One-pot platform for rapid detecting virus utilizing recombinase polymerase amplification and CRISPR/Cas12a
The livestock industry has been deeply affected by African swine fever virus (ASFV) and Capripoxvirus (CaPV), which caused an enormous economic damage. It is emergent to develop a reliable detection method. Here, we developed a rapid, ultra-sensitive, and one-pot DNA detection method combining recom...
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| Veröffentlicht in: | Applied microbiology and biotechnology 2022-06, Vol.106 (12), p.4607-4616 |
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| creator | Xiong, Yifan Cao, Gaihua Chen, Xiaolong Yang, Jun Shi, Meimei Wang, Yu Nie, Fuping Huo, Danqun Hou, Changjun |
| description | The livestock industry has been deeply affected by African swine fever virus (ASFV) and
Capripoxvirus
(CaPV), which caused an enormous economic damage. It is emergent to develop a reliable detection method. Here, we developed a rapid, ultra-sensitive, and one-pot DNA detection method combining recombinase polymerase amplification (RPA) and CRISPR/Cas12a for ASFV and CaPV, named one-pot-RPA-Cas12a (OpRCas) platform. It had the virtue of both RPA and CRISPR/Cas12a, such as high amplification efficiency, constant temperature reaction, and strict target selectivity, which made diagnosis simplified, accurate and easy to be operated without expensive equipment. Meanwhile, the reagents of RPA and CRISPR/Cas12a were added to the lid and bottom of tube in one go, which overcame the incompatibility of two reactions and aerosol contamination. To save cost, we only need a quarter of the amount of regular RPA per reaction which is enough to achieve clinical diagnosis. The OpRCas platform was 10 to 100 times more sensitive than qPCR; the limit of detection (LOD) was as low as 1.2 × 10
−6
ng/µL (3.07 copies/µL by ddPCR) of ASFV and 7.7 × 10
−5
ng/µL (1.02 copies/µL by ddPCR) of CaPV with the portable fluorometer in 40 min. In addition, the OpRCas platform combined with the lateral flow assay (LFA) strip to suit for point-of-care (POC) testing. It showed 93.3% consistency with qPCR for clinical sample analysis. Results prove that OpRCas platform is an easy-handling, ultra-sensitive, and rapid to achieve ASFV and CaPV POC testing.
Key points
• The platform realizes one-pot reaction of RPA and Cas12a.
• Sensitivity is 100 times more than qPCR.
• Three output modes are suitable to be used to quantitative test or POC testing. |
| doi_str_mv | 10.1007/s00253-022-12015-9 |
| format | Article |
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Capripoxvirus
(CaPV), which caused an enormous economic damage. It is emergent to develop a reliable detection method. Here, we developed a rapid, ultra-sensitive, and one-pot DNA detection method combining recombinase polymerase amplification (RPA) and CRISPR/Cas12a for ASFV and CaPV, named one-pot-RPA-Cas12a (OpRCas) platform. It had the virtue of both RPA and CRISPR/Cas12a, such as high amplification efficiency, constant temperature reaction, and strict target selectivity, which made diagnosis simplified, accurate and easy to be operated without expensive equipment. Meanwhile, the reagents of RPA and CRISPR/Cas12a were added to the lid and bottom of tube in one go, which overcame the incompatibility of two reactions and aerosol contamination. To save cost, we only need a quarter of the amount of regular RPA per reaction which is enough to achieve clinical diagnosis. The OpRCas platform was 10 to 100 times more sensitive than qPCR; the limit of detection (LOD) was as low as 1.2 × 10
−6
ng/µL (3.07 copies/µL by ddPCR) of ASFV and 7.7 × 10
−5
ng/µL (1.02 copies/µL by ddPCR) of CaPV with the portable fluorometer in 40 min. In addition, the OpRCas platform combined with the lateral flow assay (LFA) strip to suit for point-of-care (POC) testing. It showed 93.3% consistency with qPCR for clinical sample analysis. Results prove that OpRCas platform is an easy-handling, ultra-sensitive, and rapid to achieve ASFV and CaPV POC testing.
