Browse
Submit Data
Databases
API
Help

Dataset Information

21 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Rapid and sensitive RPA-Cas12a-fluorescence assay for point-of-care detection of African swine fever virus.

ABSTRACT: African swine fever (ASF) is a serious contagious disease that causes fatal haemorrhagic fever in domestic and wild pigs, with high morbidity. It has caused devastating damage to the swine industry worldwide, necessitating the focus of attention on detection of the ASF pathogen, the African swine fever virus (ASFV). In order to overcome the disadvantages of conventional diagnostic methods (e.g. time-consuming, demanding and unintuitive), quick detection tools with higher sensitivity need to be explored. In this study, based on the conserved p72 gene sequence of ASFV, we combined the Cas12a-based assay with recombinase polymerase amplification (RPA) and a fluorophore-quencher (FQ)-labeled reporter assay for rapid and visible detection. Five crRNAs designed for Cas12a-based assay showed specificity with remarkable fluorescence intensity under visual inspection. Within 20 minutes, with an initial concentration of two copies of DNA, the assay can produce significant differences between experimental and negative groups, indicating the high sensitivity and rapidity of the method. Overall, the developed RPA-Cas12a-fluorescence assay provides a fast and visible tool for point-of-care ASFV detection with high sensitivity and specificity, which can be rapidly performed on-site under isothermal conditions, promising better control and prevention of ASF.

SUBMITTER: Fu J

PROVIDER: S-EPMC8289064 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Development of RPA-Cas12a assay for rapid and sensitive detection of Pneumocystis jirovecii

Project description: Not available

| S-EPMC11345955 | biostudies-literature

Rapid and sensitive point-of-care detection of Leptospira by RPA-CRISPR/Cas12a targeting lipL32.

Project description:BackgroundOne of the key barriers preventing rapid diagnosis of leptospirosis is the lack of available sensitive point-of-care testing. This study aimed to develop and validate a clustered regularly-interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 12a (CRISPR/Cas12a) platform combined with isothermal amplification to detect leptospires from extracted patient DNA samples.Methodology/principal findingsA Recombinase Polymerase Amplification (RPA)-CRISPR/Cas12a-fluorescence assay was designed to detect the lipL32 gene of pathogenic Leptospira spp. The assays demonstrated a limit of detection (LOD) of 100 cells/mL, with no cross-reactivity against several other acute febrile illnesses. The clinical performance of the assay was validated with DNA extracted from 110 clinical specimens and then compared to results from qPCR detection of Leptospira spp. The RPA-CRISPR/Cas12a assay showed 85.2% sensitivity, 100% specificity, and 92.7% accuracy. The sensitivity increased on days 4-6 after the fever onset and decreased after day 7. The specificity was consistent for several days after the onset of fever. The overall performance of the RPA-CRISPR/Cas12a platform was better than the commercial rapid diagnostic test (RDT). We also developed a lateral flow detection assay (LFDA) combined with RPA-CRISPR/Cas12a to make the test more accessible and easier to interpret. The combined LFDA showed a similar LOD of 100 cells/mL and could correctly distinguish between known positive and negative clinical samples in a pilot study.Conclusions/significanceThe RPA-CRISPR/Cas12 targeting the lipL32 gene demonstrated acceptable sensitivity and excellent specificity for detection of leptospires. This assay might be an appropriate test for acute leptospirosis screening in limited-resource settings.

| S-EPMC8769300 | biostudies-literature

Rapid detection of African swine fever virus using Cas12a-based portable paper diagnostics.

Project description:African swine fever virus (ASFV) is a dsDNA virus responsible for a severe, highly contagious, and lethal disease affecting both domestic and wild pigs. ASFV has brought enormous economic loss to a number of countries, and effective vaccine and therapy are still lacking. Therefore, a rapid, sensitive, and field-deployable detection of ASFV is important for disease surveillance and control. Herein, we developed a Cas12a-mediated portable paper assay to rapidly and precisely detect ASFV. We identified a robust set of crRNAs that recognized the highly conserved region of essential ASFV genes. The Cas12a-mediated detection assay showed low tolerance for mismatch mutations, and no cross-reactivity against other common swine pathogens. We further developed a paper-based assay to allow instrument-free detection of ASFV. Specifically, we applied gold nanoparticle-antibody conjugate to engineer homemade strips and combined it with Cas12a-mediated ASFV detection. This portable paper, instrument-free diagnostics, faithfully detected ASFV in swine samples, showing comparable sensitivity to the traditionally instrument-dependent qPCR method. Taking together, we developed a highly sensitive, instant, and economic Cas12a-mediated paper diagnostics of ASFV, with a great application potential for monitoring ASFV in the field.

| S-EPMC7136273 | biostudies-literature

Establishment of RT-RPA-Cas12a assay for rapid and sensitive detection of human rhinovirus B.

