Detection of Four Porcine Enteric Coronaviruses Using CRISPR-Cas12a Combined with Multiplex Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay.
Jiajia LiuDagang TaoXinquan ChenLinyuan ShenLi ZhuBingrong XuHailong LiuShuhong ZhaoXinyun LiXiangdong LiuShengsong XieLili NiuPublished in: Viruses (2022)
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.
Keyphrases
- loop mediated isothermal amplification
- crispr cas
- sensitive detection
- sars cov
- nucleic acid
- high throughput
- real time pcr
- genome editing
- respiratory syndrome coronavirus
- gold nanoparticles
- irritable bowel syndrome
- quantum dots
- liver failure
- palliative care
- case report
- climate change
- gram negative
- circulating tumor
- physical activity
- hepatitis b virus
- drug induced
- risk assessment
- living cells
- circulating tumor cells