{"id":10992,"date":"2025-02-13T13:45:57","date_gmt":"2025-02-13T04:45:57","guid":{"rendered":"http:\/\/cell.brc.riken.jp\/en\/?page_id=10992"},"modified":"2025-02-13T13:45:57","modified_gmt":"2025-02-13T04:45:57","slug":"ebv","status":"publish","type":"page","link":"http:\/\/cell.brc.riken.jp\/en\/quality\/ebv","title":{"rendered":"EBV Test"},"content":{"rendered":"<p>Epstein-Barr virus (EBV) is a pathogenic virus belonging to the Herpesviridae family. It spreads through saliva and primarily infects B cells. Over 95% of healthy adults are infected with EBV, and it is a virus that is commonly carried throughout life in a symbiotic manner. While EBV-related disease onset is rare, it has been found to be associated with the development of certain malignant lymphomas, such as Burkitt&#8217;s lymphoma, as well as nasopharyngeal cancer.<\/p>\n<p>Our bank uses qPCR to detect EBV genomes in human cell lines derived from blood (excluding EBV-transformed B-cell lines) and in certain human cancer cell lines; oral cancer, esophageal cancer and gastric cancer that have been reported to be associated with EBV infection as a cause of cancer development. Our bank focuses on the latent infection-associated gene EBNA1<sup>*1<\/sup> and the lytic infection-associated gene BALF5<sup>*2<\/sup> as indicators for detecting the EBV genome in cell lines.<br \/>\n<sup>*1<\/sup> EBNA1: Epstein-Barr Nuclear Antigen 1<br \/>\n<sup>*2<\/sup> BALF5: the BamHI-A fragment containing the fifth leftward open reading frame<\/p>\n<p><strong>1. Preparation<\/strong><br \/>\n(1) Sample: Genomic DNA extracted from cells<br \/>\n(2) Equipment: Applied Biosystems QuantStudio3 Real-Time PCR System<br \/>\n(3) Software: QuantStudio Design &#038; Analysis Software, ThermoFisher SCIENTIFIC<br \/>\n(4) PCR Reagents: Applied Biosystems PowerUp SYBR Green Master Mix (Cat No. A25777)<br \/>\n(5) PCR Primers<br \/>\n\u3000\u3000\u2022 EBNA1 primers<br \/>\n\u3000\u3000\u3000\u3000EBNA1-F : 5\u2019-ATCAGGGCCAAGACATAGAGATG-3\u2019<br \/>\n\u3000\u3000\u3000\u3000EBNA1-R : 5\u2019-GCCAATGCAACTTGGACGTT-3\u2019<br \/>\n\u3000\u3000\u2022 BALF5 primers<br \/>\n\u3000\u3000\u3000\u3000BALF5-F : 5\u2019-CGGAAGCCCTCTGGACTTC\u20133\u2019<br \/>\n\u3000\u3000\u3000\u3000BALF5-R : 5\u2019-CCCTGTTTATCCGATGGAATG\u20133\u2019<br \/>\n(6) Standards: Synthetic DNA for each target gene<\/p>\n<p><strong>2. Method<\/strong><br \/>\n(1) Preparation of Standards: Prepare the following copy numbers for each target gene:<br \/>\n\u3000\u3000\u2022 EBNA1\uff1b1\u00d710<sup>6<\/sup>\u30011\u00d710<sup>5<\/sup>\u30011\u00d710<sup>4<\/sup>\u30011\u00d710<sup>3<\/sup>\u30011\u00d710<sup>2<\/sup>\u30011\u00d710<sup>1<\/sup> (copies)<br \/>\n\u3000\u3000\u2022 BALF5\uff1b2.5\u00d710<sup>6<\/sup>\u30012.5\u00d710<sup>5<\/sup>\u30012.5\u00d710<sup>4<\/sup>\u30012.5\u00d710<sup>3<\/sup>\u30012.5\u00d710<sup>2<\/sup>\u30012.5\u00d710<sup>1<\/sup> (copies)<br \/>\n(2) Preparation of Samples: Prepare genomic DNA solution at 2.5 ng\/uL<br \/>\n(3) Preparation of PCR Master Mix<\/p>\n<table style=\"margin-left:20px;\">\n<tbody>\n<tr>\n<td