Project description:Human rhinovirus B (HRV-B) is a major human viral pathogen that can be responsible for various kinds of infections. Due to the health risks associated with HRV-B, it is therefore crucial to explore a rapid, specific, and sensitive method for surveillance. Herein, we exploited a novel detection method for HRV-B by combining reverse-transcription recombinase polymerase amplification (RT-RPA) of nucleic acids isothermal amplification and the trans-cleavage activity of Cas12a. Our RT-RPA-Cas12a-based fluorescent assay can be completed within 35-45 min and obtain a lower detection threshold to 0.5 copies/µL of target RNA. Meanwhile, crRNA sequences without a specific protospacer adjacent motif can effectively activate the trans-cleavage activity of Cas12a. Moreover, our RT-RPA-Cas12a-based fluorescent method was examined using 30 clinical samples, and exhibited high accuracy with positive and negative predictive agreement of 90% and 100%, respectively. Taken together, a novel promising, rapid and effective RT-RPA-Cas12a-based detection method was explored and shows promising potential for on-site HRV-B infection in resource-limited settings.

| S-EPMC10640725 | biostudies-literature

Rapid and Visual RPA-Cas12a Fluorescence Assay for Accurate Detection of Dermatophytes in Cats and Dogs.

Project description:Dermatophytosis, an infectious disease caused by several fungi, can affect the hair, nails, and/or superficial layers of the skin and is of global significance. The most common dermatophytes in cats and dogs are Microsporum canis and Trichophyton mentagrophytes. Wood's lamp examination, microscopic identification, and fungal culture are the conventional clinical diagnostic methods, while PCR (Polymerase Chain Reaction) and qPCR (Quantitative PCR) are playing an increasingly important role in the identification of dermatophytes. However, none of these methods could be applied to point-of-care testing (POCT). The recent development of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) based diagnostic platform promises a rapid, accurate, and portable diagnostic tool. In this paper, we present a Cas12a-fluorescence assay to detect and differentiate the main dermatophytes in clinical samples with high specificity and sensitivity. The Cas12a-based assay was performed with a combination of recombinase polymerase amplification (RPA). The results could be directly visualized by naked eyes under blue light, and all tested samples were consistent with fungal culture and sequencing results. Compared with traditional methods, the RPA-Cas12a-fluorescence assay requires less time (about 30 min) and less complicated equipment, and the visual changes can be clearly observed with naked eyes, which is suitable for on-site clinical diagnosis.

| S-EPMC9406134 | biostudies-literature

Efficient, Rapid, and Sensitive Detection of Plant RNA Viruses With One-Pot RT-RPA–CRISPR/Cas12a Assay

Project description:Most viruses that infect plants use RNA to carry their genomic information; timely and robust detection methods are crucial for efficient control of these diverse pathogens. The RNA viruses, potexvirus (Potexvirus, family Alphaflexiviridae), potyvirus (Potyvirus, family Potyviridae), and tobamovirus (Tobamovirus, family Virgaviridae) are among the most economically damaging pathogenic plant viruses, as they are highly infectious and distributed worldwide. Their infection of crop plants, alone or together with other viruses, causes severe yield losses. Isothermal nucleic acid amplification methods, such as loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and others have been harnessed for the detection of DNA- and RNA-based viruses. However, they have a high rate of non-specific amplification and other drawbacks. The collateral activities of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease Cas systems such as Cas12 and Cas14 (which act on ssDNA) and Cas13 (which acts on ssRNA) have recently been exploited to develop highly sensitive, specific, and rapid detection platforms. Here, we report the development of a simple, rapid, and efficient RT- RPA method, coupled with a CRISPR/Cas12a-based one-step detection assay, to detect plant RNA viruses. This diagnostic method can be performed at a single temperature in less than 30 min and integrated with an inexpensive commercially available fluorescence visualizer to facilitate rapid, in-field diagnosis of plant RNA viruses. Our developed assay provides an efficient and robust detection platform to accelerate plant pathogen detection and fast-track containment strategies.

| S-EPMC7773598 | biostudies-literature

Point-of-Care Testing for Sensitive Detection of the African Swine Fever Virus Genome

