Rapid Molecular Detection of Senecavirus A Based on Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) and CRISPR/Cas12a

Chenghui Jiang ^1,2 Huibao Wang ³ Rongxia Guo ¹ Rui Yang ² Xiaoming Li ² Ping Liu ² Jing Wang ² Jincai Yang ² Yanyan Chang ^1,2* Qiaoying Zeng ^1*

• ¹ College of Veterinary Medicine, Gansu Agricultural University, Lanzhou, China
• ² Other, Lanzhou, China
• ³ Gansu Forestry Polytechnic, Academy of Environment Engineering, Tian shui, China

Senecavirus A (SVA) is an emerging picornavirus associated with porcine vesicular diseases and acute neonatal piglet mortality, threatening the global swine industry. In this study, we combined reverse transcription loop-mediated isothermal amplification (RT-LAMP) and Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein12a (CRISPR/Cas12a) using a dual-labelled fluorescence quencher or fluorescent biotin single-stranded DNA reporter molecule to develop two rapid, reliable, and portable visual SVA assays: RT-LAMP-Cas12a-FQ and RT-LAMP-Cas12a-FB. The two methods exhibited comparable detection limits, with 9.6 copies/μL achieved in 40 and 45 minutes, respectively. Furthermore, they did not cross-react with non-target nucleic acids extracted from other related viruses and showed high specificity for SVA RNA detection. The methods demonstrated satisfactory performance in detecting 69 porcine adventitious samples, with no significant differences from that of quantitative reverse transcription polymerase chain reaction (RT-qPCR). The developed RT-LAMP-Cas12a-FQ and RT-LAMP-Cas12a-FB methods are promising for early detection and routine surveillance of porcine SVA in resource-poor areas.