Establishment And Application Of A Duplex Real-Time PCR For Detection Of Mycoplasma Ovipneumoniae And Mycoplasma Mycoides Subsp.capri

Mycoplasmal pneumonia of goats and sheep（MPGS）is a kind of important infectious disease harmful to sheep and goat industry.The disease is characterized by hyperthermia,cough,progressive weight loss,fibrinous and serious inflammation of the pleura and lungs with morbidity reaching 19%-90%and mortality 40%,thus resulting in great economic losses in the goats and sheep industry in China.Mycoplasma ovipneumoniae（Mo）and Mycoplasma mycoides subsp.capri（Mmc）are two leading causes of MPGS,and there has been reports of mixed infections of Mo and Mmc in clinical practice.Mo is a pathogen causing proliferative interstitial pneumonia in sheep,goats,and particularly in lambs.The main clinical manifestations of diseased sheep are cough,runny nose,weight loss,anemia,growth retardation.The course of this disease can be up to several months even several years.Mmc belongs to filamentous Mycoplasma clusters,which is another pathogen of MPGS.Mmc can only infects goats,especially lambs.The symptoms of Mmc infection includes inflammation of the lungs,joints,mammary glands,conjunctiva,etc.,and also systemic symptoms of sick goats.The aim of this study is to establish a duplex real-time fluorescence quantitative PCR method to simultaneously detect Mo and Mmc and to provide a effective technical support for the early,rapid diagonosis,prevention and control of MPGS.According to the gene sequence of Mo p113 and Mmc MLC₁770 in GenBank,the specific primers and probes for Mo and Mmc were designed using beacon Designer 7.9 combined with NCBI Blast software analysis.Using the recombinant plasmid as positive template,through optimization of primer concentration,probe concentration and annealing temperature,the single real-time fluorescence quantitative PCR assay for Mo and Mmc detection were established.On this basis,a duplex real-time fluorescence quantitative PCR method for Mo and Mmc was established through optimization of primer concentration and probe concentration,and the specificity,sensitivity and repeatability of the method were verified.The results showed that the correlation coefficient of the standard curve（R2）was 0.998 and 0.999,respectively,and the amplification efficiency was 94.8%and 97.0%,respectively.This method only provided amplification signals for Mo and Mmc,and had no cross-amplification of Mycoplasma capricolum subsp.Capripneumoniae（Mccp）,Acholeplasmalaidlawii（AL）,Mycoplasma bovis（Mb）,Salmonella,Escherichia coli（Ec）,Staphylococcus aureus（SA）,Mannheimia haemolytica（Mh）,Orf virus（ORFV）and other common pathogens,indicating that the assay was specific.The results showed that the minimum detection limits for Mo and Mmc were all 100 copies/μL,which was 100 times higher than that of conventional PCR.Repeatability results showed that the intra-assay coefficient of variation（CV）for Mo was 0.23%to 1.98%,the inter-assay CV for Mo was 0.96%to 1.31%,the intra-assay CV for Mmc was 0.47%to 1.16%,and inter-assay CV was 0.60%to 1.76%,indicating good reproducibility.Using this duplex real-time fluorescence quantitative PCR to detect 187 clinical samples collected from Fujian province,the results showed that Mo positive rate was 70.6%（132/187）,Mmc positive rate was 11.8%15.0%（28/187）,the positive rate of mixed infection of Mo and Mmc was 11.8%（22/187）.The above data indicated that the method established firstly in this study could be used for early and rapid diagnosis of MPGS caused by Mo and Mmc.