| Mycoplasma hyopneumoniae (Mhp) is a major pathogen of Mycoplasma Pneumoniae of Swine (MPS) and it is an important causative agent of Porcine Respiratory Disease Complex (PRDC). MPS is a chronic and highly transmissible disease found in swine herds wordwide, which results in considerable economic losses due to reduced weight gain, poor feeding efficiency, and increased susceptibility to secondary infections. There are some diagnostic methods for MPS, including clinical diagnosis, pathological diagnosis and laboratory diagnosis methods, currently the main diagnosis methods of Mhp are Mhp isolated and cultured, immunofluorescence, nucleic acid probe method and polymerase chain reaction (PCR), but in the large-scale farms today, these diagnostic methods are laborious, time-consuming, sensitivity and specificity is not ideal. Recently, Real-Time PCR has been used in China and abroad for qualitative detection and identification of Mycoplasma hyopneumonie, but only a few studies have been focused on the TaqMan qualitative detection of Mhp.Recently, there are some researches to study some Mhp protein genes, for example P36, P46, P65, P97, etc. And the cilia adhesion factor P97of Mhp is the main factor caused MPS. In this study, accroding to the gene sequence of Mycoplasma hyopneumoniae (Mhp) P97reported in GenBank, primers were designed by the use of Primer5.0. Gene was amplified by PCR, then the gene was inserted into vector pMD18-T, constructed the recombinant plasmid pMD18-T/P97, transformed into E. coli Trans5a. After it was successfully identified, the TaqMan quantitative Real-Time PCR based on P97gene has been established firstly in China, after analyzing the amplification curve, standard curve, the results showed that the detection sensitivity could up to8.3copy/uL, and the linear equation between logarithm of standard sample concentration and Ct value was expressed as Y (Ct)=-3.41X+42.549. Through TaqMan quantitative PCR amplification with the other related pathogen showed that no cross-reaction was found in other mycoplasma, virus and bacteria. This method was proved to be precise, quick, sensitive and contamination-free, with a good specificity and reproducibility, which can be used for quantitative detection of Mhp in the culture medium and tissue samples.On this basis, the TaqMan quantitative PCR detection methods were used in clinical practice. Through detecting before and after inactivated Mhp content in Mycoplasma hyopneumoniae vaccine, the results showed that there were no significant differences in before and after inactivated Mhp contents. So the inactivation does not affect Mhp gene content. Through detecting the distribution of Mycoplasma hyopneumoniae in the swine tissues that attacked by Mhp. It was demonstrated that Mycoplasma hyopneumoniae gene content in the respiratory organs up to about106copy/μL, but less in the other tissues, such as small intestine, lymph nodes, lien, etc. The distribution of the Mhp in the above tissues only had102copy/μL or even less, there was no distribution in the kidney, lymph nodes. Therefore Mhp mainly caused respiratory diseases. There were43lung tissues need to detect whether they might be infected Mhp, real-time fluorescent quantitive PCR detected positive lung was100%, nested PCR detected positive lung was79%, it demonstrated that comparing to nested PCR, real-time fluorescent quantitive PCR had higher sensitivity. Through detecting the change of the Mhp content in the diseased lung tissues in the pig farms, it explained the situation of the whole pigfield infected with Mhp.It is validated that the real-time fluorescent quantitative PCR detection of mycoplasma hyopneumoniae was useful in clinical testing. |
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