Ultrasensitive and visual detection of Feline herpesvirus type-1 and Feline calicivirus using one-tube dRPA-Cas12a/Cas13a assay
Abstract Background Feline herpesvirus type 1 (FHV) and Feline calicivirus (FCV) are the primary co-infecting pathogens that cause upper respiratory tract disease in cats. However, there are currently no visual detection assays available for on-site testing. Here, we develop an ultrasensitive and vi...
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| Format: | Article |
| Language: | English |
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BMC
2024-03-01
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| Series: | BMC Veterinary Research |
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| Online Access: | https://doi.org/10.1186/s12917-024-03953-9 |
| _version_ | 1797259245944569856 |
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| author | Fumei Jiang Yunjia Liu Xiaonong Yang Yan Li Jian Huang |
| author_facet | Fumei Jiang Yunjia Liu Xiaonong Yang Yan Li Jian Huang |
| author_sort | Fumei Jiang |
| collection | DOAJ |
| description | Abstract Background Feline herpesvirus type 1 (FHV) and Feline calicivirus (FCV) are the primary co-infecting pathogens that cause upper respiratory tract disease in cats. However, there are currently no visual detection assays available for on-site testing. Here, we develop an ultrasensitive and visual detection method based on dual recombinase polymerase amplification (dRPA) reaction and the hybrid Cas12a/Cas13a trans-cleavage activities in a one-tube reaction system, referred to as one-tube dRPA-Cas12a/Cas13a assay. Results The recombinant plasmid DNAs, crRNAs, and RPA oligonucleotides targeting the FCV ORF1 gene and FHV-1 TK gene were meticulously prepared. Subsequently, dual RPA reactions were performed followed by screening of essential reaction components for hybrid CRISPR-Cas12a (targeting the FHV-1 TK gene) and CRISPR-Cas13a (targeting the FCV ORF1 gene) trans-cleavage reaction. As a result, we successfully established an ultra-sensitive and visually detectable method for simultaneous detection of FCV and FHV-1 nucleic acids using dRPA and CRISPR/Cas-powered technology in one-tube reaction system. Visual readouts were displayed using either a fluorescence detector (Fluor-based assay) or lateral flow dipsticks (LDF-based assay). As expected, this optimized assay exhibited high specificity towards only FHV-1 and FCV without cross-reactivity with other feline pathogens while achieving accurate detection for both targets with limit of detection at 2.4 × 10− 1 copies/μL for the FHV-1 TK gene and 5.5 copies/μL for the FCV ORF1 gene, respectively. Furthermore, field detection was conducted using the dRPA-Cas12a/Cas13a assay and the reference real-time PCR methods for 56 clinical samples collected from cats with URTD. Comparatively, the results of Fluor-based assay were in exceptional concordance with the reference real-time PCR methods, resulting in high sensitivity (100% for both FHV-1 and FCV), specificity (100% for both FHV-1 and FCV), as well as consistency (Kappa values were 1.00 for FHV-1 and FCV). However, several discordant results for FHV-1 detection were observed by LDF-based assay, which suggests its prudent use and interpretaion for clinical detection. In spite of this, incorporating dRPA-Cas12a/Cas13a assay and visual readouts will facilitate rapid and accurate detection of FHV-1 and FCV in resource-limited settings. Conclusions The one-tube dRPA-Cas12a/Cas13a assay enables simultaneously ultrasensitive and visual detection of FHV-1 and FCV with user-friendly modality, providing unparalleled convenience for FHV-1 and FCV co-infection surveillance and decision-making of URTD management. |
| first_indexed | 2024-04-24T23:06:22Z |
| format | Article |
| id | doaj.art-c8f0f5e5e863457da9cd76c0cf756ecb |
| institution | Directory Open Access Journal |
| issn | 1746-6148 |
| language | English |
| last_indexed | 2024-04-24T23:06:22Z |
| publishDate | 2024-03-01 |
| publisher | BMC |
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| series | BMC Veterinary Research |
