Browse
Submit Data
Databases
API
Help

Dataset Information

18 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

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

ACCESS DATA

Json Xml

Similar Datasets

Molecular Characterization and Cross-Reactivity of Feline Calicivirus Circulating in Southwestern China.

Project description:Feline calicivirus (FCV) is an important pathogen of cats that has two genogroups (GI and GII). To investigate the prevalence and molecular characteristics of FCVs in southwestern China, 162 nasal swab samples were collected from cats in animal shelters and pet hospitals. In total, 38 of the clinical samples (23.46%) were identified as FCV positive using nested RT-PCR. Phylogenetic analyses using 10 capsid protein VP1 sequences revealed that 8 GI and 2 GII strains formed two independent clusters. Additionally, three separated FCVs that were not clustered phylogenetically (two GI and one GII strains) were successfully isolated from clinical samples and their full-length genomes were obtained. Phylogenetic and recombinant analyses of a GI FCV revealed genomic breakpoints in ORF1 and ORF2 regions with evidence for recombinant events between GI sub-genogroups, which is reported in China for the first time. Furthermore, sera obtained from mice immunized independently with the three FCV isolates and a commercial vaccine were used to evaluate the cross-reactivity of neutralizing antibodies. The three separate FCVs were neutralized by each other at a 1:19 to 1:775 titer range, whereas the triple-inactivated vaccine was at a titer of 1:16, which suggested that different genogroup/sub-genogroup FCV strains exhibit significantly different titers of neutralizing antibodies, including the commercial FCV vaccine. Thus, our study revealed the genetic diversity and complex cross-reactivity levels of FCVs in southwestern China, which provides new insights for application in vaccination strategies.

| S-EPMC8473038 | biostudies-literature

Feline calicivirus

Project description:Feline calicivirus Genome sequencing and assembly

| PRJNA728105 | ENA

Feline calicivirus

Project description:Feline calicivirus Raw sequence reads

| PRJNA864891 | ENA

Feline calicivirus

Project description:Feline calicivirus Genome sequencing and assembly

| PRJNA902118 | ENA

Nucleic acid detection with CRISPR-Cas13a/C2c2.

Project description:Rapid, inexpensive, and sensitive nucleic acid detection may aid point-of-care pathogen detection, genotyping, and disease monitoring. The RNA-guided, RNA-targeting clustered regularly interspaced short palindromic repeats (CRISPR) effector Cas13a (previously known as C2c2) exhibits a "collateral effect" of promiscuous ribonuclease activity upon target recognition. We combine the collateral effect of Cas13a with isothermal amplification to establish a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity. We use this Cas13a-based molecular detection platform, termed Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify mutations in cell-free tumor DNA. Furthermore, SHERLOCK reaction reagents can be lyophilized for cold-chain independence and long-term storage and be readily reconstituted on paper for field applications.

| S-EPMC5526198 | biostudies-literature

Based on CRISPR-Cas13a system, to establish a rapid visual detection method for avian influenza viruses.

Project description:To rapidly, specifically, and sensitively detect avian influenza virus (AIV), this research established a visual detection method of recombinase-aided amplification (RAA) based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated proteins 13a (Cas13a) system. In this study, specific primers and CRISPR RNA (crRNA) were designed according to the conservative sequence of AIV Nucleprotein (NP) gene. RAA technology was used to amplify the target sequence, and the amplification products were visually detected by lateral flow dipstick (LFD). The specificity, sensitivity, and reproducibility of RAA-CRISPR-Cas13a-LFD were evaluated. At the same time, this method and polymerase chain reaction (PCR)-agarose electrophoresis method were used to detect clinical samples, and the coincidence rate of the two detection methods was calculated. The results showed that the RAA-CRISPR-Cas13a-LFD method could achieve specific amplification of the target gene fragments, and the detection results could be visually observed through the LFD. Meanwhile, there was no cross-reaction with infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), and Newcastle disease virus (NDV). The sensitivity reached 100 copies/μL, which was 1,000-fold higher than that of PCR-agarose electrophoresis method. The coincidence rate of clinical tests was 98.75 %, and the total reaction time was ~1 h. The RAA-CRISPR-Cas13a-LFD method established in this study had the advantages of rapid, simple, strong specificity, and high sensitivity, which provided a new visual method for AIV detection.

| S-EPMC10800881 | biostudies-literature

Development of CRISPR/Cas13a-based assays for the diagnosis of Schistosomiasis.

