CRISPR/Cas12b assisted loop-mediated isothermal amplification for easy, rapid and sensitive quantification of chronic HBV DNA in one-pot - Details

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Abstract：

Background:　Currently,　millions　of　people　suffer　from　undiagnosed　chronic　hepatitis　B　(CHB)　infection　each　year,　which　leads　to　high　mortality　rates　attributed　to　cirrhosis　and　hepatocellular　carcinoma.　Previously　reported　assays,　such　as　PCR-based　assays,　have　limitations　in　terms　of　convenient　for　CHB　screening　in　high-burden　regions　and　resource-limited　settings.　Recently,　diagnosis　based　on　CRISPR/Cas,　which　has　been　considered　as　a　potential　method　of　point-of-care　test　(POCT)　in　resource-limited　settings,　offers　a　significant　advantage　in　terms　of　high　sensitivity　and　specificity.　Therefore,　there　is　an　urgent　need　for　the　hepatitis　B　virus　(HBV)　detection　utilizing　CRISPR/Cas　system.　Results:　We　have　proposed　a　one-pot　of　one-step　method　for　CRISPR/Cas12b　assisted　loop-mediated　isothermal　amplification　(LAMP)　to　facilitate　the　quick,　sensitive,　and　precise　quantification　of　HBV　DNA.　This　method　is　designed　for　point-of-care　testing　following　genomic　extraction　or　sample　heat　treatment.　We　have　optimized　several　critical　factors,　such　as　the　reaction　buffer,　AapCas12b-gRNA　concentration,　reporter　and　its　concentration,　reaction　temperature,　and　chemical　additives,　to　significantly　enhance　the　performance　of　the　one-pot　assay　for　HBV.　Importantly,　it　exhibited　no　cross-reactivity　between　HBV　and　blood-borne　pathogens.　Moreover,　the　assay　is　capable　of　quantifying　HBV　DNA　within　1　h　with　a　limit　of　detection　(LOD)　of　25　copies　per　milliliter.　Additionally,　when　tested　on　236　clinical　samples,　the　assay　demonstrated　a　sensitivity　of　99.00　%　(198/200)　and　a　specificity　of　100.00　%　(36/36)　at　the　99　%　confidence　level　compared　to　real-time　quantitative　PCR.　Significance:　The　utilization　of　convenient　and　reliable　point-of-care　diagnostic　methods　is　crucial　for　reducing　the　burden　of　CHB　globally.　The　assay　we　developed　was　helpful　to　improve　the　ability　of　HBV　diagnosis　for　practical　clinical　translation,　especially　in　high-burden　regions　and　resource-limited　settings.　It　has　great　advantages　for　rapid　screening　of　CHB　as　well　as　evaluation　of　therapeutic　efficacy　as　a　companion　diagnostic　method.　©　2024

Keyword：

Chronic hepatitis B CRISPR/Cas12b HBV Isothermal amplification POCT

Community：

[ 1 ] [Xu H.]The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, 350025, China
[ 2 ] [Xu H.]Mengchao Med-X Center, Fuzhou University, Fuzhou, 350116, China
[ 3 ] [Xu H.]College of Chemical Engineering, Fuzhou University, Fuzhou, 350116, China
[ 4 ] [Lin G.]School of Basic Medical Sciences, Fujian Medical University, Fuzhou, 350122, China
[ 5 ] [Chen R.]The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, 350025, China
[ 6 ] [Chen R.]Mengchao Med-X Center, Fuzhou University, Fuzhou, 350116, China
[ 7 ] [Cai Z.]The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, 350025, China
[ 8 ] [Cai Z.]Mengchao Med-X Center, Fuzhou University, Fuzhou, 350116, China
[ 9 ] [Sun Y.]The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, 350025, China
[ 10 ] [Sun Y.]Mengchao Med-X Center, Fuzhou University, Fuzhou, 350116, China
[ 11 ] [Zhang X.]The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, 350025, China
[ 12 ] [Zhang X.]Mengchao Med-X Center, Fuzhou University, Fuzhou, 350116, China
[ 13 ] [Zhao B.]The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, 350025, China
[ 14 ] [Zhao B.]Mengchao Med-X Center, Fuzhou University, Fuzhou, 350116, China
[ 15 ] [Zeng Y.]The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, 350025, China
[ 16 ] [Zeng Y.]Mengchao Med-X Center, Fuzhou University, Fuzhou, 350116, China
[ 17 ] [Liu J.]Mengchao Med-X Center, Fuzhou University, Fuzhou, 350116, China
[ 18 ] [Liu J.]College of Chemical Engineering, Fuzhou University, Fuzhou, 350116, China
[ 19 ] [Liu J.]Department of Radiation Oncology, Fujian Medical University Cancer Hospital, Fuzhou, 350014, China
[ 20 ] [Liu X.]The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, 350025, China
[ 21 ] [Liu X.]Mengchao Med-X Center, Fuzhou University, Fuzhou, 350116, China
[ 22 ] [Liu X.]College of Chemical Engineering, Fuzhou University, Fuzhou, 350116, China
[ 23 ] [Liu X.]School of Basic Medical Sciences, Fujian Medical University, Fuzhou, 350122, China
[ 24 ] [Liu X.]School of Life Science and Technology, Xi'an Jiaotong University, Xi'an, 710049, China

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