鸡毒支原体重组酶介导等温扩增-琼脂糖凝胶电泳检测方法的建立OA

The study aimed to develop a simple,rapid,and visual recombinase-aided amplification(RAA)assay for the detection of Mycoplasma gallisepticum(MG).First,in accordance with the principles of RAA primer design,primers were designed to target the conserved regions of the mgc2 and pvpA genes of MG,respectively.Standard recombinant plasmids were constructed,and after primer screening,key pa-rameters of the RAA-agarose gel electrophoresis method were optimized,including primer concentra-tion,reaction temperature,and reaction time.Subsequently,the specificity,sensitivity and repeata-bility of the optimized method were analyzed.To further verify its clinical applicability,100 clini-cal samples suspected of MG infection were subjected to parallel detection using three methods:RAA,PCR,and real-time fluorescent quantitative PCR(RFQ-PCR).The results demonstrated that the RAA of the target genes could be completed within 15 min under isothermal conditions at 30 ℃.After subjecting the amplification products to 30 min of agarose gel electrophoresis,clear and specific bands were di-rectly observable using an ultraviolet analyzer.Methodological validation revealed that the estab-lished RAA detection system exhibited high specificity,with no cross-reactivity with other common pathogens.It also showed good repeatability and high sensitivity,with a minimum detection limit of 6.5× 101 copies/pL.Applicability test results indicated that the coincidence rate between RAA and PCR was 96％,while the coincidence rate between RAA and RFQ-PCR reached 98％.In conclusion,the RAA-agarose gel electrophoresis method established in this study is easy to operate,highly specific,and sensi-tive.It is suitable for primary institutions equipped with basic laboratories to conduct MG infection diagnosis,and thus holds promising application prospects.