Project description:BackgroundSchistosomiasis is a disease that significantly impacts human health in the developing world. Effective diagnostics are urgently needed for improved control of this disease. CRISPR-based technology has rapidly accelerated the development of a revolutionary and powerful diagnostics platform, resulting in the advancement of a class of ultrasensitive, specific, cost-effective and portable diagnostics, typified by applications in COVID-19/cancer diagnosis.MethodsWe developed CRISPR-based diagnostic platform SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) for the detection of Schistosoma japonicum and S. mansoni by combining recombinase polymerase amplification (RPA) with CRISPR-Cas13a detection, measured via fluorescent or colorimetric readouts. We evaluated SHERLOCK assays by using 150 faecal/serum samples collected from Schistosoma-infected ARC Swiss mice (female), and 189 human faecal/serum samples obtained from a S. japonicum-endemic area in the Philippines and a S. mansoni-endemic area in Uganda.FindingsThe S. japonicum SHERLOCK assay achieved 93-100% concordance with gold-standard qPCR detection across all the samples. The S. mansoni SHERLOCK assay demonstrated higher sensitivity than qPCR and was able to detect infection in mouse serum as early as 3 weeks post-infection. In human samples, S. mansoni SHERLOCK had 100% sensitivity when compared to qPCR of faecal and serum samples.InterpretationThese schistosomiasis diagnostic assays demonstrate the potential of SHERLOCK/CRISPR-based diagnostics to provide highly accurate and field-friendly point-of-care tests that could provide the next generation of diagnostic and surveillance tools for parasitic neglected tropical diseases.FundingAustralian Infectious Diseases Research Centre seed grant (2022) and National Health and Medical Research Council (NHMRC) of Australia (APP1194462, APP2008433).

| S-EPMC10382885 | biostudies-literature

Molecular epidemiology and strain diversity of circulating feline Calicivirus in Thai cats.

Project description:Feline calicivirus (FCV) is a significant viral pathogen causing upper respiratory tract and oral diseases in cats. The emergence of the virulent systemic FCV variant (VS-FCV) has raised global concern in the past decade. This study aims to explore the epidemiology, genetic characterization, and diversity of FCV strains circulating among Thai cats. Various sample types, including nasal, oral, and oropharyngeal swabs and fresh tissues, were collected from 184 cats across different regions of Thailand from 2016 to 2021. Using reverse transcription real-time polymerase chain reaction (RT-qPCR), FCV infection was investigated, with additional screening for feline herpesvirus-1 (FHV-1) by qPCR. The detection rates for FCV, FHV-1, and co-infection were 46.7, 65.8, and 31.5%, respectively. Significantly, the odds ratio (OR) revealed a strong association between the detection of a single FCV and the presence of gingivostomatitis lesions (OR: 7.15, 95% CI: 1.89-26.99, p = 0.004). In addition, FCV detection is notably less likely in vaccinated cats (OR: 0.22, 95% CI: 0.07-0.75, p = 0.015). Amino acid sequence analysis based on the VP1 major capsid protein gene of the 14 FCV-Thai (FCV-TH) strains revealed genetic diversity compared to the other 43 global strains (0 to 86.6%). Intriguingly, a vaccine-like FCV variant was detected in one cat. In summary, this study provides insights into the epidemiology and molecular characteristics of FCV diversity within the Thai cat population for the first time. The identification of unique physicochemical characteristics in the capsid hypervariable region of some FCV-TH strains challenges previous hypotheses. Therefore, further exploration of vaccine-like FCV variants is crucial for a comprehensive understanding and to improve viral prevention and control strategies.

| S-EPMC11180889 | biostudies-literature

Molecular epidemiology and phylogenetic analysis of feline calicivirus in Kunshan, China.

Project description:Feline calicivirus (FCV) is a highly contagious virus in cats, which typically causes respiratory tract and oral infections. Despite vaccination against FCV being a regular practice in China, new FCV cases still occur. Antigenic diversity of FCV hinders the effective control by vaccination. This is first report which aims to investigate the molecular epidemiology and molecular characteristics of FCV in Kunshan, China. The nasopharyngeal swabs were collected from cats showing variable clinical signs from different animal clinics in Kunshan from 2022 to 2023. Preliminary detection and sequencing of the FCV capsid gene were performed to study genetic diversity and evolutionary characteristics. FCV-RNA was identified in 52 (26%) of the samples using RT-PCR. A significant association was found between FCV-positive detection rate, age, gender, vaccination status and living environment, while a non-significant association was found with breed of cats. Nucleotide analysis revealed two genotypes, GI and GII. GII predominated in Kunshan, with diverse strains and amino acid variations potentially affecting vaccination efficacy and FCV detection. Notably, analysis pinpointed certain strains' association with FCV-virulent systemic disease pathotypes. This investigation sheds light on FCV dynamics, which may aid in developing better prevention strategies and future vaccine designs against circulating FCV genotypes.

| S-EPMC10900597 | biostudies-literature

CRISPR-Cas13a-Based Detection for Bovine Viral Diarrhea Virus.

Project description:Bovine Viral Diarrhea Virus (BVDV) is the main pathogen of bovine viral diarrhea disease (BVD), which leads to enormous economic losses in the cattle industry. A sensitive and specific detection for BVDV is advantageous to the control of BVDV. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have been used for detecting virus RNA. In this study, the expression and purification of LwCas13a protein was optimized and the RNase activity of LwCas13a in vitro was verified. CRISPR-LwCas13a system could detect BVDV virus and BVDV RNA with high specificity and simplicity. The detection limit of the LwCas13a system was 103 pM, and there were no cross-reactions with HEK293T and MDBK. In summary, a sensitive, specific, and simple nucleic acid detection method based on CRISPR-Cas13a was developed for BVDV. This method provides a new detection strategy for early diagnosis of BVDV.

| S-EPMC8219879 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data