{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T19:38:35Z","timestamp":1792784315762,"version":"4.5.0"},"reference-count":20,"publisher":"Springer Science and Business Media LLC","issue":"2","license":[{"start":{"date-parts":[[2022,3,17]],"date-time":"2022-03-17T00:00:00Z","timestamp":1647475200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2022,3,17]],"date-time":"2022-03-17T00:00:00Z","timestamp":1647475200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"funder":[{"DOI":"10.13039\/501100001809","name":"the National Natural Science Foundation of China","doi-asserted-by":"crossref","award":["81770611"],"award-info":[{"award-number":["81770611"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100001809","name":"the National Natural Science Foundation of China","doi-asserted-by":"crossref","award":["82002243"],"award-info":[{"award-number":["82002243"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Key Projects of the Beijing Municipal Education Commission's Science and Technology Plan","award":["KZ202010025035"],"award-info":[{"award-number":["KZ202010025035"]}],"id":[{"id":"https:\/\/ror.org\/04bpn6s66","id-type":"ROR","asserted-by":"crossref"}]},{"name":"Special key research project of capital health development scientific research","award":["2020-1-1151"],"award-info":[{"award-number":["2020-1-1151"]}]},{"name":"the Demonstrating Application and Research of Clinical Diagnosis and Treatment Technology in Beijing","award":["Z191100006619096"],"award-info":[{"award-number":["Z191100006619096"]}]},{"name":"the Demonstrating Application and Research of Clinical Diagnosis and Treatment Technology in Beijing","award":["Z191100006619097"],"award-info":[{"award-number":["Z191100006619097"]}]},{"DOI":"10.13039\/501100017616","name":"Beijing Talents foundation","doi-asserted-by":"crossref","award":["2018000021469G289"],"award-info":[{"award-number":["2018000021469G289"]}],"id":[{"id":"10.13039\/501100017616","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Beijing Hospitals Authority Youth Programme","award":["QML20201702"],"award-info":[{"award-number":["QML20201702"]}]}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Hepatol Int"],"published-print":{"date-parts":[[2022,4]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:sec>\n                    <jats:title>Background and aims<\/jats:title>\n                    <jats:p>The formation of an intranuclear pool of covalently closed circular DNA (cccDNA) in the liver is the main cause of persistent hepatitis B virus (HBV) infection. Here, we established highly sensitive and specific methods to detect cccDNA based on CRISPR-Cas13a technology.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>We used plasmid-safe ATP-dependent DNase (PSAD) enzymes and HindIII to digest loose circle rcDNA and double-stranded linear DNA, amplify specific HBV cccDNA fragments by rolling circle amplification (RCA) and PCR, and detect the target gene using CRISPR-Cas13a technology. The CRISPR-Cas13a-based assay for the detection of cccDNA was further clinically validated using HBV-related liver tissues, plasma, whole blood and peripheral blood mononuclear cells (PBMCs).<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>Based on the sample pretreatment step, the amplification step and the detection step, we established a new CRISPR-Cas13a-based assay for the detection of cccDNA. After the amplification of RCA and PCR, 1 copy\/\u03bcl HBV cccDNA could be detected by CRISPR\/Cas13-assisted fluorescence readout. We used ddPCR, qPCR, RCA-qPCR, PCR-CRISPR and RCA-PCR-CRISPR methods to detect 20, 4, 18, 14 and 29 positive samples in liver tissue samples from 40 HBV-related patients, respectively. HBV cccDNA was almost completely undetected in the 20 blood samples of HBV patients (including plasma, whole blood and PBMCs) by the above 5 methods.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>We developed a novel CRISPR-based assay for the highly sensitive and specific detection of HBV cccDNA, presenting a promising alternative for accurate detection of HBV infection, antiviral therapy evaluation and treatment guidance.