Detection of Four Porcine Enteric Coronaviruses Using CRISPR-Cas12a Combined with Multiplex Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay

Abstract

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.

Clustered Regularly Interspaced Short Palindromic Repeat/Cas12a Mediated Multiplexable and Portable Detection Platform for GII Genotype Porcine Epidemic Diarrhoea Virus Rapid Diagnosis

“…Because of the high conservation between the N gene sequences, the detection methods that target the N gene have a more extensive detection range compared with the S gene, but cannot distinguish between the GI and GII strains ( Miller et al, 2016 ). Liu et al (2022) developed a CRISPR-/Cas12a-based detection system combined with multiplex reverse transcriptase loop-mediated isothermal amplification, which allows the detection of PEDV, TGEV, PDCoV, and SADS-CoV with the naked eye. However, they do not perform an experiment to identify different types of PEDV strains.…”

Current State of Molecular and Serological Methods for Detection of Porcine Epidemic Diarrhea Virus

“…This newly developed assay achieved single-copy sensitivity with no cross-reactivity. The results were visible to the naked eye using a ROX-labeled ssDNA-FQ reporter [ 116 ]. Generally, the methods described above are sensitive, capable of rapid detection, do not require expensive equipment or long training and can be performed at the point of care.…”

Development of CRISPR-Mediated Nucleic Acid Detection Technologies and Their Applications in the Livestock Industry

“…So far, a multiplex RT-PCR assay [ 124 ] is available to test these viruses simultaneously, but it requires specialized instruments and skilled personnel. The multiplex isothermal amplification in combination with CRISPR/Cas12a assay, which has successfully distinguished PEDV, TGEV, PDCoV, and SADS-CoV [ 83 ] would have the potential to simultaneously and differentially detect these three viruses in the field.…”

“…To decide if a pig should be immunized with the CV777 vaccine, an RT-RAA-CRISPR/Cas12a platform targeting the S gene was created for the detection of GII PEDV [ 82 ]. Additionally, Liu et al (2022) developed a single-tube multiplex RT-LAMP-Cas12a diagnostics to simultaneously detect TGEV, PDCoV, SADS-CoV, and PEDV, although it cannot recognize different PEDV strains [ 83 ]. With naked-eye colorimetric detection, it has a LOD of one copy and takes only 25 min [ 83 ].…”

“…Additionally, Liu et al (2022) developed a single-tube multiplex RT-LAMP-Cas12a diagnostics to simultaneously detect TGEV, PDCoV, SADS-CoV, and PEDV, although it cannot recognize different PEDV strains [ 83 ]. With naked-eye colorimetric detection, it has a LOD of one copy and takes only 25 min [ 83 ]. These CRISPR/Cas12a-based assays, with different detection purposes, have promising potential for the prevent and control of PEDV worldwide.…”