Key points
• The platform realizes one-pot reaction of RPA and Cas12a.
• Sensitivity is 100 times more than qPCR.
• Three output modes are suitable to be used to quantitative test or POC testing.</description><identifier>ISSN: 0175-7598</identifier><identifier>EISSN: 1432-0614</identifier><identifier>DOI: 10.1007/s00253-022-12015-9</identifier><identifier>PMID: 35708748</identifier><language>eng</language><publisher>Berlin/Heidelberg: Springer Berlin Heidelberg</publisher><subject>African swine fever ; African Swine Fever Virus - genetics ; African Swine Fever Virus - metabolism ; Air pollution ; Amplification ; Animals ; Applied Genetics and Molecular Biotechnology ; Asfarviridae ; Biomedical and Life Sciences ; Biotechnology ; Contamination ; CRISPR ; CRISPR-Cas Systems ; Diagnosis ; Fluorometers ; Identification and classification ; Incompatibility ; Life Sciences ; Livestock ; Livestock industry ; Microbial Genetics and Genomics ; Microbiology ; Nucleic Acid Amplification Techniques - methods ; Nucleotidyltransferases ; Reagents ; Real-Time Polymerase Chain Reaction ; Recombinase ; Recombinases ; Recombinases - genetics ; Selectivity ; Sensitivity and Specificity ; Swine ; Viruses</subject><ispartof>Applied microbiology and biotechnology, 2022-06, Vol.106 (12), p.4607-4616</ispartof><rights>The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022</rights><rights>2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.</rights><rights>COPYRIGHT 2022 Springer</rights><rights>The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c575t-e17a1e0eb2ea882ab7cdecc07f7dd772b05b02a1de83b01d7111838729f260183</citedby><cites>FETCH-LOGICAL-c575t-e17a1e0eb2ea882ab7cdecc07f7dd772b05b02a1de83b01d7111838729f260183</cites><orcidid>0000-0001-8550-036X</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s00253-022-12015-9$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s00253-022-12015-9$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>230,314,777,781,882,27905,27906,41469,42538,51300</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/35708748$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Xiong, Yifan</creatorcontrib><creatorcontrib>Cao, Gaihua</creatorcontrib><creatorcontrib>Chen, Xiaolong</creatorcontrib><creatorcontrib>Yang, Jun</creatorcontrib><creatorcontrib>Shi, Meimei</creatorcontrib><creatorcontrib>Wang, Yu</creatorcontrib><creatorcontrib>Nie, Fuping</creatorcontrib><creatorcontrib>Huo, Danqun</creatorcontrib><creatorcontrib>Hou, Changjun</creatorcontrib><title>One-pot platform for rapid detecting virus utilizing recombinase polymerase amplification and CRISPR/Cas12a</title><title>Applied microbiology and biotechnology</title><addtitle>Appl Microbiol Biotechnol</addtitle><addtitle>Appl Microbiol Biotechnol</addtitle><description>The livestock industry has been deeply affected by African swine fever virus (ASFV) and
Capripoxvirus
(CaPV), which caused an enormous economic damage. It is emergent to develop a reliable detection method. Here, we developed a rapid, ultra-sensitive, and one-pot DNA detection method combining recombinase polymerase amplification (RPA) and CRISPR/Cas12a for ASFV and CaPV, named one-pot-RPA-Cas12a (OpRCas) platform. It had the virtue of both RPA and CRISPR/Cas12a, such as high amplification efficiency, constant temperature reaction, and strict target selectivity, which made diagnosis simplified, accurate and easy to be operated without expensive equipment. Meanwhile, the reagents of RPA and CRISPR/Cas12a were added to the lid and bottom of tube in one go, which overcame the incompatibility of two reactions and aerosol contamination. To save cost, we only need a quarter of the amount of regular RPA per reaction which is enough to achieve clinical diagnosis. The OpRCas platform was 10 to 100 times more sensitive than qPCR; the limit of detection (LOD) was as low as 1.2 × 10
−6
ng/µL (3.07 copies/µL by ddPCR) of ASFV and 7.7 × 10
−5
ng/µL (1.02 copies/µL by ddPCR) of CaPV with the portable fluorometer in 40 min. In addition, the OpRCas platform combined with the lateral flow assay (LFA) strip to suit for point-of-care (POC) testing. It showed 93.3% consistency with qPCR for clinical sample analysis. Results prove that OpRCas platform is an easy-handling, ultra-sensitive, and rapid to achieve ASFV and CaPV POC testing.