| spelling | doaj.art-c8f0f5e5e863457da9cd76c0cf756ecb2024-03-17T12:29:07ZengBMCBMC Veterinary Research1746-61482024-03-0120111210.1186/s12917-024-03953-9Ultrasensitive and visual detection of Feline herpesvirus type-1 and Feline calicivirus using one-tube dRPA-Cas12a/Cas13a assayFumei Jiang0Yunjia Liu1Xiaonong Yang2Yan Li3Jian Huang4Key Laboratory of Animal Medicine, Southwest Minzu UniversityKey Laboratory of Animal Medicine, Southwest Minzu UniversityKey Laboratory of Animal Medicine, Southwest Minzu UniversityKey Laboratory of Animal Medicine, Southwest Minzu UniversityKey Laboratory of Animal Medicine, Southwest Minzu UniversityAbstract Background Feline herpesvirus type 1 (FHV) and Feline calicivirus (FCV) are the primary co-infecting pathogens that cause upper respiratory tract disease in cats. However, there are currently no visual detection assays available for on-site testing. Here, we develop an ultrasensitive and visual detection method based on dual recombinase polymerase amplification (dRPA) reaction and the hybrid Cas12a/Cas13a trans-cleavage activities in a one-tube reaction system, referred to as one-tube dRPA-Cas12a/Cas13a assay. Results The recombinant plasmid DNAs, crRNAs, and RPA oligonucleotides targeting the FCV ORF1 gene and FHV-1 TK gene were meticulously prepared. Subsequently, dual RPA reactions were performed followed by screening of essential reaction components for hybrid CRISPR-Cas12a (targeting the FHV-1 TK gene) and CRISPR-Cas13a (targeting the FCV ORF1 gene) trans-cleavage reaction. As a result, we successfully established an ultra-sensitive and visually detectable method for simultaneous detection of FCV and FHV-1 nucleic acids using dRPA and CRISPR/Cas-powered technology in one-tube reaction system. Visual readouts were displayed using either a fluorescence detector (Fluor-based assay) or lateral flow dipsticks (LDF-based assay). As expected, this optimized assay exhibited high specificity towards only FHV-1 and FCV without cross-reactivity with other feline pathogens while achieving accurate detection for both targets with limit of detection at 2.4 × 10− 1 copies/μL for the FHV-1 TK gene and 5.5 copies/μL for the FCV ORF1 gene, respectively. Furthermore, field detection was conducted using the dRPA-Cas12a/Cas13a assay and the reference real-time PCR methods for 56 clinical samples collected from cats with URTD. Comparatively, the results of Fluor-based assay were in exceptional concordance with the reference real-time PCR methods, resulting in high sensitivity (100% for both FHV-1 and FCV), specificity (100% for both FHV-1 and FCV), as well as consistency (Kappa values were 1.00 for FHV-1 and FCV). However, several discordant results for FHV-1 detection were observed by LDF-based assay, which suggests its prudent use and interpretaion for clinical detection. In spite of this, incorporating dRPA-Cas12a/Cas13a assay and visual readouts will facilitate rapid and accurate detection of FHV-1 and FCV in resource-limited settings. Conclusions The one-tube dRPA-Cas12a/Cas13a assay enables simultaneously ultrasensitive and visual detection of FHV-1 and FCV with user-friendly modality, providing unparalleled convenience for FHV-1 and FCV co-infection surveillance and decision-making of URTD management.https://doi.org/10.1186/s12917-024-03953-9FHV-1FCVSimultaneous detectionRPAHybrid CRISPR/Cas effectorsVisual modality |
| spellingShingle | Fumei Jiang Yunjia Liu Xiaonong Yang Yan Li Jian Huang Ultrasensitive and visual detection of Feline herpesvirus type-1 and Feline calicivirus using one-tube dRPA-Cas12a/Cas13a assay BMC Veterinary Research FHV-1 FCV Simultaneous detection RPA Hybrid CRISPR/Cas effectors Visual modality |
| title | Ultrasensitive and visual detection of Feline herpesvirus type-1 and Feline calicivirus using one-tube dRPA-Cas12a/Cas13a assay |
| title_full | Ultrasensitive and visual detection of Feline herpesvirus type-1 and Feline calicivirus using one-tube dRPA-Cas12a/Cas13a assay |
| title_fullStr | Ultrasensitive and visual detection of Feline herpesvirus type-1 and Feline calicivirus using one-tube dRPA-Cas12a/Cas13a assay |
| title_full_unstemmed | Ultrasensitive and visual detection of Feline herpesvirus type-1 and Feline calicivirus using one-tube dRPA-Cas12a/Cas13a assay |
| title_short | Ultrasensitive and visual detection of Feline herpesvirus type-1 and Feline calicivirus using one-tube dRPA-Cas12a/Cas13a assay |
| title_sort | ultrasensitive and visual detection of feline herpesvirus type 1 and feline calicivirus using one tube drpa cas12a cas13a assay |
| topic | FHV-1 FCV Simultaneous detection RPA Hybrid CRISPR/Cas effectors Visual modality |
| url | https://doi.org/10.1186/s12917-024-03953-9 |
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