Project description:African swine fever (ASF) is a contagious viral hemorrhagic disease that affects domestic pigs and wild boar. The disease is notifiable to the World Organization of Animal Health (WOAH), and causes significant deaths and economic losses. There is currently no fully licensed vaccine available. As a result, early identification of the causative agent, ASF virus (ASFV), is crucial for the implementation of control measures. PCR and real-time PCR are the WOAH-recommended standard methods for the direct detection of ASFV. However, under special field conditions or in simple or remote field laboratories, there may be no sophisticated equipment or even stable electricity available. Under these circumstances, point-of-care systems can be put in place. Along these lines, a previously published, rapid, reliable, and electricity-free extraction method (TripleE) was used to isolate viral nucleic acid from diagnostic specimens. With this tool, nucleic acid extraction from up to eight diagnostic samples can be realized in one run in less than 10 min. In addition, the possibility of completely omitting viral DNA extraction was analyzed with so-called direct real-time PCR protocols using ASFV original samples diluted to 1:40 in RNase-free water. Furthermore, three real-time PCR cyclers, developed for use under field conditions (IndiField, Liberty16 and UF-300 GenecheckerTM), were comparatively applied for the sensitive high-speed detection of ASFV genomes, with overall PCR run times between 20 and 54 min. Depending on the viral DNA extraction/releasing method used and the point-of-care cycler applied, a total time for detection of 30 to 60 min for up to eight samples was feasible. As expected, the limitations in analytical sensitivity were positively correlated to the analysis time. These limitations are acceptable for ASFV diagnostics due to the expected high ASFV genome loads in diseased animals or carcasses.

| S-EPMC9781289 | biostudies-literature

A recombinase polymerase amplification-based assay for rapid detection of African swine fever virus.

Project description:A recombinase polymerase amplification (RPA)-based method was developed for rapid and specific detection of African swine fever virus (ASFV), the etiologic agent of African swine fever, a devastating disease of swine. Primers and the exo probe targeting the conserved region of the P72 gene of ASFV were designed and the reaction was run on the Genie III scanner device. Using recombinant plasmid DNA containing the P72 gene as template, we showed that the amplified product could be detected in less than 10 min and that the detection limit was 102 copies DNA/reaction [same detection limit as real-time polymerase chain reaction (PCR)]. The RPA assay did not cross-detect CSFV, PCV-2, PRV, PRRSV, or FMDV, common viruses seen in pigs. Tests of recombinant plasmid-spiked serum samples revealed that RPA and real-time PCR had the same diagnostic rate. The RPA assay, which is simple, cost-effective, and fast, is a promising alternative to real-time PCR for ASFV detection.

| S-EPMC5644446 | biostudies-literature

One-Pot Visual Detection of African Swine Fever Virus Using CRISPR-Cas12a.

Project description:African swine fever virus (ASFV) is a leading cause of worldwide agricultural loss. ASFV is a highly contagious and lethal disease for both domestic and wild pigs, which has brought enormous economic losses to a number of countries. Conventional methods, such as general polymerase chain reaction and isothermal amplification, are time-consuming, instrument-dependent, and unsatisfactorily accurate. Therefore, rapid, sensitive, and field-deployable detection of ASFV is important for disease surveillance and control. Herein, we created a one-pot visual detection system for ASFV with CRISPR/Cas12a technology combined with LAMP or RPA. A mineral oil sealing strategy was adopted to mitigate sample cross-contamination between parallel vials during high-throughput testing. Furthermore, the blue fluorescence signal produced by ssDNA reporter could be observed by the naked eye without any dedicated instrument. For CRISPR-RPA system, detection could be completed within 40 min with advantageous sensitivity. While CRISPR-LAMP system could complete it within 60 min with a high sensitivity of 5.8 × 102 copies/μl. Furthermore, we verified such detection platforms display no cross-reactivity with other porcine DNA or RNA viruses. Both CRISPR-RPA and CRISPR-LAMP systems permit highly rapid, sensitive, specific, and low-cost Cas12a-mediated visual diagnostic of ASFV for point-of-care testing (POCT) applications.

| S-EPMC9339671 | biostudies-literature

Development and clinical application of a novel CRISPR-Cas12a based assay for the detection of African swine fever virus.

Project description:BACKGROUND:As no treatment or effective vaccine for African swine fever virus (ASFV) is currently available, a rapid, highly sensitive diagnostic is urgently needed to curb the spread of ASFV. RESULTS:Here we designed a novel CRISPR-Cas12a based assay for ASFV detection. To detect different ASFV genotypes, 19 crRNAs were designed to target the conserved p72 gene in ASFV, and several crRNAs with high activity were identified that could be used as alternatives in the event of new ASFV variants. The results showed that the sensitivity of the CRISPR-Cas12a based assay is about ten times higher than either the commercial quantitative PCR (qPCR) kit or the OIE-recommended qPCR. CRISPR-Cas12a based assay could also detect ASFV specifically without cross-reactivity with other important viruses in pigs and various virus genotypes. We also found that longer incubation times increased the detection limits, which could be applied to improve assay outcomes in the detection of weakly positive samples and new ASFV variants. In addition, both the CRISPR-Cas12a based assay and commercial qPCR showed very good consistency. CONCLUSIONS:In summary, the CRISPR-Cas12a based assay offers a feasible approach and a new diagnostic technique for the diagnosis of ASFV, particularly in resource-poor settings.

| S-EPMC7491166 | 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