| Porcine epidemic diarrhea virus(PEDV)is an enteric coronavirus that causes acute watery diarrhea and vomiting in pigs and unweaned piglets,with high pathogenicity and mortality,making the swine production industry suffer from a serious economic crisis,and achieving rapid field detection of PEDV is a major industrial need.The existing PEDV detection methods mainly include virus isolation and identification,enzyme-linked immunosorbent assay,polymerase chain reaction,etc.Although these methods have been of great help in the detection of PEDV,they still have the defects of long detection time,tedious operation,high requirements for instrumentation and equipment,and more importantly,the PEDV vaccine is widely used in commercial pig farms in China,but pigs are prone to continuous However,pigs are prone to continuous detoxification after injection and are not fully immune to all mutated wild-type strains,and laboratory tests are often unable to quickly identify wild strains from vaccine strains.In addition to the inability to accurately identify PEDV wild-type strains from vaccine strains,the shortcomings of these techniques have hindered the application and popularity of rapid field testing for PEDV.Therefore,it is important to establish a rapid,easy-to-use,sensitive and low-equipment clinical test method.The CRISPR/Cas system is a defense mechanism present in most bacteria with all archaeato destroy foreign plasmids or phage DNA and is widely used in genetic engineering.In recent years,scientists have discovered that class II Cas protein Cas12a has cr RNA-directed target DNA-excited non-specific ss DNA cleavage activity and applied the CRISPR-Cas system to the field of nucleic acid detection,which has become a hot research topic in the field of in vitro diagnostics.Reverse transcriptase-promoted recombinase amplification(RT-ERA)is the exponential amplification of target genes on templates by reverse transcriptase,recombinase and single-stranded binding proteins at a constant temperature setting,which breaks the limits of precision temperature-controlled equipment.In addition,the chromatographic double antibody sandwich strip technology is fast and portable,and combining it with CRISPR/Cas12a technology is expected to overcome the shortcomings of existing pathogen detection technologies,which are time-consuming,cumbersome and require high instrumentation,and bring more sensitive,specific,immediate and easy detection.The use of CRISPR/Cas12a system to identify PEDV wild strains from vaccine strains has not been reported,so this study was conducted to construct a rapid assay to identify wild strains from vaccine strains using CRISPR/Cas12a system,RT-ERA isothermal amplification technique,fluorescence analysis and test strip signal output for ORF3sequences of PEDV.The main contents of this study are as follows:1.Genetic evolutionary analysis of ORF3 gene of four PEDV wild strainsIn this study,four disease materials were selected from those tested positive,and four sets of ORF3 gene sequences were obtained by sequencing and comparison,and the sequences were uploaded to the NCBI database.After comparing with 24 PEDV strains,it was found that all four strains tested in this study belonged to G2 type and were distantly related to the vaccine strain CV777.This study provides a reference for the evolutionary trends of the ORF3 gene of PEDV wild strains.2.Establishment of a CRISPR/Cas12a visualization system assayIn this paper,a CRISPR/Cas12a-based assay was developed to identify PEDV wild strains from vaccine strains.Based on the PEDV ORF3 gene sequence published in Gen Bank,full-length primers were designed using Prime Premier 5.0 software to construct the recombinant plasmid p MD-19T-ORF3;2 pairs of cr RNA primer pairs,3 pairs of RT-ERA primers and probes were designed for the deletion sites on the ORF3 sequence between the PEDV wild strain and the vaccine strain,and these The primers were screened,and the reaction temperature and time were optimized with the screened primers;sensitivity and specificity tests were performed using the recombinant plasmid as the template.The test results showed that the 2nd cr RNA primer pair and the first RT-ERA primer pair were more effective and used in downstream tests;the optimal reaction time and temperature were 30 min and 37°C,respectively,after optimization of reaction conditions;the sensitivity of this test reached 2copies/μL,which was 10 times higher than q PCR,and there was no interaction with other pathogens,and the detection of clinical samples in The sensitivity of the test was 2 copies/μL,which was 10 times higher than q PCR,and there was no interaction with other pathogens.3.Establishment of CRISPR-LF detection methodIn this study,a CRISPR-Lateral flow strip(CRISPR-LF)assay was established based onthe above CRISPR visualization detection system by introducing a chromatographic double antibody sandwich strip technique,which can be detected by naked eye,with cr RNA primers and RT-ERA primers after screening with CRISPR fluorescence detection system for reaction Optimization of temperature and time;sensitivity and specificity tests were performed using recombinant plasmids as templates.After optimization of the reaction conditions,the optimal reaction time and temperature were 30 min and 37°C,respectively;the sensitivity of this test reached 2×10~2 copies/μL,and showed good accuracy and specificity in the detection of clinical samples.In summary,the CRISPR/Cas12a-based system established in this study has high sensitivity,strong specificity,rapidity and efficiency compared with traditional PCR and q PCR,while improving the portability of the assay,which is suitable for field testing where test resources are scarce,and has a good development prospect in the field of molecular POCT detection,which is important for early diagnosis and preventive control of PEDV. |
