Establishment Of A Method For Detection Of Actinobacillus Pleuropneumoniae Based On LAMP-PfAgo Technology

Actinobacillus pleuropneumoniae(APP),the causative agent of Porcine contagious pleuropneumonia(PCP),can cause respiratory disease in pigs and has a serious impact on the pig industry worldwide.APP serotypes are numerous and require the addition of factor V for growth.The pathogenic factors of this bacterium can interact with each other to cause serious damage to the organism,and the perforated exotoxins Apx I,Apx II and Apx III produced by them are important damage inducing pathogenic factors.In this study,a recombinant vector p MD19-T-Apx IV was constructed based on the conserved region of Apx IV gene,and the template was amplified using Loop-mediated isothermal amplification(LAMP).Five primer pairs were designed using online software,and the LAMP system was optimized.We designed the guide DNA and molecular beacons required for PfAgo(Pyrococcus furiosus Argonaute)cleavage by primer screening and optimized the detection system,and analyzed its feasibility by designing tests for specificity,sensitivity and clinical sample detection.1.Construction and expression of PfAgo protein expression vector and activity verificationPfAgo high temperature resistant protein can be used as an artificial restriction enzyme to recognize and cleave the DNA sequence of the target site.After protein purification and high temperature centrifugation,a 90.4 k Da high temperature resistant protein was obtained,but it did not achieve the desired effect when compared with commercial protein.2.Construction of p MD-19T-Apx IV recombinant plasmid and optimization of LAMP reaction systemThe p MD-19T-Apx IV recombinant plasmid was constructed by comparing the published sequences of Apx IV gene in Gen Bank,finding its highly conserved region,and designing specific primers for LAMP system according to this target gene.The amplification of the target sequences was carried out by using the Bst DNA polymerase strand-switching function.3.Establishment and optimization of the PfAgo protein-fluorescence-based detection methodThe target sequence of PAND(PfAgo-mediated Nucleic acid Detection)was determined based on the conserved sequence of the conserved region of Apx IV,and the molecular beacon was designed and optimized by primer screening and system optimization(temperature,guide DNA concentration,Mn2+ and other key factors)to determine the detection results by fluorometric method.The optimized PAND system with LAMP amplification showed no cross-reactivity with several common respiratory pathogens by specificity test;the sensitivity test with LAMP amplification showed the lowest detection limit of 5.29×102 copies/μL for recombinant plasmid p MD19-T-Apx IV.The results were 100% positive for 17 clinical samples using the established method.