Ultrasensitive and visual detection of Feline herpesvirus type-1 and Feline calicivirus using one-tube dRPA-Cas12a/Cas13a assay.

Autor: Jiang F; Key Laboratory of Animal Medicine, Southwest Minzu University, Chengdu City, Sichuan Province, China., Liu Y; Key Laboratory of Animal Medicine, Southwest Minzu University, Chengdu City, Sichuan Province, China., Yang X; Key Laboratory of Animal Medicine, Southwest Minzu University, Chengdu City, Sichuan Province, China., Li Y; Key Laboratory of Animal Medicine, Southwest Minzu University, Chengdu City, Sichuan Province, China. lyvet2004@163.com.; Department of Clinical Veterinary Medicine, College of Animal Science and Veterinary Medicine, Southwest Minzu University, No. 16, South 4th Section, 1st-Ring Road, Wuhou, Chengdu, Sichuan, 610041, China. lyvet2004@163.com., Huang J; Key Laboratory of Animal Medicine, Southwest Minzu University, Chengdu City, Sichuan Province, China. hjvet03@sina.cn.; Veterinary Teaching Hospital, Southwest Minzu University, Chengdu, China. hjvet03@sina.cn.; Department of Clinical Veterinary Medicine, College of Animal Science and Veterinary Medicine, Southwest Minzu University, No. 16, South 4th Section, 1st-Ring Road, Wuhou, Chengdu, Sichuan, 610041, China. hjvet03@sina.cn.
Jazyk: angličtina
Zdroj: BMC veterinary research [BMC Vet Res] 2024 Mar 16; Vol. 20 (1), pp. 106. Date of Electronic Publication: 2024 Mar 16.
DOI: 10.1186/s12917-024-03953-9
Abstrakt: 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.
(© 2024. The Author(s).)
Databáze: MEDLINE
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