A Novel Typing Method for Clostridium perfringens Using Multiplex Recombinase Polymerase Amplification and CRISPR/Cas12a

Siying Li^1,2Qinghong Zhou¹Qingxun Zhang³Ziqin Lin⁴Qianyi Zhang¹Sihong Wu⁵Luying Wang¹Sheng Ye¹Xingxing Xiao¹Shuai Gao^1*

• ¹Wenzhou Medical University, Wenzhou, China
• ²The Second Xiangya Hospital of Central South University, Changsha, China
• ³Beijing Academy of Science and Technology Beijing Milu Ecological Research Center, Beijing, China
• ⁴Yangtze University Jingzhou Hospital, Jingzhou, China
• ⁵First Hospital of Jiaxing, Jiaxing, China

Distinct toxinotypes of Clostridium perfringens cause different diseases in animals and humans. Rapid and accurate typing methods remain essential for early diagnosis, effective intervention, and reduced mortality. In this study, we designed specific primer pairs and crRNA sequences targeting the α, β, ε, and ι toxin genes of C. perfringens and constructed a rapid, sensitive, accurate and instrument-free method for typing of C. perfringens. This typing method of C. perfringens based on the Multiplex Recombinase Polymerase Amplification (RPA)-assisted CRISPR/Cas12a system, termed Cp-MRC12a, can be completed in one hour. The Cp-MRC12a assay shows high sensitivity with a detection limit of 10 copies/µL for the type A strain and 100 copies/µL for the type B-E strains and high specificity without cross-reactivity to non-target bacteria, and demonstrates a reliable performance in detecting clinical and spiked samples. Collectively, Cp-MRC12a provides a robust and practical approach for the typing of C. perfringens strains, offering substantial advances for early disease diagnosis and pathogen identification.