIDS ; Candida ; Candida spp ; Clinical microbiology ; Evolution ; Fungi ; Genetic analysis ; Genetic diversity ; high resolution melting ; HIV ; HIV/AIDS patients ; Human immunodeficiency virus ; Laboratories ; Melting curve ; non‐HIV persons ; Nucleotide sequence ; Patients ; Phylogeny ; Polymerase chain reaction ; Sequence analysis ; Species</subject><ispartof>Journal of clinical laboratory analysis, 2020-10, Vol.34 (10), p.e23444-n/a</ispartof><rights>2020 The Authors. Published by Wiley Periodicals LLC</rights><rights>2020. This work is published under http://creativecommons.org/licenses/by-nc-nd/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c4254-f7f22c6907efe19273c7d738daa4114424a4cda6e9a2b6477f33960d5d5e50a93</citedby><cites>FETCH-LOGICAL-c4254-f7f22c6907efe19273c7d738daa4114424a4cda6e9a2b6477f33960d5d5e50a93</cites><orcidid>0000-0001-7241-5246 ; 0000-0002-9066-1034</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC7595915/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC7595915/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,1411,11541,27901,27902,45550,45551,46027,46451,53766,53768</link.rule.ids></links><search><creatorcontrib>Eghtedar Nejad, Esmaeel</creatorcontrib><creatorcontrib>Ghasemi Nejad Almani, Pooya</creatorcontrib><creatorcontrib>Mohammadi, Mohammad Ali</creatorcontrib><creatorcontrib>Salari, Samira</creatorcontrib><title>Molecular identification of Candida isolates by Real‐time PCR‐high‐resolution melting analysis and investigation of the genetic diversity of Candida species</title><title>Journal of clinical laboratory analysis</title><description>Background
Candida species are considered as the cause of one of the most important opportunistic fungal diseases. Accurate identification of Candida species is important because of antifungal susceptibility patterns are different among these species, so proper identification helps in the selection of antifungal drugs for the prevention and treatment. Phenotypic methods for identification of Candida species, which are widely used in clinical microbiology laboratories, have some limitations. Real‐time PCR followed by the high‐resolution melting analysis (HRMA) is a novel approach for the rapid recognition of pathogenic fungi. Molecular phylogeny is essential for obtaining a better understanding of the evolution of the genus Candida and the identification of the relative degree of the Candida species. The purpose of this study was molecular identification of Candida isolates by Real‐time PCR‐high‐resolution melting analysis and investigation of the genetic diversity of Candida species.
Methods
Two hundred and thirty‐two Candida isolates including 111 Candida isolates obtained from 96 HIV/AIDS patients and 121 Candida isolates obtained from 98 non‐HIV persons were identified by real‐time PCR and high‐resolution melting curve analysis. To evaluate genetic diversity and relationships among Candida species, PCR products of nine clinical Candida isolates, as a representative of each kind of species, were randomly selected for DNA sequence analysis.
Results
In HIV/AIDS patients, six species of Candida spp. were identified as follows: C albicans (n = 64; 57.7%), C glabrata (n = 31; 27.92%), C parapsilosis (n = 9; 8.1%), C tropicalis (n = 4; 3.6%), C krusei (n = 2; 1.8%), and C kefyr (n = 1; 0.90%). In non‐HIV persons, we identified eight species of Candida including C albicans (n = 46; 38.33%) followed by C glabrata and C krusei (each one, n = 18; 15%), C tropicalis (n = 13; 10.83%), C lusitaniae (n = 12; 5.17%), C parapsilosis (n = 10; 4.31%), and C kefyr and C guillermondii (each one, n = 2; 1.66%). Also, the phylogenetic analysis showed the presence of two main clades and six separate subclades. Accordingly, about 88.9% of the isolates were located in clade I and 11.10% of the studied isolates were in clade II.
Conclusions
Real‐time PCR followed by high‐resolution melting analysis (HRMA) is known as a reliable, fast, and simple approach for detection and accurate identification of Candida species, especially in clinical samples.
