Establishment Of A CRISPR/Cas12a-based One-pot Method For The Detection Of Porcine Parvovirus 7

Porcine Parvovirus 7(PPV7)can cause reproductive disorders in sows and stunted piglets,resulting in economic losses in pig farms.Since no specific vaccine has been developed for this virus,early detection is needed to facilitate the culling of diseased pigs for prevention purposes,and none of the currently available detection methods can achieve on-site detection.Therefore,in this study,we established a one-pot detection method for PPV7 based on a CRISPR/Cas12a detection system combined with RPA amplification technology to achieve field-based detection of PPV7.The main results are as follows:(1)The more conserved PPV7-NS1 gene was selected by sequence comparison,and the p UC57-NS1 plasmid was constructed as a DNA template for screening the best sg RNA and the best RPA primer pair,a fluorescent reporter with a hairpin-like structure,Hairbin-reporter,was also introduced,which is capable of generating fluorescence faster than the common ss DNA-reporter,creating a faster visual method for the detection of PPV7.(2)Determine the optimal amplification and detection time.When the reaction temperature was 38℃,using 10~4 copies of p UC57-NS1 plasmid as the substrate,the RPA amplification reaction could produce sufficient nucleic acid products in 15 min,and the CRISPR detection reaction could observe significant fluorescence of the system in 15 min.However,considering the situation when the substrate concentration is low,20 min was chosen as the optimal RPA amplification time and30 min as the optimal CRISPR detection time.(3)Introduce PC-sg RNA that can be broken by UV irradiation instead of normal sg RNA to ensure better detection by isolating RPA amplification and CRISPR detection in one tube without opening the cap for pipetting operation.(4)Through sensitivity and specificity validation,it was determined that the method established in this study has a minimum detection limit of 10 copies and high specificity,and does not fluoresce when other viruses are detected.In summary,this study successfully established a CRISPR/Cas12a-based one-tube assay for the detection of PPV7.The method can greatly reduce the possibility of aerosol contamination and ensure the reliability of the results,while reducing the time required for the test,allowing the results to be determined by visual inspection in a shorter time,providing technical support for the field testing of PPV7 in pig farms,and also helping to develop other disease detection methods.