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1007\/s12072-022-10311-0","type":"journal-article","created":{"date-parts":[[2022,3,17]],"date-time":"2022-03-17T04:04:41Z","timestamp":1647489881000},"page":"306-315","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":45,"title":["CRISPR\/Cas13-assisted hepatitis B virus covalently closed circular DNA detection"],"prefix":"10.1007","volume":"16","author":[{"given":"Xiangying","family":"Zhang","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuan","family":"Tian","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ling","family":"Xu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zihao","family":"Fan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yaling","family":"Cao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yingmin","family":"Ma","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hao","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Feng","family":"Ren","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2022,3,17]]},"reference":[{"key":"10311_CR1","doi-asserted-by":"publisher","first-page":"773","DOI":"10.15585\/mmwr.mm6728a2","volume":"67","author":"Y Hutin","year":"2018","unstructured":"Hutin Y, Nasrullah M, Easterbrook P, Nguimfack BD, Burrone E, Averhoff F, et al. Access to treatment for hepatitis B virus infection-worldwide, 2016. MMWR Morb Mortal Wkly Rep. 2018;67:773\u2013777","journal-title":"MMWR Morb Mortal Wkly Rep"},{"key":"10311_CR2","first-page":"308","volume":"388","author":"L The","year":"2016","unstructured":"The L. Towards elimination of viral hepatitis by 2030. Lancet. 2016;388:308","journal-title":"Lancet"},{"key":"10311_CR3","doi-asserted-by":"publisher","first-page":"2313","DOI":"10.1016\/S0140-6736(18)31865-8","volume":"392","author":"WK Seto","year":"2018","unstructured":"Seto WK, Lo YR, Pawlotsky JM, Yuen MF. Chronic hepatitis B virus infection. Lancet. 2018;392:2313\u20132324","journal-title":"Lancet"},{"key":"10311_CR4","doi-asserted-by":"publisher","first-page":"1560","DOI":"10.1002\/hep.29800","volume":"67","author":"NA Terrault","year":"2018","unstructured":"Terrault NA, Lok A, McMahon BJ, Chang KM, Hwang JP, Jonas MM, et al. Update on prevention, diagnosis, and treatment of chronic hepatitis B: AASLD 2018 hepatitis B guidance. Hepatology. 2018;67:1560\u20131599","journal-title":"Hepatology"},{"key":"10311_CR5","doi-asserted-by":"publisher","first-page":"1972","DOI":"10.1136\/gutjnl-2015-309809","volume":"64","author":"M Nassal","year":"2015","unstructured":"Nassal M. HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B. Gut. 2015;64:1972\u20131984","journal-title":"Gut"},{"key":"10311_CR6","doi-asserted-by":"publisher","first-page":"S41","DOI":"10.1016\/j.jhep.2016.02.009","volume":"64","author":"J Lucifora","year":"2016","unstructured":"Lucifora J, Protzer U. Attacking hepatitis B virus cccDNA\u2014the holy grail to hepatitis B cure. J Hepatol. 2016;64:S41\u2013S48","journal-title":"J Hepatol"},{"key":"10311_CR7","doi-asserted-by":"publisher","first-page":"843","DOI":"10.1053\/j.gastro.2007.06.057","volume":"133","author":"T Volz","year":"2007","unstructured":"Volz T, Lutgehetmann M, Wachtler P, Jacob A, Quaas A, Murray JM, et al. Impaired intrahepatic hepatitis B virus productivity contributes to low viremia in most HBeAg-negative patients. Gastroenterology. 2007;133:843\u2013852","journal-title":"Gastroenterology"},{"issue":"4","key":"10311_CR8","doi-asserted-by":"publisher","first-page":"471","DOI":"10.1016\/j.jmii.2014.08.008","volume":"49","author":"LT Sai","year":"2016","unstructured":"Sai LT, Yao YY, Guan YY, Shao LH, Ma RP, Ma LX. Hepatitis B virus infection and replication in a new cell culture system established by fusing HepG2 cells with primary human hepatocytes. J Microbiol Immunol Infect. 2016;49(4):471\u2013476","journal-title":"J Microbiol Immunol Infect"},{"key":"10311_CR9","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.00539-17","author":"J Luo","year":"2017","unstructured":"Luo J, Cui X, Gao L, Hu J. Identification of an intermediate in hepatitis B virus covalently closed circular (CCC) DNA formation and sensitive and selective CCC DNA detection. J Virol. 2017. https:\/\/doi.org\/10.1128\/JVI.00539-17","journal-title":"J Virol"},{"key":"10311_CR10","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.01117-18","author":"B Qu","year":"2018","unstructured":"Qu B, Ni Y, Lempp FA, Vondran F, Urban S. T5 exonuclease hydrolysis of hepatitis B virus replicative intermediates allows reliable quantification and fast drug efficacy testing of covalently closed circular DNA by PCR. J Virol. 