Key points
• The platform realizes one-pot reaction of RPA and Cas12a.
• Sensitivity is 100 times more than qPCR.
• Three output modes are suitable to be used to quantitative test or POC testing.</description><subject>African swine fever</subject><subject>African Swine Fever Virus - genetics</subject><subject>African Swine Fever Virus - metabolism</subject><subject>Air pollution</subject><subject>Amplification</subject><subject>Animals</subject><subject>Applied Genetics and Molecular Biotechnology</subject><subject>Asfarviridae</subject><subject>Biomedical and Life Sciences</subject><subject>Biotechnology</subject><subject>Contamination</subject><subject>CRISPR</subject><subject>CRISPR-Cas Systems</subject><subject>Diagnosis</subject><subject>Fluorometers</subject><subject>Identification and classification</subject><subject>Incompatibility</subject><subject>Life Sciences</subject><subject>Livestock</subject><subject>Livestock industry</subject><subject>Microbial Genetics and Genomics</subject><subject>Microbiology</subject><subject>Nucleic Acid Amplification Techniques - methods</subject><subject>Nucleotidyltransferases</subject><subject>Reagents</subject><subject>Real-Time Polymerase Chain Reaction</subject><subject>Recombinase</subject><subject>Recombinases</subject><subject>Recombinases - genetics</subject><subject>Selectivity</subject><subject>Sensitivity and Specificity</subject><subject>Swine</subject><subject>Viruses</subject><issn>0175-7598</issn><issn>1432-0614</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2022</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><recordid>eNp9kl1vFCEUhonR2LX6B7wwk3ijF9MCMwzMjUmz8WOTJjVbvSYMnFmpMzAC01h_vYxbW9cYQwIHeM5LzuFF6DnBJwRjfhoxpqwqMaUloZiwsn2AVqSuaIkbUj9EK0w4KzlrxRF6EuMVxoSKpnmMjirGseC1WKGvFw7KyadiGlTqfRiLPBVBTdYUBhLoZN2uuLZhjsWc7GB_LPsA2o-ddSpCMfnhZoSwhGqcBttbrZL1rlDOFOvt5vLj9nStIqHqKXrUqyHCs9v1GH1-9_bT-kN5fvF-sz47LzXjLJVAuCKAoaOghKCq49qA1pj33BjOaYdZh6kiBkTVYWI4IURUgtO2pw3O4TF6s9ed5m4Eo8GloAY5BTuqcCO9svLwxtkvcuevZZu7SJtF4NWtQPDfZohJjjZqGAblwM9R0oZzxismaEZf_oVe-Tm4XF6mBKO0yV9zT-3UANK63ud39SIqzzhua17VbZ2pk39QeRgYrfYOepvPDxJeHyRkJsH3tFNzjHJzuT1k6Z7VwccYoL_rB8FysZPc20lmO8lfdpJtTnrxZyfvUn77JwPVHoj5yu0g3Jf_H9mfp8vUcg</recordid><startdate>20220601</startdate><enddate>20220601</enddate><creator>Xiong, Yifan</creator><creator>Cao, Gaihua</creator><creator>Chen, Xiaolong</creator><creator>Yang, Jun</creator><creator>Shi, Meimei</creator><creator>Wang, Yu</creator><creator>Nie, Fuping</creator><creator>Huo, Danqun</creator><creator>Hou, Changjun</creator><general>Springer Berlin Heidelberg</general><general>Springer</general><general>Springer Nature B.V</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>ISR</scope><scope>3V.</scope><scope>7QL</scope><scope>7T7</scope><scope>7WY</scope><scope>7WZ</scope><scope>7X7</scope><scope>7XB</scope><scope>87Z</scope><scope>88A</scope><scope>88E</scope><scope>88I</scope><scope>8AO</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>8FL</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BEZIV</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FRNLG</scope><scope>FYUFA</scope><scope>F~G</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>K60</scope><scope>K6~</scope><scope>K9.</scope><scope>L.