| Porcine Pavovirus(PPV)and Porcine reproductive and respiratory syndrome(PRRSV)are two common clinical agents of reproductive disorders in pigs and are extremely widespread.PPV has few clinical signs,but when it does,it can cause huge losses,while PRRSV can cause immunosuppression in the body,making the herd less immune and leading to other infections and other diseases.A highly sensitive and specific test is therefore important for the differential diagnosis of PPV and PRRSV in clinical practice.Recently,a new restriction endonuclease PfAgo(Pyrococcus furiosus Argonaute)has been of interest to a wide range of researchers because of its high sensitivity and flexibility.In this study,we screened g DNA for conserved sequences of porcine reproductive and respiratory syndrome virus and Porcine Pavovirus and designed the corresponding molecular beacons.The method was then combined to investigate the lower limit of detection and specificity.The optimised method was then used to test the clinical samples in parallel with the established SYBR Green I Fluorometric method in the literature to verify the accuracy of the method.The test methods and results are as follows:1.Establishment of a fluorometric assay for porcine reproductive and respiratory syndrome virusThe p ET28a-PfAgo recombinant plasmid stored in the laboratory was transformed into E. coli for induced expression of the protein and purified to obtain PfAgo protein at a concentration of 242.68μg/m L with a size of about 93.6 k Da,verifying the presence of cleavage activity of the protein.The reaction product of the second primer pair showed an amplified band at 1133 bp,which was consistent with the expected result.Subsequently,ORF6 was selected as the conserved sequence of PRRSV to construct a plasmid with a fragment size of 412 bp.After determining the target sequence and establishing the fluorometric method,the reaction conditions were optimized and tested,and the results were found to be not as good as expected when compared with the relevant literature reports.2.Establishment of the Porcine Pavovirus PfAgo fluorescence assayPCR identified the viral nucleic acid as PPV.The VP2 gene was selected to successfully construct the viral plasmid,and the fragment size of 1711 bp was identified by PCR.The primer g DNA-Q3 was selected based on its cleavage effect on the target,and its corresponding two g DNA and molecular beacons were designed to establish the fluorometric method to screen the optimal reaction conditions:The system was cleaved using three primers,the optimum reaction temperature was 95°C,the optimum primer usage was 750pmol,the optimum molecular beacon usage was 15 pmol,the optimum reaction time was 30min,the specificity test showed that the method was highly specific,so this study successfully constructed a method based on PfAgo technology to detect PPV.3.Establishment of a fluorescence binding LAMP assay for Porcine Pavovirus PfAgoThe optimal amplification temperature was 61℃,the optimal reaction time was 40 min,dNTP optimal final concentration is 1.2 mm,the optimal final concentration of manganese ion was 10 m M,and the optimal internal and external primer ratio was 4:1,with a sensitivity of 10~1 copies/μL.The specificity test showed no cross-reactivity with other samples and good specificity.The combination of LAMP amplification products and fluorescence assay results in a minimum detection limit of 10~1 copies/μL and specificity assay results indicate that the method is highly specific.The PRRSV-NACD30-like fluorometric method established in this study was not effective;however,the optimized PPV-PfAgo fluorometric method was able to combine with LAMP technology to successfully establish the PPV-LAMP-PfAgo fluorometric method.The method has strong sensitivity and specificity,with the advantages of sensitivity,rapidity and low cost of detection,providing a new technical means for the detection of PPV at the clinical grassroots level. |
