Non-nucleic acid extraction and ultra-sensitive detection of African swine fever virus via CRISPR/Cas12a.

Early diagnosis of the African swine fever virus (ASFV) is the main preventive measure for ASFV. Here, we developed a fluorescent biosensor and lateral flow assay (LFA) strip based on direct PCR combined with CRISPR/Cas12a system for ASF. Direct PCR can simultaneously split samples and efficiently amplify without sacrificing sensitivity, which eliminated the steps of nucleic acid extraction. Furthermore, by the CRISPR/Cas12a, the biosensor addressed false positives caused by non-specific amplification and had high sensitivity with the actual limit of detection (LOD) of 7.6×10-4 ng·μL-1 (4 copies·μL-1). In addition, the strategy was built on the lateral flow assay (LFA) strip to achieve visual and portable detection for point-of-care testing. Moreover, the biosensor by a fluorometer and LFA strip showed a high accuracy to rival qPCR in actual sample detection. Therefore, the biosensor is an ultra-sensitive and specific tool that can replace traditional methods. KEY POINTS: • No nucleic acid extraction, direct PCR-simplified steps, and reduced time and cost • CRISPR/Cas12a solved the false positives caused by nonspecific amplification • The combination of the LFA strip and biosensor is more convenient for POC detection.

Cao G ，Xiong Y ，Nie F ，Chen X ，Peng L ，Li Y ，Yang M ，Huo D ，Hou C ... -  《-》

One-pot platform for rapid detecting virus utilizing recombinase polymerase amplification and CRISPR/Cas12a.

The livestock industry has been deeply affected by African swine fever virus (ASFV) and Capripoxvirus (CaPV), which caused an enormous economic damage. It is emergent to develop a reliable detection method. Here, we developed a rapid, ultra-sensitive, and one-pot DNA detection method combining recombinase polymerase amplification (RPA) and CRISPR/Cas12a for ASFV and CaPV, named one-pot-RPA-Cas12a (OpRCas) platform. It had the virtue of both RPA and CRISPR/Cas12a, such as high amplification efficiency, constant temperature reaction, and strict target selectivity, which made diagnosis simplified, accurate and easy to be operated without expensive equipment. Meanwhile, the reagents of RPA and CRISPR/Cas12a were added to the lid and bottom of tube in one go, which overcame the incompatibility of two reactions and aerosol contamination. To save cost, we only need a quarter of the amount of regular RPA per reaction which is enough to achieve clinical diagnosis. The OpRCas platform was 10 to 100 times more sensitive than qPCR; the limit of detection (LOD) was as low as 1.2 × 10-6 ng/µL (3.07 copies/µL by ddPCR) of ASFV and 7.7 × 10-5 ng/µL (1.02 copies/µL by ddPCR) of CaPV with the portable fluorometer in 40 min. In addition, the OpRCas platform combined with the lateral flow assay (LFA) strip to suit for point-of-care (POC) testing. It showed 93.3% consistency with qPCR for clinical sample analysis. Results prove that OpRCas platform is an easy-handling, ultra-sensitive, and rapid to achieve ASFV and CaPV POC testing. KEY POINTS: • The platform realizes one-pot reaction of RPA and Cas12a. • Sensitivity is 100 times more than qPCR. • Three output modes are suitable to be used to quantitative test or POC testing.

Xiong Y ，Cao G ，Chen X ，Yang J ，Shi M ，Wang Y ，Nie F ，Huo D ，Hou C ... -  《-》

KP177R-based visual assay integrating RPA and CRISPR/Cas12a for the detection of African swine fever virus.

Early detection of the virus in the environment or in infected pigs is a critical step to stop African swine fever virus (ASFV) transmission. The p22 protein encoded by ASFV KP177R gene has been shown to have no effect on viral replication and virulence and can serve as a molecular marker for distinguishing field virus strains from future candidate KP177R deletion vaccine strains. This study established an ASFV detection assay specific for the highly conserved ASFV KP177R gene based on recombinase polymerase amplification (RPA) and the CRISPR/Cas12 reaction system. The KP177R gene served as the initial template for the RPA reaction to generate amplicons, which were recognized by guide RNA to activate the trans-cleavage activity of Cas12a protein, thereby leading to non-specific cleavage of single-stranded DNA as well as corresponding color reaction. The viral detection in this assay could be determined by visualizing the results of fluorescence or lateral flow dipstick (LFD) biotin blotting for color development, and was respectively referred to as fluorescein-labeled RPA-CRISPR/Cas12a and biotin-labeled LFD RPA-CRISPR/Cas12a. The clinical samples were simultaneously subjected to the aforementioned assay, while real-time quantitative PCR (RT-qPCR) was employed as a control for determining the diagnostic concordance rate between both assays. The results showed that fluorescein- and biotin-labeled LFD KP177R RPA-CRISPR/Cas12a assays specifically detected ASFV, did not cross-react with other swine pathogens including PCV2, PEDV, PDCoV, and PRV. The detection assay established in this study had a limit of detection (LOD) of 6.8 copies/μL, and both assays were completed in 30 min. The KP177R RPA-CRISPR/Cas12a assay demonstrated a diagnostic coincidence rate of 100% and a kappa value of 1.000 (p < 0.001), with six out of ten clinical samples testing positive for ASFV using both KP177R RPA-CRISPR/Cas12a and RT-qPCR, while four samples tested negative in both assays. The rapid, sensitive and visual detection assay for ASFV developed in this study is suitable for field application in swine farms, particularly for future differentiation of field virus strains from candidate KP177R gene-deleted ASFV vaccines, which may be a valuable screening tool for ASF eradication.

Luan H ，Wang S ，Ju L ，Liu T ，Shi H ，Ge S ，Jiang S ，Wu J ，Peng J ... -  《Frontiers in Immunology》

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

Wang X ，He S ，Zhao N ，Liu X ，Cao Y ，Zhang G ，Wang G ，Guo C ... -  《BMC MICROBIOLOGY》

Dipstick-based rapid nucleic acids purification and CRISPR/Cas12a-mediated isothermal amplification for visual detection of African swine fever virus.

African swine fever virus (ASFV) can cause highly contagious and fatal disease among domestic pigs, resulting in considerable economic losses for swine breeders. There is a strong demand for accurate, rapid, and simple detection methods especially for on-site application. Nucleic acid testing is the most commonly used method for ASFVdetection. However, traditional nucleic acid purification step is time- and labor-consuming. The nucleic acid purification, amplification and amplicons detection rely on laboratory settings which limits the on-site detection. Here, we proposed a simple and cost-effective detection method that utilized filter paper to purify nucleic acids from swine blood and employed CRISPR/Cas12a-mediated loop-mediated isothermal amplification (LAMP) reaction to detect ASFV. The filter paper which was made into dipsticks could effectively purify nucleic acids from whole blood in 2 min. This simple and low-cost purification method avoided multiple pipetting steps and potential amplification inhibitors (e.g., ethanol) that were generally used in traditional nucleic acids extraction processes. After nucleic acid purification, the lyophilized LAMP reagent dissolved by elution solution was employed to perform isothermal amplification reaction on a portable heating block. The CRISPR/Cas12a system was designed to specifically detect amplicons. Assisted by a portable homemade device, the fluorescent signals produced by positive samples could be observed by the naked eye, while negative samples remained colorless. The whole detection procedure could be finished within 50 min with a detection limit of one copies/μL. This established method provided a novel strategy for rapid visualized detection and showed great potential for on-site application.

Qian S ，Chen Y ，Peng C ，Wang X ，Wu H ，Che Y ，Wang H ，Xu J ，Wu J ... -  《-》