AUTHOR=Qin Chao , Liu Jiajia , Zhu Wenqi , Zeng Muchu , Xu Ke , Ding Jinmei , Zhou Hao , Zhu Jianshen , Ke Yuqing , Li Lai Yan , Sheng Gaoyuan , Li Zhuoru , Luo Huaixi , Jiang Shengyao , Chen Kangchun , Ding Xianting , Meng He TITLE=One-Pot Visual Detection of African Swine Fever Virus Using CRISPR-Cas12a JOURNAL=Frontiers in Veterinary Science VOLUME=9 YEAR=2022 URL=https://www.frontiersin.org/journals/veterinary-science/articles/10.3389/fvets.2022.962438 DOI=10.3389/fvets.2022.962438 ISSN=2297-1769 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.