| Mycoplasma ovipneumoniae(M.ovipneumoniae)is the causative agent of chronic nonprogressive pneumonia in sheep,goat,bighorn and small ruminants.Cough,gasping,progression wasting,interstitial fibrosis in lung are some of the characteristic symptoms of the disease.The progression of the disease is usual y slow and may span from a few months to years,and the disease could onset in four seasons.In China,this pathogen was first found in sichuan's Border Leicester sheep brought from New Zealand and the UK and then found in Gansu,Ningxia,Inner Mongolia,Hebei,Xinjiang and other provinces.This worldwide epidemic has caused huge economic loss to the sheep industry for decades.Therefore,it is of great significance to establish a rapid,sensitive detection method for rapid screening and effective prevention of the disease.This study preliminarily explored the establishment of a rapid detection method based on the lateral chromatography from the serological and etiology detection.This study expressed and purified infA,Met,MO-1 and MO-2 prokaryotic expression protein of M.ovipneumoniae,and extracted whole-cell proteins and membrane protein of M.ovipneumoniae.These proteins that as detect antigen assembled into gold immunochro matographic strip of capture method in which colloidal gold marked staphylococcus aureus protein A(SPA),and test line and control line was separately coated detect antigen and Chicken SPA antibody.The four prokaryotic expression proteins that as detect antigen assembled into gold immunochromatographic strip of double antigen sandwich method in which colloidal gold marked prokaryotic expression protein and test line coated it too.The serum samples add on the sample pad,and read the results within 10~15 minutes.When the infA,Met recombina nt protein was used as detection antigen of capture method,the reaction results of positive samples were satisfactory,but negative samples have seriously false positive phenomenon with about 50 %.When the infA,Met recombinant protein was used as detection antigen of double antigen sandwich method,the reaction results of positive samples were very weak and negative samples have false positive phenomenon.When the MO-1,MO-2 protein was used as detection antigen of capture method,positive samples' reaction results were weak and negatives have false positive phenomenon,which were consistent with the results of double antigen sandwich method.When whole-cell proteins and membrane protein as detection antigen of capture method,the reaction had good specificity and the former's detectable rate was higher.So,a serological gold immunochromatographic assay was developed based on the whole-cell proteins of M.ovipneumoniae.This assay was specific and sensitive to M.ovipneumoniae,and showed good repetition and stability.After further optimization,it can be used widely in anima l husbandry and veterinary station and scattered farmers.The study combined touchdown PCR(TD PCR)with gold nanoparticle(GN)based lateral flow assay by conjugating GNs with anti-digoxin antibody,pre-immobilizing streptavidin and goat anti-mouse IgG on the test line and control line respectively,which was conducted to establish a simple,rapid and effective assay,TD PCR-LFA for M.ovipneumoniae detection.The 5' termina of specific primers labled Digoxin,biotin respectively,and,TD PCR was carried out with templates of M.ovipneumoniae-whole genome.The results was easily achievable by visual observation within 15 minutes after loading of the PCR products onto the LFA device.This assay was highly specific to M.ovipneumoniae without cross reactions,and showed good repetition and stability both in inter and intro assay.In addition,the detection limit of the assay was 20.6 ng/ml,suggesting that the assay has a high sensibility to M.ovipneumoniae.The detection for M.ovipneumoniae can be completed within 2 hours and this assay provides the possible foundation for clinic detection at grassroots and veterinary station. |
PDF Full Text Request