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

Abstract

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.

Recent Advances in CRISPR-Cas Technologies for Synthetic Biology

“…It can also distinguish a single nucleotide polymorphism (SNP) locus in the target DNA of the human genome. Through this method, SARS‑CoV‑2 variant (Najjar et al, 2022 ; Rossetti et al, 2022 ; Wu et al, 2022 ) and African swine fever virus (Qin et al, 2022 ) are diagnosed.…”

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“…And the preamplification also enhanced the sensitivity, as the LOD of the synthesized dsDNA template was calculated to be 7 × 10 4 copies/μL and 7 × 10 2 copies/μL for RPA and LAMP-assisted detection systems, respectively. 104 Meanwhile, utilizing similar strategies, Xiong et al have also achieved one-step and sensitive detection of ASFV and Capripoxvirus (CaPV) via combining RPA amplification and CRISPR/Cas12a detection within one tube. 105 In addition to spatial isolation, temporal separation is another serviceable method to integrate preamplification and CRISPR/ Cas12a cleavage-based detection within one pot.…”

“…This one-pot design successfully saved operation time, as the total detection can be finished within 40 min. And the preamplification also enhanced the sensitivity, as the LOD of the synthesized dsDNA template was calculated to be 7 × 10 4 copies/μL and 7 × 10 2 copies/μL for RPA and LAMP-assisted detection systems, respectively . Meanwhile, utilizing similar strategies, Xiong et al have also achieved one-step and sensitive detection of ASFV and Capripoxvirus (CaPV) via combining RPA amplification and CRISPR/Cas12a detection within one tube …”

“…For instance, Ameen et al have developed a deep learning model for precise prediction of the crRNA activity in SARS-CoV-2 detection . Except for the off-target effect, contaminants from the environment may also lead to a false-positive. , To avoid contamination, more work needs to be done on developing integrated devices and simplifying the workflow of CRISPR/Cas12a POC biosensors.…”

“…129 Except for the off-target effect, contaminants from the environment may also lead to a falsepositive. 87,104 To avoid contamination, more work needs to be done on developing integrated devices and simplifying the workflow of CRISPR/Cas12a POC biosensors.…”