猪源嗜血支原体三重TaqMan探针荧光定量PCR方法建立和应用  

Establishment and Application of a Triplex TaqMan Probe Real-time Fluorescene Quantitative PCR Method for Detection of Hemotrophic mycoplasma from Swine(Sus Scorfa)

作　　者：付媛[1] 石团员[1] 袁秀芳[1] 徐丽华[1] 孙洪超 韦强[1] 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)

机构地区：[1]浙江省农业科学院畜牧兽医研究所,杭州310021

出　　处：《农业生物技术学报》2022年第6期1228-1236,共9页Journal of Agricultural Biotechnology

基　　金：浙江省重点研发计划(2019C02052-4);浙江省公益性项目(2017C32018)。

摘　　要：猪附红细胞体病是由猪支原体(Mycoplasma suis)、微小支原体(M.parvum)和浙江分离到的待定新种嗜血支原体(Candidatus M.haemosuis,CMh)引起猪(Sus Scorfa)传染性贫血、黄疸和母猪流产等症状的疾病。3种病原在我国猪群中的流行和传播情况不明,为鉴别诊断3种猪源嗜血支原体和了解该类病原在我国猪群中的流行特点,本研究建立了1种基于甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)基因的三重TaqMan探针荧光定量PCR(qPCR)鉴别检测方法。根据已知的3种猪源嗜血支原体GAPDH基因序列,利用Beacon Designer 4.0软件分别设计特异性引物和探针。通过分别优化Mg^(2+)浓度、热启动酶浓度、引物和探针浓度等反应条件,建立了三重TaqMan探针qPCR方法,并验证了该法的特异性、敏感性和重复性。结果表明,建立的三重TaqMan探针qPCR方法标准曲线相关系数R^(2)在0.9992~0.9996之间,扩增效率在99.9%~103.2%之间;该法对其他常见猪病无扩增信号,具有良好的特异性;该法对3种猪源嗜血支原体的最低检测限均为1 copy/μL;组内和组间变异系数在0.31%~1.75%之间,表明重复性好。应用该法对浙江省148份猪抗凝血样品检测并与二重PCR符合比较,结果显示三重Taqman探针qPCR检测M.suis、M.parvum和CMh的阳性率分别为50%(74/148)、48.6%(72/148)和64.9%(96/148),3种病原的混合感染率为29.7%(44/148);二重PCR方法检测M.suis/M.parvum和CMh阳性率分别为29.1%(43/148)和15.5%(23/148),二重PCR阳性样品三重qPCR检测均阳性。本研究建立的方法可用于临床上M.suis、M.parvum和CMh的鉴别诊断,为猪源嗜血支原体病的流行病学调查和防控提供技术支撑。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 Mg^(2+),hot-start r Taq,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(R^(2))of the established triplex Taqman probe qPCR method was between 0.9992~0.9996,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

关 键 词：猪支原体 微小支原体 新种嗜血支原体 嗜血支原体 荧光定量PCR 附红细胞体病 

分 类 号：S855[农业科学—临床兽医学]