ITS1 Copy Number Varies among Batrachochytrium dendrobatidis Strains: Implications for qPCR Estimates of Infection Intensity from Field-Collected Amphibian Skin Swabs
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{"title"=>"ITS1 Copy Number Varies among Batrachochytrium dendrobatidis Strains: Implications for qPCR Estimates of Infection Intensity from Field-Collected Amphibian Skin Swabs", "type"=>"journal", "authors"=>[{"first_name"=>"Ana V.", "last_name"=>"Longo", "scopus_author_id"=>"22953960000"}, {"first_name"=>"David", "last_name"=>"Rodriguez", "scopus_author_id"=>"56984124400"}, {"first_name"=>"Domingos", "last_name"=>"da Silva Leite", "scopus_author_id"=>"6506592589"}, {"first_name"=>"Luís Felipe", "last_name"=>"Toledo", "scopus_author_id"=>"7003557544"}, {"first_name"=>"Cinthya", "last_name"=>"Mendoza Almeralla", "scopus_author_id"=>"55630561700"}, {"first_name"=>"Patricia A.", "last_name"=>"Burrowes", "scopus_author_id"=>"8633014600"}, {"first_name"=>"Kelly R.", "last_name"=>"Zamudio", "scopus_author_id"=>"6603683897"}], "year"=>2013, "source"=>"PLoS ONE", "identifiers"=>{"issn"=>"19326203", "pui"=>"368580172", "doi"=>"10.1371/journal.pone.0059499", "sgr"=>"84875284656", "scopus"=>"2-s2.0-84875284656", "isbn"=>"1932-6203 (Electronic)\\n1932-6203 (Linking)", "pmid"=>"23555682"}, "id"=>"97bf1435-a2ae-3762-9c14-0552d55240dc", "abstract"=>"Genomic studies of the amphibian-killing fungus (Batrachochytrium dendrobatidis, [Bd]) identified three highly divergent genetic lineages, only one of which has a global distribution. Bd strains within these linages show variable genomic content due to differential loss of heterozygosity and recombination. The current quantitative polymerase chain reaction (qPCR) protocol to detect the fungus from amphibian skin swabs targets the intergenic transcribed spacer 1 (ITS1) region using a TaqMan fluorescent probe specific to Bd. We investigated the consequences of genomic differences in the quantification of ITS1 from eight distinct Bd strains, including representatives from North America, South America, the Caribbean, and Australia. To test for potential differences in amplification, we compared qPCR standards made from Bd zoospore counts for each strain, and showed that they differ significantly in amplification rates. To test potential mechanisms leading to strain differences in qPCR reaction parameters (slope and y-intercept), we: a) compared standard curves from the same strains made from extracted Bd genomic DNA in equimolar solutions, b) quantified the number of ITS1 copies per zoospore using a standard curve made from PCR-amplicons of the ITS1 region, and c) cloned and sequenced PCR-amplified ITS1 regions from these same strains to verify the presence of the probe site in all haplotypes. We found high strain variability in ITS1 copy number, ranging from 10 to 144 copies per single zoospore. Our results indicate that genome size might explain strain differences in ITS1 copy number, but not ITS1 sequence variation because the probe-binding site and primers were conserved across all haplotypes. For standards constructed from uncharacterized Bd strains, we recommend the use of single ITS1 PCR-amplicons as the absolute standard in conjunction with current quantitative assays to inform on copy number variation and provide universal estimates of pathogen zoospore loads from field-caught amphibians.", "link"=>"http://www.mendeley.com/research/its1-copy-number-varies-among-batrachochytrium-dendrobatidis-strains-implications-qpcr-estimates-inf", "reader_count"=>111, "reader_count_by_academic_status"=>{"Unspecified"=>2, "Professor > Associate Professor"=>4, "Student > Doctoral Student"=>6, "Researcher"=>24, "Student > Ph. D. Student"=>22, "Student > Postgraduate"=>4, "Student > Master"=>28, "Other"=>5, "Student > Bachelor"=>14, "Professor"=>2}, "reader_count_by_user_role"=>{"Unspecified"=>2, "Professor > Associate Professor"=>4, "Student > Doctoral Student"=>6, "Researcher"=>24, "Student > Ph. D. Student"=>22, "Student > Postgraduate"=>4, "Student > Master"=>28, "Other"=>5, "Student > Bachelor"=>14, "Professor"=>2}, "reader_count_by_subject_area"=>{"Engineering"=>1, "Unspecified"=>2, "Environmental Science"=>9, "Biochemistry, Genetics and Molecular Biology"=>9, "Agricultural and Biological Sciences"=>82, "Medicine and Dentistry"=>3, "Veterinary Science and Veterinary Medicine"=>1, "Computer Science"=>1, "Immunology and Microbiology"=>2, "Earth and Planetary