Rapid detection of FAdV-4 by one-tube RPA-CRISPR/Cas12a assay
IntroductionFowl adenovirus serotype 4 (FAdV-4) is a highly contagious viral pathogen of global significance that affects various avian species. It primarily infects poultry and wild birds, leading to avian inclusion body hepatitis (IBH) and hepatitis-hydropericardium syndrome (HHS). The development...
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Frontiers Media S.A.
2025-02-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2025.1541943/full |
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| author | Lei Ma Xueping Wang Mingliang Zhang Mengjie Zhu |
| author_facet | Lei Ma Xueping Wang Mingliang Zhang Mengjie Zhu |
| author_sort | Lei Ma |
| collection | DOAJ |
| description | IntroductionFowl adenovirus serotype 4 (FAdV-4) is a highly contagious viral pathogen of global significance that affects various avian species. It primarily infects poultry and wild birds, leading to avian inclusion body hepatitis (IBH) and hepatitis-hydropericardium syndrome (HHS). The development of rapid diagnostic tools for detecting FAdV-4 is crucial for effective disease control and eradication efforts.MethodsIn this study, we developed a recombinase polymerase amplification (RPA) combined with CRISPR/Cas12a assay, specifically targeting the FAdV-4 Hexon gene. RPA and CRISPR/Cas12a reagents were added to the bottom and lid of the test tube at once, allowing the detection process to occur within a single reaction tube. This approach reduced contamination.ResultsThe RPA-CRISPR/Cas12a detection method can identify as few as 10 copies of the genome per reaction, demonstrating 100% sensitivity comparable to that of fluorescence PCR (qPCR). This approach exhibits high specificity for FAdV-4, with no cross-reactivity observed with other FAdV serotypes or common avian pathogens. Additionally, the agreement rate between the results of RPA-CRISPR/Cas12a and qPCR for detecting clinical samples is as high as 97.5%.DiscussionTherefore, the RPA-CRISPR/Cas12a assay presents a promising alternative for the simple, sensitive, and specific identification of FAdV-4. |
| format | Article |
| id | doaj-art-cf07bf4c87b44e7e90776e91da9579c9 |
| institution | Kabale University |
| issn | 1664-302X |
| language | English |
| publishDate | 2025-02-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Microbiology |
| spelling | doaj-art-cf07bf4c87b44e7e90776e91da9579c92025-02-03T06:33:53ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2025-02-011610.3389/fmicb.2025.15419431541943Rapid detection of FAdV-4 by one-tube RPA-CRISPR/Cas12a assayLei Ma0Xueping Wang1Mingliang Zhang2Mengjie Zhu3School of Biotechnology and Food Engineering, Anyang Institute of Technology, Anyang, ChinaCollege of Animal Science and Technology, Tarim University, AIar, ChinaSchool of Biotechnology and Food Engineering, Anyang Institute of Technology, Anyang, ChinaSchool of Biotechnology and Food Engineering, Anyang Institute of Technology, Anyang, ChinaIntroductionFowl adenovirus serotype 4 (FAdV-4) is a highly contagious viral pathogen of global significance that affects various avian species. It primarily infects poultry and wild birds, leading to avian inclusion body hepatitis (IBH) and hepatitis-hydropericardium syndrome (HHS). The development of rapid diagnostic tools for detecting FAdV-4 is crucial for effective disease control and eradication efforts.MethodsIn this study, we developed a recombinase polymerase amplification (RPA) combined with CRISPR/Cas12a assay, specifically targeting the FAdV-4 Hexon gene. RPA and CRISPR/Cas12a reagents were added to the bottom and lid of the test tube at once, allowing the detection process to occur within a single reaction tube. This approach reduced contamination.ResultsThe RPA-CRISPR/Cas12a detection method can identify as few as 10 copies of the genome per reaction, demonstrating 100% sensitivity comparable to that of fluorescence PCR (qPCR). This approach exhibits high specificity for FAdV-4, with no cross-reactivity observed with other FAdV serotypes or common avian pathogens. Additionally, the agreement rate between the results of RPA-CRISPR/Cas12a and qPCR for detecting clinical samples is as high as 97.5%.DiscussionTherefore, the RPA-CRISPR/Cas12a assay presents a promising alternative for the simple, sensitive, and specific identification of FAdV-4.https://www.frontiersin.org/articles/10.3389/fmicb.2025.1541943/fullavian adenovirus type 4Cas12arecombinase polymerase amplificationdetectionsensitivity |
| spellingShingle | Lei Ma Xueping Wang Mingliang Zhang Mengjie Zhu Rapid detection of FAdV-4 by one-tube RPA-CRISPR/Cas12a assay Frontiers in Microbiology avian adenovirus type 4 Cas12a recombinase polymerase amplification detection sensitivity |
| title | Rapid detection of FAdV-4 by one-tube RPA-CRISPR/Cas12a assay |
| title_full | Rapid detection of FAdV-4 by one-tube RPA-CRISPR/Cas12a assay |
| title_fullStr | Rapid detection of FAdV-4 by one-tube RPA-CRISPR/Cas12a assay |
| title_full_unstemmed | Rapid detection of FAdV-4 by one-tube RPA-CRISPR/Cas12a assay |
| title_short | Rapid detection of FAdV-4 by one-tube RPA-CRISPR/Cas12a assay |
| title_sort | rapid detection of fadv 4 by one tube rpa crispr cas12a assay |
| topic | avian adenovirus type 4 Cas12a recombinase polymerase amplification detection sensitivity |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2025.1541943/full |
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