{"id":1126,"date":"2013-01-11T16:07:20","date_gmt":"2013-01-11T15:07:20","guid":{"rendered":"http:\/\/sppadbase.ipp.cnr.it\/?p=1126"},"modified":"2013-01-11T16:09:32","modified_gmt":"2013-01-11T15:09:32","slug":"fungiquant-a-broad-coverage-fungal-quantitative-real-time-pcr-assay","status":"publish","type":"post","link":"https:\/\/sppadbase.ipsp.cnr.it\/?p=1126","title":{"rendered":"FungiQuant: A broad-coverage fungal quantitative real-time PCR assay"},"content":{"rendered":"<div class=\"textimage\">\n<p class=\"textimage\">\n<p><strong>Background<\/strong><br \/>\nFungal load quantification is a critical component of fungal community analyses. Limitation of current approaches for quantifying the fungal component in the human microbiome suggests the need for new broad-coverage techniques.<br \/>\n<strong>Methods<\/strong><br \/>\nWe analyzed 2,085 18S rRNA gene sequences from the SILVA database for assay design. We generated and quantified plasmid standards using a qPCR-based approach. We evaluated assay coverage against 4,968 sequences and performed assay validation following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments MIQE guidelines.<br \/>\n<strong>Results<\/strong><br \/>\nWe designed FungiQuant, a TaqManR qPCR assay targeting a 351 bp region in the fungal 18S rRNA gene. Our in silico analysis showed that FungiQuant is a perfect sequence match to 90.0% of the 2,617 fungal species analyzed. We showed that FungiQuants is 100% sensitive and its amplification efficiencies ranged from 76.3% to 114.5%, with r2-values of &gt;0.99 against the 69 fungal species tested. Additionally, FungiQuant inter- and intra-run coefficients of variance ranged from &lt;10% and &lt;20%, respectively. We further showed that FungiQuant has a limit of quantification 25 copies and a limit of detection at 5 copies. Lastly, by comparing results from human-only background DNA with low-level fungal DNA, we showed that amplification in two or three of a FungiQuant performed in triplicate is statistically significant for true positive fungal detection.<br \/>\n<strong>Conclusions<\/strong><br \/>\nFungiQuant has comprehensive coverage against diverse fungi and is a robust quantification and detection tool for delineating between true fungal detection and non-target human DNA.<\/p>\n<div class=\"clearer\"><\/div>\n<p><strong>Cindy M Liu, Sergey Kachur, Michael G Dwan, Alison G Abraham, Maliha Aziz, Po-Ren Hsueh, Yu-Tsung Huang, Joseph D Busch, Louis J Lamit, Catherine A Gehring, Paul Keim and Lance B Price<\/strong><br \/>\nBMC Microbiology 2012, 12:255<br \/>\ndoi: 10.1186\/1471-2180-12-255<\/p>\n<p>via <a href='http:\/\/www.biomedcentral.com\/1471-2180\/12\/255\/abstract'>BMC Microbiology | Abstract | FungiQuant: A broad-coverage fungal quantitative real-time PCR assay<\/a>.<\/p>\n","protected":false},"excerpt":{"rendered":"\n<p class=\"textimage\">\n<p>Background Fungal load quantification is a critical component of fungal community analyses. Limitation of current approaches for quantifying the fungal component in the human microbiome suggests the need for new broad-coverage techniques. Methods We analyzed 2,085 18S rRNA gene sequences from the SILVA database for assay design. We generated and quantified plasmid <span style=\"color:#777\"> . . . &rarr; Read More: <a href=\"https:\/\/sppadbase.ipsp.cnr.it\/?p=1126\">FungiQuant: A broad-coverage fungal quantitative real-time PCR assay<\/a><\/span><\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":[],"categories":[15,3,21],"tags":[16,58],"_links":{"self":[{"href":"https:\/\/sppadbase.ipsp.cnr.it\/index.php?rest_route=\/wp\/v2\/posts\/1126"}],"collection":[{"href":"https:\/\/sppadbase.ipsp.cnr.it\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/sppadbase.ipsp.cnr.it\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/sppadbase.ipsp.cnr.it\/index.php?rest_route=\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/sppadbase.ipsp.cnr.it\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=1126"}],"version-history":[{"count":9,"href":"https:\/\/sppadbase.ipsp.cnr.it\/index.php?rest_route=\/wp\/v2\/posts\/1126\/revisions"}],"predecessor-version":[{"id":1184,"href":"https:\/\/sppadbase.ipsp.cnr.it\/index.php?rest_route=\/wp\/v2\/posts\/1126\/revisions\/1184"}],"wp:attachment":[{"href":"https:\/\/sppadbase.ipsp.cnr.it\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=1126"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/sppadbase.ipsp.cnr.it\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=1126"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/sppadbase.ipsp.cnr.it\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=1126"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}