2018. https:\/\/doi.org\/10.1128\/JVI.01117-18","journal-title":"J Virol"},{"key":"10311_CR11","doi-asserted-by":"publisher","first-page":"608","DOI":"10.1186\/s12879-014-0608-y","volume":"14","author":"Y Zhong","year":"2014","unstructured":"Zhong Y, Hu S, Xu C, Zhao Y, Xu D, Zhao Y, et al. A novel method for detection of HBV cccDNA in hepatocytes using rolling circle amplification combined with in situ PCR. BMC Infect Dis. 2014;14:608","journal-title":"BMC Infect Dis"},{"key":"10311_CR12","doi-asserted-by":"publisher","first-page":"2063","DOI":"10.1007\/s10529-015-1890-5","volume":"37","author":"D Mu","year":"2015","unstructured":"Mu D, Yan L, Tang H, Liao Y. A sensitive and accurate quantification method for the detection of hepatitis B virus covalently closed circular DNA by the application of a droplet digital polymerase chain reaction amplification system. Biotechnol Lett. 2015;37:2063\u20132073","journal-title":"Biotechnol Lett"},{"key":"10311_CR13","doi-asserted-by":"publisher","first-page":"301","DOI":"10.1016\/j.jhep.2018.03.021","volume":"69","author":"GP Caviglia","year":"2018","unstructured":"Caviglia GP, Abate ML, Tandoi F, Ciancio A, Amoroso A, Salizzoni M, et al. Quantitation of HBV cccDNA in anti-HBc-positive liver donors by droplet digital PCR: a new tool to detect occult infection. J Hepatol. 2018;69:301\u2013307","journal-title":"J Hepatol"},{"key":"10311_CR14","doi-asserted-by":"publisher","first-page":"36","DOI":"10.1016\/j.coi.2014.12.008","volume":"32","author":"R Barrangou","year":"2015","unstructured":"Barrangou R. The roles of CRISPR-Cas systems in adaptive immunity and beyond. 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Methods Mol Biol. 2012;903:113\u2013128","journal-title":"Methods Mol Biol"},{"key":"10311_CR20","first-page":"41","volume":"95","author":"S Bowden","year":"2004","unstructured":"Bowden S, Jackson K, Littlejohn M, Locarnini S. Quantification of HBV covalently closed circular DNA from liver tissue by real-time PCR. Methods Mol Med. 2004;95:41\u201350","journal-title":"Methods Mol Med"}],"container-title":["Hepatology International"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1007\/s12072-022-10311-0.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/article\/10.1007\/s12072-022-10311-0\/fulltext.html","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1007\/s12072-022-10311-0.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,4,17]],"date-time":"2022-04-17T06:04:37Z","timestamp":1650175477000},"score":1,"resource":{"primary":{"URL":"https:\/\/link.springer.com\/10.1007\/s12072-022-10311-0"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,3,17]]},"references-count":20,"journal-issue":{"issue":"2","published-print":{"date-parts":[[2022,4]]}},"alternative-id":["10311"],"URL":"https:\/\/doi.org\/10.1007\/s12072-022-10311-0","relation":{},"ISSN":["1936-0533","1936-0541"],"issn-type":[{"value":"1936-0533","type":"print"},{"value":"1936-0541","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,3,17]]},"assertion":[{"value":"29 November 2021","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"6 February 2022","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"17 March 2022","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Declarations"}},{"value":"Xiangying Zhang, Yuan Tian, Ling Xu, Zihao Fan, Yaling Cao, Yingmin Ma, Hao Li and Feng Ren have declared that no competing interest exists.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Conflict of interest"}},{"value":"The study protocol satisfied the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the medical ethics committee of Beijing Youan Hospital, Capital Medical University.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval"}},{"value":"Not applicable.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Animal research (ethics)"}},{"value":"The requirement for written informed consent was waived because of the retrospective nature of this study.","order":5,"name":"Ethics","group":{"name":"EthicsHeading","label":"Consent to participate"}},{"value":"All the authors consented to publish work.","order":6,"name":"Ethics","group":{"name":"EthicsHeading","label":"Consent to publish"}},{"value":"Not applicable.","order":7,"name":"Ethics","group":{"name":"EthicsHeading","label":"Plant reproducibility"}},{"value":"All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008 (5). Informed consent was obtained from all patients for being included in the study.","order":8,"name":"Ethics","group":{"name":"EthicsHeading","label":"Human Participants"}}]}}