-</scope><scope>LK8</scope><scope>M0C</scope><scope>M0S</scope><scope>M1P</scope><scope>M2P</scope><scope>M7N</scope><scope>M7P</scope><scope>P64</scope><scope>PQBIZ</scope><scope>PQBZA</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>Q9U</scope><scope>7X8</scope><scope>5PM</scope><orcidid>https://orcid.org/0000-0001-8550-036X</orcidid></search><sort><creationdate>20220601</creationdate><title>One-pot platform for rapid detecting virus utilizing recombinase polymerase amplification and CRISPR/Cas12a</title><author>Xiong, Yifan ; Cao, Gaihua ; Chen, Xiaolong ; Yang, Jun ; Shi, Meimei ; Wang, Yu ; Nie, Fuping ; Huo, Danqun ; Hou, Changjun</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c575t-e17a1e0eb2ea882ab7cdecc07f7dd772b05b02a1de83b01d7111838729f260183</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2022</creationdate><topic>African swine fever</topic><topic>African Swine Fever Virus - genetics</topic><topic>African Swine Fever Virus - metabolism</topic><topic>Air pollution</topic><topic>Amplification</topic><topic>Animals</topic><topic>Applied Genetics and Molecular Biotechnology</topic><topic>Asfarviridae</topic><topic>Biomedical and Life Sciences</topic><topic>Biotechnology</topic><topic>Contamination</topic><topic>CRISPR</topic><topic>CRISPR-Cas Systems</topic><topic>Diagnosis</topic><topic>Fluorometers</topic><topic>Identification and classification</topic><topic>Incompatibility</topic><topic>Life Sciences</topic><topic>Livestock</topic><topic>Livestock industry</topic><topic>Microbial Genetics and Genomics</topic><topic>Microbiology</topic><topic>Nucleic Acid Amplification Techniques - methods</topic><topic>Nucleotidyltransferases</topic><topic>Reagents</topic><topic>Real-Time Polymerase Chain Reaction</topic><topic>Recombinase</topic><topic>Recombinases</topic><topic>Recombinases - genetics</topic><topic>Selectivity</topic><topic>Sensitivity and Specificity</topic><topic>Swine</topic><topic>Viruses</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Xiong, Yifan</creatorcontrib><creatorcontrib>Cao, Gaihua</creatorcontrib><creatorcontrib>Chen, Xiaolong</creatorcontrib><creatorcontrib>Yang, Jun</creatorcontrib><creatorcontrib>Shi, Meimei</creatorcontrib><creatorcontrib>Wang, Yu</creatorcontrib><creatorcontrib>Nie, Fuping</creatorcontrib><creatorcontrib>Huo, Danqun</creatorcontrib><creatorcontrib>Hou, Changjun</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Gale In Context: Science</collection><collection>ProQuest Central (Corporate)</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>ABI/INFORM Collection</collection><collection>ABI/INFORM Global (PDF only)</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>ABI/INFORM Global (Alumni Edition)</collection><collection>Biology Database (Alumni Edition)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Science Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ABI/INFORM Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest One Sustainability</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Business Premium Collection</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Business Premium Collection (Alumni)</collection><collection>Health Research Premium Collection</collection><collection>ABI/INFORM Global (Corporate)</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Business Collection (Alumni Edition)</collection><collection>ProQuest Business Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ABI/INFORM Professional Advanced</collection><collection>ProQuest Biological Science Collection</collection><collection>ABI/INFORM Global</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Science Database</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biological Science Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>ProQuest One Business</collection><collection>ProQuest One Business (Alumni)</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central Basic</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Applied microbiology and biotechnology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Xiong, Yifan</au><au>Cao, Gaihua</au><au>Chen, Xiaolong</au><au>Yang, Jun</au><au>Shi, Meimei</au><au>Wang, Yu</au><au>Nie, Fuping</au><au>Huo, Danqun</au><au>Hou, Changjun</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>One-pot