Two hundred and thirty‐two Candida isolates including 111 Candida isolates from 96 HIV/AIDS patients and 121 Candida isolates from 98 non‐HIV persons were used in this study. Clinical Candida isolates and eight Candida reference strains were grown on Sabouraud dextrose agar media for 18‐24 hours at 37°C. The DNA of Candida isolates was extracted. Real‐time PCR followed by HRMA was performed on clinical Candida isolates. The mean Tm ± 4 SD and the dMelt curve shape of clinical Candida isolates were compared to the Candida reference strains panel. To evaluate genetic diversity and relationships among Candida species, PCR products of nine clinical Candida isolates, as a representative of any species, were randomly selected for DNA sequence analysis.</description><subject>Acquired immune deficiency syndrome</subject><subject>AIDS</subject><subject>Candida</subject><subject>Candida spp</subject><subject>Clinical microbiology</subject><subject>Evolution</subject><subject>Fungi</subject><subject>Genetic analysis</subject><subject>Genetic diversity</subject><subject>high resolution melting</subject><subject>HIV</subject><subject>HIV/AIDS patients</subject><subject>Human immunodeficiency virus</subject><subject>Laboratories</subject><subject>Melting curve</subject><subject>non‐HIV persons</subject><subject>Nucleotide sequence</subject><subject>Patients</subject><subject>Phylogeny</subject><subject>Polymerase chain reaction</subject><subject>Sequence analysis</subject><subject>Species</subject><issn>0887-8013</issn><issn>1098-2825</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2020</creationdate><recordtype>article</recordtype><sourceid>24P</sourceid><sourceid>BENPR</sourceid><recordid>eNp9kc2K1EAQx4Mo7rh68QkavIiQtT-T9EVYwvrFiLLouenprmRq6CRjOhnJzUfwGXw0n8SenWVRD56qoH71r49_lj1l9IJRyl_uXLAXXEgp72UrRnWV84qr-9mKVlWZV5SJs-xRjDtKaaVZ8TA7E7xQhRZylf38MARwc7AjQQ_9hA06O-HQk6Ehte09ekswDsFOEMlmIddgw6_vPybsgHyqr1O6xXabwgiJmm9aOwgT9i2xvQ1LxJgST7A_QJywvVOftkBa6GFCRzweYIw4LX-OjXtwCPFx9qCxIcKT23iefXl99bl-m68_vnlXX65zJ7mSeVM2nLtC0xIaYJqXwpW-FJW3VjImJZdWOm8L0JZvClmWjRC6oF55BYpaLc6zVyfd_bzpwLv0jdEGsx-xs-NiBovm70qPW9MOB1MqrTRTSeD5rcA4fJ3TsabD6CAE28MwR8MlF4opKsqEPvsH3Q3zmN51pJQqZEErnqgXJ8qNQ4wjNHfLMGqO1puj9ebG-gSzE_wNAyz_Ic37en156vkN8nS2IQ</recordid><startdate>202010</startdate><enddate>202010</enddate><creator>Eghtedar Nejad, Esmaeel</creator><creator>Ghasemi Nejad Almani, Pooya</creator><creator>Mohammadi, Mohammad Ali</creator><creator>Salari, Samira</creator><general>John Wiley & Sons, Inc</general><general>John Wiley and Sons Inc</general><scope>24P</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7QP</scope><scope>7T5</scope><scope>7U9</scope><scope>7X7</scope><scope>7XB</scope><scope>8C1</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M7P</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>7X8</scope><scope>5PM</scope><orcidid>https://orcid.org/0000-0001-7241-5246</orcidid><orcidid>https://orcid.org/0000-0002-9066-1034</orcidid></search><sort><creationdate>202010</creationdate><title>Molecular identification of Candida isolates by Real‐time PCR‐high‐resolution melting analysis and investigation of the genetic diversity of Candida species</title><author>Eghtedar Nejad, Esmaeel ; Ghasemi Nejad Almani, Pooya ; Mohammadi, Mohammad Ali ; Salari, Samira</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4254-f7f22c6907efe19273c7d738daa4114424a4cda6e9a2b6477f33960d5d5e50a93</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2020</creationdate><topic>Acquired immune deficiency syndrome</topic><topic>AIDS</topic><topic>Candida</topic><topic>Candida spp</topic><topic>Clinical microbiology</topic><topic>Evolution</topic><topic>Fungi</topic><topic>Genetic analysis</topic><topic>Genetic diversity</topic><topic>high resolution melting</topic><topic>HIV</topic><topic>HIV/AIDS patients</topic><topic>Human immunodeficiency virus</topic><topic>Laboratories</topic><topic>Melting curve</topic><topic>non‐HIV persons</topic><topic>Nucleotide sequence</topic><topic>Patients</topic><topic>Phylogeny</topic><topic>Polymerase chain reaction</topic><topic>Sequence analysis</topic><topic>Species</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Eghtedar Nejad, Esmaeel</creatorcontrib><creatorcontrib>Ghasemi Nejad Almani, Pooya</creatorcontrib><creatorcontrib>Mohammadi, Mohammad Ali</creatorcontrib><creatorcontrib>Salari, Samira</creatorcontrib><collection>Wiley Online Library Open Access</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Immunology Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Public Health Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Biological Science Database</collection><collection>Publicly Available Content Database</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Journal of clinical laboratory analysis</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Eghtedar Nejad, Esmaeel</au><au>Ghasemi Nejad Almani, Pooya</au><au>Mohammadi, Mohammad Ali</au><au>Salari, Samira</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Molecular identification of Candida isolates by Real‐time PCR‐high‐resolution melting analysis and investigation of the genetic diversity of Candida species</atitle><jtitle>Journal of clinical laboratory analysis</jtitle><date>2020-10</date><risdate>2020</risdate><volume>34</volume><issue>10</issue><spage>e23444</spage><epage>n/a</epage><pages>e23444-n/a</pages><issn>0887-8013</issn><eissn>1098-2825</eissn><abstract>Background
