Establishment and Application of a Triplex TaqMan Probe Real-time Fluorescene Quantitative PCR Method for Detection of Hemotrophic mycoplasma from Swine (Sus Scorfa)
FU Yuan*, SHI Tuan-Yuan, YUAN Xiu-Fang, XU Li-Hua, SUN Hong-Chao, WEI Qiang
Institute of Animal Husbandry and Veterinary Medicine, Zhejiang Academy of Agricultural Science, Hangzhou 310021, China
Abstract：Porcine eperythrozoonosis is caused by Mycoplasma suis, M. parvum and a new species Candidatus M. haemosuis (CMh) isolated from Zhejiang province,which leads to anemia, jaundice and abortion of swine (Sus Scorfa). The prevalence and transmission of pig's hemoplasmas is not clear in China. In order to differential diagnose the main pathogens prevalence of porcine eperythrozoonosis, a triplex TaqMan probe fluorescence quantitative PCR (qPCR) was established based on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. According to the published GAPDH gene sequences of pig's hemotrophic mycoplasma, the primers and probes were designed by Beacon Designer 4.0 software for the 3 porcine hemoplasmas. By optimizing the concentration of Mg2+, hot-start rTaq, primers and probes, the triplex Taqman probe qPCR method was established, and the specificity, sensitivity, reproducibility of the method were verified. The results showed that the correlation coefficient (R2) of the established triplex Taqman probe qPCR method was between 0.999 2~0.999 6, and the amplification efficiency was between 99.9%~103.2%. No amplification curve were found from DNA that had been extracted from the pig's samples of others common pathogens. The lowest detection limit of the method for Mycoplasma suis, M. parvum and CMh was 1 copy/μL. The coefficient of intra- and inter-group variations was between 0.31%~1.75%, indicating good repeatability. 148 blood samples from Zhejiang province pig farm, were detected by triplex Taqman probe qPCR and compared with double PCR, the results showed that the positive rates of M. suis, M. parvum and CMh were 50% (74/148) , 48.6% (72/148) and 64.9% (96/148), respectively, the mixed infection rate of the three pathogens was 29.7% (44/148). The positive rates of M. suis/M. parvum and CMh detected by duplex PCR were 29.1% (43/148) and 15.5% (23/148), respectively. The positive samples detected by duplex PCR were all positive by triplex Taqman probe qPCR. The method established in this study can be used for the clinical diagnosis of M. suis, M. parvum and CMh, which is a technical support for the epidemiological investigation, prevention and control of swine Hemotrophic mycoplasma.
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