platform for rapid detecting virus utilizing recombinase polymerase amplification and CRISPR/Cas12a</atitle><jtitle>Applied microbiology and biotechnology</jtitle><stitle>Appl Microbiol Biotechnol</stitle><addtitle>Appl Microbiol Biotechnol</addtitle><date>2022-06-01</date><risdate>2022</risdate><volume>106</volume><issue>12</issue><spage>4607</spage><epage>4616</epage><pages>4607-4616</pages><issn>0175-7598</issn><eissn>1432-0614</eissn><abstract>The livestock industry has been deeply affected by African swine fever virus (ASFV) and
Capripoxvirus
(CaPV), which caused an enormous economic damage. It is emergent to develop a reliable detection method. Here, we developed a rapid, ultra-sensitive, and one-pot DNA detection method combining recombinase polymerase amplification (RPA) and CRISPR/Cas12a for ASFV and CaPV, named one-pot-RPA-Cas12a (OpRCas) platform. It had the virtue of both RPA and CRISPR/Cas12a, such as high amplification efficiency, constant temperature reaction, and strict target selectivity, which made diagnosis simplified, accurate and easy to be operated without expensive equipment. Meanwhile, the reagents of RPA and CRISPR/Cas12a were added to the lid and bottom of tube in one go, which overcame the incompatibility of two reactions and aerosol contamination. To save cost, we only need a quarter of the amount of regular RPA per reaction which is enough to achieve clinical diagnosis. The OpRCas platform was 10 to 100 times more sensitive than qPCR; the limit of detection (LOD) was as low as 1.2 × 10
−6
ng/µL (3.07 copies/µL by ddPCR) of ASFV and 7.7 × 10
−5
ng/µL (1.02 copies/µL by ddPCR) of CaPV with the portable fluorometer in 40 min. In addition, the OpRCas platform combined with the lateral flow assay (LFA) strip to suit for point-of-care (POC) testing. It showed 93.3% consistency with qPCR for clinical sample analysis. Results prove that OpRCas platform is an easy-handling, ultra-sensitive, and rapid to achieve ASFV and CaPV POC testing.
Key points
• The platform realizes one-pot reaction of RPA and Cas12a.
• Sensitivity is 100 times more than qPCR.
• Three output modes are suitable to be used to quantitative test or POC testing.</abstract><cop>Berlin/Heidelberg</cop><pub>Springer Berlin Heidelberg</pub><pmid>35708748</pmid><doi>10.1007/s00253-022-12015-9</doi><tpages>10</tpages><orcidid>https://orcid.org/0000-0001-8550-036X</orcidid><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 0175-7598 |
| ispartof | Applied microbiology and biotechnology, 2022-06, Vol.106 (12), p.4607-4616 |
| issn | 0175-7598 1432-0614 |
| language | eng |
| recordid | cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_9201268 |
| source | MEDLINE; SpringerLink Journals |
| subjects | African swine fever African Swine Fever Virus - genetics African Swine Fever Virus - metabolism Air pollution Amplification Animals Applied Genetics and Molecular Biotechnology Asfarviridae Biomedical and Life Sciences Biotechnology Contamination CRISPR CRISPR-Cas Systems Diagnosis Fluorometers Identification and classification Incompatibility Life Sciences Livestock Livestock industry Microbial Genetics and Genomics Microbiology Nucleic Acid Amplification Techniques - methods Nucleotidyltransferases Reagents Real-Time Polymerase Chain Reaction Recombinase Recombinases Recombinases - genetics Selectivity Sensitivity and Specificity Swine Viruses |
| title | One-pot platform for rapid detecting virus utilizing recombinase polymerase amplification and CRISPR/Cas12a |
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