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CRISPR-Cas13a Based Visual Detection Assays for Feline Calicivirus Circulating in Southwest China.

ABSTRACT: Feline calicivirus (FCV) is a well-known causative pathogen for upper respiratory infection in cats. Its high genetic variability challenges existing molecular diagnostic methods in clinical settings. Thus, we developed two sensitive and visual assays for FCV nucleic acid detection based on RPA reaction and CRISPR-Cas13a trans-cleavage activity. Recombinant plasmid DNA, crRNAs, and RPA primers were designed and prepared, respectively, targeting to FCV ORF1 gene. Besides, purified LwCas13a protein was produced by E.coli prokaryotic expression system. To confirm the validity of FCV-Cas13a assays, seven reaction systems (RSs) with different components were tested, and visual readouts were displayed by lateral flow dipstick (FCV-Cas13a-LFD) and fluorescence detector (FCV-Cas13a-FLUOR), respectively. The established FCV-Cas13a assays were capable of detecting FCV nucleic acid in presetting RSs without cross-reaction with other feline-associated pathogens, and the detection limit was as low as 5.5 copies/μl for both visual methods. Moreover, the positive rate of 56 clinical specimens detected by FCV-Cas13a assays (67.9%, 38/56) was notably higher than that of RT-qPCR (44.6%, 25/56) (p < 0.001), including 13 presumptive positive specimens. Taken together, FCV-Cas13a assays provided reliable and visual diagnostic alternatives for FCV field detection.

SUBMITTER: Huang J

PROVIDER: S-EPMC9310557 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundFeline 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.ResultsThe 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.ConclusionsThe 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.

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CRISPR-Cas13a Based Visual Detection Assays for Feline Calicivirus Circulating in Southwest China.

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