id=\"t1\">DDW<\/td>\n<td id=\"t1\" style=\"text-align: right;\">4 uL<\/td>\n<\/tr>\n<tr>\n<td id=\"t1\">Primer FW (10 pmol\/uL)<\/td>\n<td id=\"t1\" style=\"text-align: right;\">1 uL<\/td>\n<\/tr>\n<tr>\n<td id=\"t1\">Primer RV (10 pmol\/uL)<\/td>\n<td id=\"t1\" style=\"text-align: right;\">1 uL<\/td>\n<\/tr>\n<tr>\n<td id=\"t1\">PowerUp SYBR Green Master Mix (2x)<\/td>\n<td id=\"t1\" style=\"text-align: right;\">10 uL<\/td>\n<\/tr>\n<tr>\n<td colspan=\"2\" id=\"t1\">\n<hr \/>\n<\/td>\n<\/tr>\n<tr>\n<td id=\"t1\">Total<\/td>\n<td id=\"t1\" style=\"text-align: right;\">16 uL \/Sample<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<p>(4) Dispense 16 uL of master mix into each well of a PCR 96-well plate.<br \/>\n(5) Add 4 uL of the prepared sample to each well.<br \/>\n(6) PCR Program:<br \/>\n\u2022\tUDG Activation: 50\u00b0C for 2 minutes (hold)<br \/>\n\u2022\tDual-Lock DNA Polymerase: 95\u00b0C for 2 minutes (hold)<br \/>\n\u2022\tReaction Cycles (Denaturation \u2192 Annealing\/Extension): 95\u00b0C for 15 seconds \u2192 60\u00b0C for 1 minute * (40 cycles)<br \/>\n\u2022\tMelt Curve Analysis: 95\u00b0C for 15 seconds \u2192 60\u00b0C for 1 minute \u2192 95\u00b0C for 15 seconds <sup>*<\/sup> (1 cycle)<br \/>\n<sup>*<\/sup> Fluorescent data collection points<br \/>\n(7) Result Analysis<\/p>\n<p><strong>3. Interpretation of the result<\/strong><br \/>\nAfter confirming the validity of the test (Efficiency, R<sup>2<\/sup>, and Melt Curve TM values of the standards must be within valid conditions), the results are assessed.<\/p>\n<p><strong>4. References<\/strong><br \/>\n\u2022\tReferences on PCR<br \/>\n\u3000\u3000\u3000\u3000QuantStudio 3\/QuantStudio 5 \u30ea\u30a2\u30eb\u30bf\u30a4\u30e0 PCR \u30b7\u30b9\u30c6\u30e0 \u7c21\u6613\u64cd\u4f5c\u30ac\u30a4\u30c9(Japanese only)<br \/>\n\u2022\tReferences on determining detection limits<br \/>\n\u3000\u3000\u3000\u3000\u8840\u6db2\u88fd\u5264\u306e\u30a6\u30a4\u30eb\u30b9\u306b\u5bfe\u3059\u308b\u5b89\u5168\u6027\u78ba\u4fdd\u3092\u76ee\u7684\u3068\u3057\u305f\u6838\u9178\u5897\u5e45\u691c\u67fb(NAT)\u306e\u5b9f\u65bd\u306b\u95a2\u3059\u308b\u30ac\u30a4\u30c9\u30e9\u30a4\u30f3 (\u539a\u751f\u52b4\u50cd\u7701)(Japanese only)<br \/>\n\u2022\tReferences on primers and standards<br \/>\n\u3000\u3000\u3000\u3000Generation of two human induced pluripotent stem cell lines derived from two juvenile nephronophthisis patients with NPHP1 deletion. <em>Stem Cell Res.<\/em> 2020 May; 45:101815. <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/32361464\/\">PMID: 32361464<\/a>.<\/p>\n","protected":false},"excerpt":{"rendered":"<p>Epstein-Barr virus (EBV) is a pathogenic virus belonging to the Herpesviridae family. It spreads through saliva and primarily infects B cells. Over 95% of healthy adults are infected with EBV, and it is a virus that is commonly carried throughout life in a symbiotic manner. While EBV-related disease onset is rare, it has been found to be associated with the development of certain malignant lymphomas, such as Burkitt&#8217;s lymphoma, as well as nasopharyngeal cancer. Our bank uses qPCR to