Establishment Of RPA Assay For Detection Of Mycoplasma Ovipneumoniae

Mycoplasma ovipneumoniae(MO)is the main pathogen causing mycoplasma pneumonia,which can infect sheep and goats.The current detection methods for MO mainly include:identification of pathogen isolation and culture,serological detection,PCR detection,etc.Although the above methods can specifically detect MO,they are not suitable for rapid on-site detection.Recombinase Polymerase Amplification(RPA)is an in vitro nucleic acid isothermal amplification detection technology,which can detect both DNA and RNA.It has the characteristics of simplicity,high efficiency and sensitivity.In this project,MO specific marker gene was screened through bioinformatics analysis combined with PCR technology,and on this base,an RPA detection method was established which can provide a new technology for the clinical detection of MO and support for the prevention and control of mycoplasma pneumonia.The specific experimental results are as follows:1.Through reviewing literature and patents,bioinformatics analysis and comparison,specific primers were further designed,and 6 MO specific marker genes including p80,p102,hsp70,pdhA,dihydrollpoyl dehydrogenase and transketolase were screened by PCR.All of the above genes can accurately distinguish MO from other pathogenic bacteria related to pneumonia.The transketolase gene has good specificity,no primer dimer,and the detection of MO based on this gene has not been reported,so transketolase is identified as a new marker gene for MO.2.Based on the transketolase gene to establish a basic RPA detection method for MO.Firstly,The optimal reaction time was determined to be 20 min and the optimal reaction temperature was 39?;the results of specific identification showed that this method could accurately distinguish MO strain from other strains associated with sheep pneumonia,and have good specificity;gradient dilution of positive standard qualityplasmid of transketolase gene to detect the minimum copy number,and the sensitivity of this method is 1.9x 102 copies/?L,10 times more sensitive than PCR;intra-group and inter-group repeatability verification experiments show that the method has good repeatability.3.Based on the Basic RPA,the nfo RPA method for detecting MO was established.The optimal reaction time was 15 minutes and the optimal reaction temperature was 39?;this method can accurately distinguish sheep mycoplasma pneumoniae strains with other pneumonia-related strains;gradient dilution of positive standard-quality plasmid of transketolase gene to detect the minimum copy number,the sensitivity of this method is 19 copies/?L,which is more sensitive than PCR 100 times higher;repeated experiments within and between groups show good repeatability.4.A total of 52 sheep-nose swabs were collected from Ningxia.Basic RPA and nfo RPA were used to detect the clinical samples.PCR method have detected 24 MO positive samples,with a positive rate of 46.2%(24/52).Basic RPA detected 17 MO positive samples,with a positive rate of 32.7%(17/52).It shows that MO Basic RPA method established in this experiment can be used for clinical sample detection.26 MO positive samples were detected by nfo RPA,with a positive rate of 50.0%(26/52).Further comparative analysis showed that the detection rate of nfo RPA was 8.3%higher than that of PCR,indicating that the MO nfo RPA established in this experiment had a higher positive rate than PCR and Basic RPA.Conclusion:After bioinformatics analysis and comparison,PCR was used to screen 6 MO specific marker genes,and the target genes for establishing MO RPA detection method were identified.MO basic RPA and nfo RPA detection methods were established based on transketolase gene.Preliminary test results of clinical samples show that the established RPA method has good specificity,sensitivity and repeatability,and the detection rate of MO nfo RPA is higher than that of PCR and Basic RPA,which can be used as a candidate detection method for clinical MO.