Candida species are considered as the cause of one of the most important opportunistic fungal diseases. Accurate identification of Candida species is important because of antifungal susceptibility patterns are different among these species, so proper identification helps in the selection of antifungal drugs for the prevention and treatment. Phenotypic methods for identification of Candida species, which are widely used in clinical microbiology laboratories, have some limitations. Real‐time PCR followed by the high‐resolution melting analysis (HRMA) is a novel approach for the rapid recognition of pathogenic fungi. Molecular phylogeny is essential for obtaining a better understanding of the evolution of the genus Candida and the identification of the relative degree of the Candida species. The purpose of this study was molecular identification of Candida isolates by Real‐time PCR‐high‐resolution melting analysis and investigation of the genetic diversity of Candida species.
Methods
Two hundred and thirty‐two Candida isolates including 111 Candida isolates obtained from 96 HIV/AIDS patients and 121 Candida isolates obtained from 98 non‐HIV persons were identified by real‐time PCR and high‐resolution melting curve analysis. To evaluate genetic diversity and relationships among Candida species, PCR products of nine clinical Candida isolates, as a representative of each kind of species, were randomly selected for DNA sequence analysis.
Results
In HIV/AIDS patients, six species of Candida spp. were identified as follows: C albicans (n = 64; 57.7%), C glabrata (n = 31; 27.92%), C parapsilosis (n = 9; 8.1%), C tropicalis (n = 4; 3.6%), C krusei (n = 2; 1.8%), and C kefyr (n = 1; 0.90%). In non‐HIV persons, we identified eight species of Candida including C albicans (n = 46; 38.33%) followed by C glabrata and C krusei (each one, n = 18; 15%), C tropicalis (n = 13; 10.83%), C lusitaniae (n = 12; 5.17%), C parapsilosis (n = 10; 4.31%), and C kefyr and C guillermondii (each one, n = 2; 1.66%). Also, the phylogenetic analysis showed the presence of two main clades and six separate subclades. Accordingly, about 88.9% of the isolates were located in clade I and 11.10% of the studied isolates were in clade II.
Conclusions
Real‐time PCR followed by high‐resolution melting analysis (HRMA) is known as a reliable, fast, and simple approach for detection and accurate identification of Candida species, especially in clinical samples.
Two hundred and thirty‐two Candida isolates including 111 Candida isolates from 96 HIV/AIDS patients and 121 Candida isolates from 98 non‐HIV persons were used in this study. Clinical Candida isolates and eight Candida reference strains were grown on Sabouraud dextrose agar media for 18‐24 hours at 37°C. The DNA of Candida isolates was extracted. Real‐time PCR followed by HRMA was performed on clinical Candida isolates. The mean Tm ± 4 SD and the dMelt curve shape of clinical Candida isolates were compared to the Candida reference strains panel. To evaluate genetic diversity and relationships among Candida species, PCR products of nine clinical Candida isolates, as a representative of any species, were randomly selected for DNA sequence analysis.</abstract><cop>New York</cop><pub>John Wiley & Sons, Inc</pub><pmid>32656934</pmid><doi>10.1002/jcla.23444</doi><tpages>8</tpages><orcidid>https://orcid.org/0000-0001-7241-5246</orcidid><orcidid>https://orcid.org/0000-0002-9066-1034</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | Acquired immune deficiency syndrome AIDS Candida Candida spp Clinical microbiology Evolution Fungi Genetic analysis Genetic diversity high resolution melting HIV HIV/AIDS patients Human immunodeficiency virus Laboratories Melting curve non‐HIV persons Nucleotide sequence Patients Phylogeny Polymerase chain reaction Sequence analysis Species |
| title | Molecular identification of Candida isolates by Real‐time PCR‐high‐resolution melting analysis and investigation of the genetic diversity of Candida species |
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