detect EBV genomes in human cell lines derived from blood (excluding EBV-transformed B-cell lines) and in certain human cancer cell lines; oral cancer, esophageal cancer and gastric cancer that have been reported to be associated with EBV infection as a cause of cancer development. Our bank focuses on the latent infection-associated gene EBNA1*1 and the lytic infection-associated gene BALF5*2 as indicators for detecting the EBV genome in cell lines. *1 EBNA1: Epstein-Barr Nuclear Antigen 1 *2 BALF5: the BamHI-A fragment containing the fifth leftward open reading frame 1. Preparation (1) Sample: Genomic DNA extracted from cells (2) Equipment: Applied Biosystems QuantStudio3 Real-Time PCR System (3) Software: QuantStudio Design &#038; Analysis Software, ThermoFisher SCIENTIFIC (4) PCR Reagents: Applied Biosystems PowerUp SYBR Green [&hellip;]<\/p>\n","protected":false},"author":14,"featured_media":0,"parent":9319,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_seopress_titles_title":"","_seopress_titles_desc":"","_seopress_robots_index":"","_seopress_robots_follow":"","_seopress_robots_imageindex":"","_seopress_robots_snippet":"","_seopress_robots_primary_cat":"","_seopress_robots_breadcrumbs":"","_seopress_robots_freeze_modified_date":"","_seopress_robots_custom_modified_date":"","_seopress_robots_canonical":"","_seopress_social_fb_title":"","_seopress_social_fb_desc":"","_seopress_social_fb_img":"","_seopress_social_fb_img_attachment_id":0,"_seopress_social_fb_img_width":0,"_seopress_social_fb_img_height":0,"_seopress_social_twitter_title":"","_seopress_social_twitter_desc":"","_seopress_social_twitter_img":"","_seopress_social_twitter_img_attachment_id":0,"_seopress_social_twitter_img_width":0,"_seopress_social_twitter_img_height":0,"_seopress_redirections_value":"","_seopress_redirections_enabled":"","_seopress_redirections_enabled_regex":"","_seopress_redirections_logged_status":"both","_seopress_redirections_param":"","_seopress_redirections_type":301,"_seopress_analysis_target_kw":"","footnotes":"","_wp_rev_ctl_limit":""},"class_list":["post-10992","page","type-page","status-publish","hentry"],"_links":{"self":[{"href":"http:\/\/cell.brc.riken.jp\/en\/wp-json\/wp\/v2\/pages\/10992","targetHints":{"allow":["GET"]}}],"collection":[{"href":"http:\/\/cell.brc.riken.jp\/en\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"http:\/\/cell.brc.riken.jp\/en\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"http:\/\/cell.brc.riken.jp\/en\/wp-json\/wp\/v2\/users\/14"}],"replies":[{"embeddable":true,"href":"http:\/\/cell.brc.riken.jp\/en\/wp-json\/wp\/v2\/comments?post=10992"}],"version-history":[{"count":1,"href":"http:\/\/cell.brc.riken.jp\/en\/wp-json\/wp\/v2\/pages\/10992\/revisions"}],"predecessor-version":[{"id":10995,"href":"http:\/\/cell.brc.riken.jp\/en\/wp-json\/wp\/v2\/pages\/10992\/revisions\/10995"}],"up":[{"embeddable":true,"href":"http:\/\/cell.brc.riken.jp\/en\/wp-json\/wp\/v2\/pages\/9319"}],"wp:attachment":[{"href":"http:\/\/cell.brc.riken.jp\/en\/wp-json\/wp\/v2\/media?parent=10992"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}