Sciences"=>1}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>1}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>3}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>2}, "Earth and Planetary Sciences"=>{"Earth and Planetary Sciences"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>82}, "Computer Science"=>{"Computer Science"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>9}, "Unspecified"=>{"Unspecified"=>2}, "Environmental Science"=>{"Environmental Science"=>9}, "Veterinary Science and Veterinary Medicine"=>{"Veterinary Science and Veterinary Medicine"=>1}}, "reader_count_by_country"=>{"Cambodia"=>1, "Hungary"=>1, "United States"=>8, "Brazil"=>1}, "group_count"=>7}

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  • {"files"=>["https://ndownloader.figshare.com/files/994476"], "description"=>"<p>Standard curve regression lines for multiple isolates of <i>Batrachochytrium dendrobatidis</i> based on zoospore counts (Standard Set A).</p>", "links"=>[], "tags"=>["regression", "lines", "isolates", "zoospore", "counts"], "article_id"=>657596, "categories"=>["Microbiology", "Genetics", "Biotechnology", "Plant Biology"], "users"=>["Ana V. Longo", "David Rodriguez", "Domingos da Silva Leite", "Luís Felipe Toledo", "Cinthya Mendoza Almeralla", "Patricia A. Burrowes", "Kelly R. Zamudio"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0059499.g002", "stats"=>{"downloads"=>1, "page_views"=>33, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Standard_curve_regression_lines_for_multiple_isolates_of_Batrachochytrium_dendrobatidis_based_on_zoospore_counts_Standard_Set_A_/657596", "title"=>"Standard curve regression lines for multiple isolates of <i>Batrachochytrium dendrobatidis</i> based on zoospore counts (Standard Set A).", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-03-22 07:42:18"}
  • {"files"=>["https://ndownloader.figshare.com/files/1007934"], "description"=>"<p>Standard curve coefficients (slopes and intercepts) were estimated from the linear regression equation between cycle threshold (Ct) and log-transformed zoospore counts. Average (± SD) DNA concentration of extracts is based on 10<sup>7</sup> zoospores in 200 µL extraction buffer.</p>*<p>10<sup>6</sup> zoospores.</p>", "links"=>[], "tags"=>["efficiencies", "dna", "concentrations"], "article_id"=>668554, "categories"=>["Microbiology", "Genetics", "Biotechnology", "Plant Biology"], "users"=>["Ana V. Longo", "David Rodriguez", "Domingos da Silva Leite", "Luís Felipe Toledo", "Cinthya Mendoza Almeralla", "Patricia A. Burrowes", "Kelly R. Zamudio"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0059499.t001", "stats"=>{"downloads"=>2, "page_views"=>19, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Standard_curve_equations_reaction_efficiencies_and_starting_DNA_concentrations_of_eight_Batrachochytrium_dendrobatidis_strains_/668554", "title"=>"Standard curve equations, reaction efficiencies and starting DNA concentrations of eight <i>Batrachochytrium dendrobatidis</i> strains.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-03-21 02:22:34"}
  • {"files"=>["https://ndownloader.figshare.com/files/994489", "https://ndownloader.figshare.com/files/994493", "https://ndownloader.figshare.com/files/994495", "https://ndownloader.figshare.com/files/994497", "https://ndownloader.figshare.com/files/994499"], "description"=>"<div><p>Genomic studies of the amphibian-killing fungus (<i>Batrachochytrium dendrobatidis</i>, [<i>Bd</i>]) identified three highly divergent genetic lineages, only one of which has a global distribution. <i>Bd</i> strains within these linages show variable genomic content due to differential loss of heterozygosity and recombination. The current quantitative polymerase chain reaction (qPCR) protocol to detect the fungus from amphibian skin swabs targets the intergenic transcribed spacer 1 (ITS1) region using a TaqMan fluorescent probe specific to <i>Bd</i>. We investigated the consequences of genomic differences in the quantification of ITS1 from eight distinct <i>Bd</i> strains, including representatives from North America, South America, the Caribbean, and Australia. To test for potential differences in amplification, we compared qPCR standards made from <i>Bd</i> zoospore counts for each strain, and showed that they differ significantly in amplification rates. To test potential mechanisms leading to strain differences in qPCR reaction parameters (slope and y-intercept), we: a) compared standard curves from the