| Caprine mycoplasma pneumoniae(CMP) is a kind of contagious disease of goats caused by mycoplasmas, which characterize is cough, fever, pleurisy and cellulose pneumonia.Many kinds of mycoplasma can cause CMP in goat, such as M.capricolum subsp.capripneumoniae(MCCP),M.capricolumsubsp. Capricolum(Mmc),M. mycoides subsp.Capri(Mcc),M.mycoidessubsp.mycoidesLC(MmmLC)andM.ovipneumoniae(MO). Numerous publications have documented the presence of CMP throughout more than forty countries and districts, which has become one of the most serious threats to goat breeding industry. CMP is widely popular in china which caused by Mmc,MmmLC,MO. In Sichuan province, sporadic outbreaks of CMP have been recorded since 1982, but there is no a systemic epidemiological investigation about CMP. The polymerase chain reaction (PCR) has become the most commonly method for rapid detection of disease, due to specificity, high sensitivity, and the advantages of fast. So far, a duplex PCR for the detection of MO and Mycoplasma mycoides cluster, and a real-time PCR for the detection of MO had not been reported at home and abroad. So, it is significant for us to carry out an epidemiological investagation and develop PCR assay for rapid detection of mycoplasma infection.In this study, epidemiological investagation of CMP was performed in Sichuan province and two kinds of PCR methods were developed and applied in clinical.1. Epidemiological investagation of CMP in main caprine production regions of Sichuan provinceA systematically epidemiological investigation of CMP was carry out using isolation and PCR identification method in Sichuan province including 7 regions such as Zigong, Jianyang, Chengdu, Leshan, Lezhi, Nanjiang, Meishan. 36 strains of mycoplasma isolated from the 135 clinical samples (lung and nasal swab) through the medium of choice and optimization of culture conditions, which were recognized as MO after PCR identification.The results confirmed that the main pathogen of CMP was MO in Sichuan province, which provided a scientific basis for prevention and control of CMP2. The real-time PCR assays detecting MO were established successfullyBecause of the small size genome and low guanine+cytosine (G + C) content, specific primers of MO was designed difficultly. In this study, a pair of primers was selected successfully for the real-time PCR assays from 21 pairs MO-specific primers which were designed using Primer Premier 5.0 and Oligo 7.0 based on nine target genes selected from genome of MO(swmo15) through selecting high guanine+cytosine (G+C) content gene as target genes. The real-time PCR assays based on SYBR GreenIfor detecting MO were developed by optimization of real-time PCR conditions,establishing the standard curves, and evaluating of specificity and sensitivity. The following results were obtained: this real-time PCR assay can amplify a 146bp fragment from genome of MO; The linear range were 3×10~3～3×10~7; the correlation coefficient of standard curve was 0.998; the amplification efficiency of the real-time PCR assay were 91.2%; the standard equation was y=-3.551x+33.052; the detection limits were 3 copies, more sensitive (1,000-fold) than conventional methods; the melting curve of the real-time PCR assays presented a single peak without non-specific amplification and primer dimmer; the method has better specificity which positive rate of 36 Clinical isolates of MO was 100%,but all negative for the unrelated pathogens,such as Mmc,MmmLC,Mccp,Mcc,mycoplasma bovis,Mannheimia haemolytica,Pasteurella multocida,Staphylococcus aureus,E. coli. This real-time PCR assay established in this study would be a rapid, specific and sensitive new method for the diagnosis of mycoplasmal infection ,epidemiological investigation and Pharmacodynamic evaluation.3. A duplex PCR assays detecting MO and Mycoplasma mycoides cluster were established successfullyA duplex PCR assay for the detection of MO and Mycoplasma mycoides cluster has been developed in this research using the recorded specific primers of MO and Mycoplasma mycoides cluster by optimization of duplex PCR assays conditions , evaluating of specificity and sensitivity. The following results were obtained: this duplex PCR assay can simultaneous amplify a 548bp fragment from genome of MmmLC(Y-goat) and 361 bp fragment from genome of MO(Y98),but no DNA fragment were amplified from other bacteria such as Mannheimia haemolytica,E. coli,Pasteurella multocida,Staphylococcus aureus. The detection limits suggested that it can deteced 1.0pg DNA of Y-goat and 10.0pg DNA of Y98 respectively, or 10.0pg mixed DNA of Y-goat and Y98.The positive rate of clinical samples of Mycoplasma mycoides and MO culturing were 2/6 and 14/36 respectively. This duplex PCR assays can detect Mycoplasma mycoides and MO culturing in one PCR reaction which significantly shortened the time of diagnosis and improved the efficiency of diagnosis. It provides a powerful tool for large-scale epidemiological investigations of CMP... |
PDF Full Text Request