Establishment and Application of a Duplex TaqMan Probe Real-time Fluorescence Quantitative PCR Method for Detection of Mycoplasma ovipneumoniae and M.mycoides subsp.capri
LIN Yu-Sheng, LI Sha-Sha, JIANG Jin-Xiu, ZHANG Jing-Peng, YOU Wei HU Qi-Lin*
Institute of Animal Husbandry & Veterinary Medicine, Fujian Academy of Agricultural Sciences, Fuzhou 350013, China
Abstract：Mycoplasma ovipneumoniae (Mo) and M.mycoides subsp.capri (Mmc) are the main pathogens of M.pneumonia of sheep and goats (MPGS).In order to rapidly identify the main pathogens of MPGS, a duplex TaqMan probe Real-time fluorescence quantitative PCR (qRT-PCR) detection method was established for simultaneous detection of Mo and Mmc.Using Beacon Designer 7.9 combined with NCBI Blast software analysis, the specific primers and probes were designed based on the p113 sequence of Mo and the MLC_1770 (hypothetical protein gene) sequence of Mmc.The duplex TaqMan probe qRT-PCR method was established by optimizing the reaction conditions such as primer concentration, probe concentration and annealing temperature, and the specificity, sensitivity and repeatability of the method were validated.The result showed that the correlation coefficient (R2) of Mo and Mmc were 0.998 and 0.999, respectively, and the amplification efficiency of Mo and Mmc were 94.8% and 97%, respectively.The assay showed a good specificity without cross reaction from other common pathogens of sheep and goats.The lowest detectable limit (LDL) of the method for Mo and Mmc was the same, which was 100 copies/μL, and its sensitivity was 100 times more than conventional PCR.The inter- and intra-variation coefficients of the assay were less than 2%.Applying the assay to detect 187 cases of clinical samples which were collected from different areas of Fujian, the result showed that the positive rates of Mo and Mmc were 47.6% (89/187) and 11.8% (22/187), respectively, and the duplex positive rate of infection was 10.2% (19/187).The above results indicated that the assay could be used for the accurate and rapid detection of Mo and Mmc in clinic.The study provided technical support for rapid detection and epidemiology of Mo and Mmc.
林裕胜, 李莎莎, 江锦秀, 张靖鹏, 游伟, 胡奇林. 绵羊肺炎支原体和丝状支原体山羊亚种双重TaqMan探针荧光定量PCR检测方法的建立及应用[J]. 农业生物技术学报, 2019, 27(5): 943-950.
LIN Yu-Sheng, LI Sha-Sha, JIANG Jin-Xiu, ZHANG Jing-Peng, YOU Wei HU Qi-Lin. Establishment and Application of a Duplex TaqMan Probe Real-time Fluorescence Quantitative PCR Method for Detection of Mycoplasma ovipneumoniae and M.mycoides subsp.capri. 农业生物技术学报, 2019, 27(5): 943-950.
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