Detection of Four Porcine Enteric Coronaviruses Using CRISPR-Cas12a Combined with Multiplex Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay
Jiajia Liu,
Dagang Tao,
Xinquan Chen,
Linyuan Shen,
Li Zhu,
Bingrong Xu,
Hailong Liu,
Shuhong Zhao,
Xinyun Li,
Xiangdong Liu,
Shengsong Xie,
Lili Niu
Affiliations
Jiajia Liu
College of Animal Science and Technology, Sichuan Agricultural University, Chengdu 611130, China
Dagang Tao
Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education and Key Lab of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan 430070, China
Xinquan Chen
College of Animal Science and Technology, Sichuan Agricultural University, Chengdu 611130, China
Linyuan Shen
College of Animal Science and Technology, Sichuan Agricultural University, Chengdu 611130, China
Li Zhu
College of Animal Science and Technology, Sichuan Agricultural University, Chengdu 611130, China
Bingrong Xu
Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education and Key Lab of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan 430070, China
Hailong Liu
Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education and Key Lab of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan 430070, China
Shuhong Zhao
Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education and Key Lab of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan 430070, China
Xinyun Li
Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education and Key Lab of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan 430070, China
Xiangdong Liu
Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education and Key Lab of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan 430070, China
Shengsong Xie
Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education and Key Lab of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan 430070, China
Lili Niu
College of Animal Science and Technology, Sichuan Agricultural University, Chengdu 611130, China
Porcine enteric coronaviruses have caused immense economic losses to the global pig industry, and pose a potential risk for cross-species transmission. The clinical symptoms of the porcine enteric coronaviruses (CoVs) are similar, making it difficult to distinguish between the specific pathogens by symptoms alone. Here, a multiplex nucleic acid detection platform based on CRISPR/Cas12a and multiplex reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) was developed for the detection of four diarrhea CoVs: porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV). With this strategy, we realized a visual colorimetric readout visible to the naked eye without specialized instrumentation by using a ROX-labeled single-stranded DNA-fluorescence-quenched (ssDNA-FQ) reporter. Our method achieved single-copy sensitivity with no cross-reactivity in the identification and detection of the target viruses. In addition, we successfully detected these four enteric CoVs from RNA of clinical samples. Thus, we established a rapid, sensitive, and on-site multiplex molecular differential diagnosis technology for porcine enteric CoVs.
