Establishment of Visual Detection Method for Porcine (Sus scrofa) Senecavirus A RT-RAA-CRISPR/Cas12a
JIANG Cheng-Hui1,2,3, GUO Rong-Xia1, LI Xiao-Ming2, CHANG Yan-Yan2,3, WANG Hui-Bao4, LIU Ping2, WANG Jing2, YANG Jin-Cai2,3, ZENG Qiao-Ying1,*
1 College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730030, China; 2 China Agricultural Veterinary Biological Science and Technology Co., Ltd., Lanzhou 730046, China; 3 Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences,Lanzhou 730046, China; 4 College of Environmental Engineering, Gansu Forestry Voctech University, Tianshui 741020, China
Abstract：The porcine Senecavirus A (SVA) is the sole member of the Senecavirus genus in the family of small RNA viruses. It has been demonstrated that the virus can cause vesicular diseases in pigs (Sus scrofa) and lead to the death of newborn piglets. This poses a threat to the development of the global pig farming industry. The objective of this study was to establish a novel method based on reverse transcription-recombinase polymerase amplification (RT-RAA) and CRISPR/Cas12a techniques for the visual detection of SVA. The methodology involved the design of 3 pairs of RT-RAA primers and guide RNA targeting sequence (crRNAs) according to the conserved sequence of the SVA genome. Furthermore, a single-strand DNA probe (ssDNA) was designed. The optimal reaction concentration ratio of crRNA and LbCas12a was then optimized by orthogonal experiment. Concurrently, the specificity, sensitivity and repeatability of the RT-RAA-CRISPR/Cas12a method were evaluated. Finally, 110 clinical samples with known background were examined. The results showed that the optimal reaction concentrations for crRNA and LbCas12a were 100 and 200 nmol/L, respectively. Utilizing standard RNA (SD-RNA) as the template, the sensitivity of the RT-RAA-CRISPR/Cas12a method was determined to be 15.1 copies/μL. The established RT-RAA-CRISPR/Cas12a method demonstrated no cross-reactivity with Classical swine fever virus (CSFV), Porcine epidemic diarrhea virus (PEDV), Porcine circovirus type 2 (PCV2), Porcine reproductive and respiratory syndrome virus (PRRSV) and Foot-and-mouth disease virus (FMDV). The repeatability test demonstrated that the coefficient of variation within batches was less than 5%, and the coefficient of variation between batches was less than 10%, indicating that the method exhibited excellent repeatability. The clinical sample results were consistent with those of qPCR, exhibiting a 100% coincidence rate. In summary, the study successfully established a visual RT-RAA-CRISPR/Cas12a detection method with strong specificity, high sensitivity and good repeatability, which can be used for early, effective surveillance and epidemiological investigation of SVA.
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