Establishment and Application of SYBR Green I Real-time Fluorescence Quantitative PCR Assay for Mycoplasma hyopneumoniae
In order to establish a rapid and efficient method to detect Mycoplasma hyopneumoniae(Mhp),4 pairs of specific primers were designed based on the conserved sequence of Mhp published by NCBI,and the optimal primer(SX4)was screened after correlation analysis based on the test results of the primers by fluorescence quantitative PCR and fitting curves to establish a Mhp SYBR Green I real-time fluorescence quantitative PCR assay.The method could be used to specifically detect Mhp,without specific amplification to porcine circovirus type 2,porcine parvovirus,pseudorabies virus,Haemophilus parasuis serotype 4 and Streptococcus suis serotype 2.The sensitivity of SX4 was up to 4.9×101 copies/μL,and inter/intra-group variable coefficient was less than 2%.The method could be used to detect the amount of Mhp from produced antigen and commercial vaccines.In conclusion,the Mhp SYBR Green I real-time fluorescence quantitative PCR assay was successfully established,and could be used to detect the amount of Mhp from produced antigen and clinical vaccines with the advantages of strong specificity,high sensitivity and good repeatability.
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