same strains made from extracted <i>Bd</i> genomic DNA in equimolar solutions, b) quantified the number of ITS1 copies per zoospore using a standard curve made from PCR-amplicons of the ITS1 region, and c) cloned and sequenced PCR-amplified ITS1 regions from these same strains to verify the presence of the probe site in all haplotypes. We found high strain variability in ITS1 copy number, ranging from 10 to 144 copies per single zoospore. Our results indicate that genome size might explain strain differences in ITS1 copy number, but not ITS1 sequence variation because the probe-binding site and primers were conserved across all haplotypes. For standards constructed from uncharacterized <i>Bd</i> strains, we recommend the use of single ITS1 PCR-amplicons as the absolute standard in conjunction with current quantitative assays to inform on copy number variation and provide universal estimates of pathogen zoospore loads from field-caught amphibians.</p> </div>", "links"=>[], "tags"=>["its1", "varies", "implications", "qpcr", "estimates", "field-collected", "amphibian", "swabs"], "article_id"=>657605, "categories"=>["Microbiology", "Genetics", "Biotechnology", "Plant Biology"], "users"=>["Ana V. Longo", "David Rodriguez", "Domingos da Silva Leite", "Luís Felipe Toledo", "Cinthya Mendoza Almeralla", "Patricia A. Burrowes", "Kelly R. Zamudio"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0059499.s001", "https://dx.doi.org/10.1371/journal.pone.0059499.s002", "https://dx.doi.org/10.1371/journal.pone.0059499.s003", "https://dx.doi.org/10.1371/journal.pone.0059499.s004", "https://dx.doi.org/10.1371/journal.pone.0059499.s005"], "stats"=>{"downloads"=>20, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/ITS1_Copy_Number_Varies_among_Batrachochytrium_dendrobatidis_Strains_Implications_for_qPCR_Estimates_of_Infection_Intensity_from_Field_Collected_Amphibian_Skin_Swabs__/657605", "title"=>"ITS1 Copy Number Varies among <em>Batrachochytrium dendrobatidis</em> Strains: Implications for qPCR Estimates of Infection Intensity from Field-Collected Amphibian Skin Swabs", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2013-03-22 07:44:35"}
  • {"files"=>["https://ndownloader.figshare.com/files/994484"], "description"=>"<p>Upper and lower diagonals are pairwise <i>F</i><sub>ST</sub> values estimated from ITS1 haplotypes obtained by cloning/Sanger sequencing, and from Illumina sequencing, respectively. Darker shades represent greater genetic differentiation.</p>", "links"=>[], "tags"=>["its1", "haplotype", "frequencies"], "article_id"=>657604, "categories"=>["Microbiology", "Genetics", "Biotechnology", "Plant Biology"], "users"=>["Ana V. Longo", "David Rodriguez", "Domingos da Silva Leite", "Luís Felipe Toledo", "Cinthya Mendoza Almeralla", "Patricia A. Burrowes", "Kelly R. Zamudio"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0059499.g006", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Pairwise_F_ST_values_based_on_ITS1_haplotype_frequencies_in_each_Bd_strain_/657604", "title"=>"Pairwise <i>F</i><sub>ST</sub> values based on ITS1 haplotype frequencies in each <i>Bd</i> strain.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-03-22 07:43:43"}
  • {"files"=>["https://ndownloader.figshare.com/files/994481"], "description"=>"<p>Estimated ITS1 copy number variation for each <i>Batrachochytrium dendrobatidis</i> isolate using PCR amplicons in (A) one zoospore, and (B) in 1.5 ng DNA.</p>", "links"=>[], "tags"=>["its1", "pcr", "amplicons", "ng"], "article_id"=>657601, "categories"=>["Microbiology", "Genetics", "Biotechnology", "Plant Biology"], "users"=>["Ana V. Longo", "David Rodriguez", "Domingos da Silva Leite", "Luís Felipe Toledo", "Cinthya Mendoza Almeralla", "Patricia A. Burrowes", "Kelly R. Zamudio"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0059499.g004", "stats"=>{"downloads"=>4, "page_views"=>60, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Estimated_ITS1_copy_number_variation_for_each_Batrachochytrium_dendrobatidis_isolate_using_PCR_amplicons_in_A_one_zoospore_and_B_in_1_5_ng_DNA_/657601", "title"=>"Estimated ITS1 copy number variation for each <i>Batrachochytrium dendrobatidis</i> isolate using PCR amplicons in (A) one zoospore, and (B) in 1.5 ng DNA.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-03-22 07:43:11"}

PMC Usage Stats | Further Information

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Relative Metric

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