Application of CRISPR-Cas12a Enhanced Fluorescence Assay Coupled with Nucleic Acid Amplification for the Sensitive Detection of African Swine Fever Virus

AbstractHere we report the development of a high throughput, all-solution phase, and isothermal detection system to detect African Swine Fever Virus (ASFV). CRISPR-Cas12a programmed with a CRISPR RNA (crRNA) is used to detect ASFV target DNA. Upon ASFV DNA binding, the Cas12a/crRNA/ASFV DNA complex becomes activated and degrades a fluorescent single stranded DNA (ssDNA) reporter present in the assay. We combine this powerful CRISPR-Cas assay with fluorescence-based point-of-care (POC) system we developed for rapid and accurate virus detection. Without nucleic acid amplification, a detection limit of 1 pM is achieved within 2 hrs. In addition, the ternary Cas12a/crRNA/ASFV DNA complex is highly stable at physiological temperature and continues to cleave the ssDNA reporter even after 24 hrs of incubation, resulting in an improvement of the detection limit to 100 fM. We show that this system is very specific and can differentiate nucleic acid targets with closely matched sequences. The high sensitivity and selectivity of our system enables the detection of ASFV in femtomolar range. Importantly, this system features a disposable cartridge and a sensitive custom designed fluorometer, enabling compact, multiplexing, and simple ASFV detection, intended for low resource settings.

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Application of an AlphaLISA method for rapid sensitive detection of African swine fever virus in porcine serum

Applied Microbiology and Biotechnology ◽

10.1007/s00253-021-11339-2 ◽

2021 ◽

Author(s):

Dongjie Chen ◽

Di Wang ◽

Caixia Wang ◽

Fang Wei ◽

Hongyuan Zhao ◽

...

Keyword(s):

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Sensitive Detection ◽

Porcine Serum

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

PLoS ONE ◽

10.1371/journal.pone.0254815 ◽

2021 ◽

Vol 16 (7) ◽

pp. e0254815

Author(s):

Jinyu Fu ◽

Yueping Zhang ◽

Guang Cai ◽

Geng Meng ◽

Shuobo Shi

Keyword(s):

African Swine Fever Virus ◽

Point Of Care ◽

African Swine Fever ◽

High Sensitivity ◽

Fluorescence Assay ◽

Fever Virus ◽

Diagnostic Methods ◽

High Morbidity ◽

Swine Industry ◽

Control And Prevention

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.

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Evidence for the type of nucleic acid in African swine fever virus

Archives of Virology ◽

10.1007/bf01241851 ◽

1966 ◽

Vol 19 (3) ◽

pp. 289-304 ◽

Cited By ~ 15

Author(s):

W. Plowright ◽

F. Brown ◽

J. Parker

Keyword(s):

Nucleic Acid ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus

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Optimizing Release of Nucleic Acids of African Swine Fever Virus and Influenza A Virus from FTA Cards

International Journal of Molecular Sciences ◽

10.3390/ijms222312915 ◽

2021 ◽

Vol 22 (23) ◽

pp. 12915

Author(s):

Ahmed Elnagar ◽

Timm C. Harder ◽

Sandra Blome ◽

Martin Beer ◽

Bernd Hoffmann

Keyword(s):

Nucleic Acid ◽

Influenza A Virus ◽

Influenza A ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Universal Method ◽

Viral Dna ◽

Dna And Rna ◽

Fta Cards

FTA cards and related products simplify the collection, transport, and transient storage of biological sample fluids. Here, we have compared the yield and quality of DNA and RNA released from seven different FTA cards using seven releasing/extraction methods with eleven experimental eluates. For the validation, dilution series of African swine fever virus (ASFV) positive EDTA blood and Influenza A virus (IAV) positive allantoic fluid were used. Based on our data, we conclude that direct PCR amplification without the need for additional nucleic acid extraction and purification could be suitable and more convenient for ASFV DNA release from FTA cards. In contrast, IAV RNA loads can be amplified from FTA card punches if a standard extraction procedure including a lysis step is applied. These differences between the amplifiable viral DNA and RNA after releasing and extraction are not influenced by the type of commercial FTA card or the eleven different nucleic acid releasing procedures used for the comparative analyses. In general, different commercial FTA cards were successfully used for the storage and recovery of the ASFV and IAV genetic material suitable for PCR. Nevertheless, the usage of optimized nucleic acid releasing protocols could improve the recovery of the viral genome of both viruses. Here, the application of Chelex® Resin 100 buffer mixed with 1 × Tris EDTA buffer (TE, pH 8.0) or with TED 10 (TE buffer and Dimethylsulfoxid) delivered the best results and can be used as a universal method for releasing viral DNA and RNA from FTA cards.

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