摘要
牛传染性胸膜肺炎和牛支原体病均为重要的牛传染病,两者均可致牛出现呼吸系统症状,临床上难以区分。为建立鉴别诊断牛传染性胸膜肺炎和牛支原体病的双重Taq Man荧光定量PCR检测方法,本研究根据这两种病的病原丝状支原体丝状亚种和牛支原体的基因组保守区域(丝状支原体丝状亚种nt826-nt1742,牛支原体nt189-nt618)分别设计引物与探针,通过优化反应体系与反应条件,建立同时检测这两种病原的Taq Man荧光定量PCR检测方法。特异性试验结果显示,该方法仅对丝状支原体丝状亚种模拟样品和牛支原体检测结果为阳性,而巴氏杆菌、牛传染性鼻气管炎病毒、无乳支原体、山羊支原体山羊肺炎亚种、丝状支原体山羊亚种、关节炎支原体Leachii株检测结果均为阴性,特异性较强。分别以1.0×10~7拷贝/μL~1.0×10~1拷贝/μL的p EASY-Mmm和p EASY-Mb质粒标准品为模板,进行敏感性试验,结果显示,该方法对丝状支原体丝状亚种和牛支原体重组质粒标准品的检测限均为1.0×10~1拷贝/μL,敏感性较高。对不同浓度质粒标准品混合物的重复性试验结果显示,组内与组间重复性试验变异系数均小于2.5%,重复性较好。利用该方法对112份临床样品(104份鼻拭子、8份肺组织样品)检测,结果显示,丝状支原体丝状亚种检测结果和已发表PCR方法检测结果一致,均为阴性,而本实验建立的方法检测出4份牛支原体阳性样品,且经测序证实检出样品为真实阳性样品,而已发表的多重PCR方法检测该病原结果均为阴性。本研究建立了能够同时检测丝状支原体丝状亚种和牛支原体的双重Taq Man荧光定量PCR方法,其特异性强、敏感性高、重复性好,可用于各种临床样品的检测,为这两种病原的快速检测和流行病学调查提供了技术手段。
Bovine contagious pleuropneumonia and Mycoplasma bovis pneumonia are important bovine infectious diseases with respiratory symptoms that are difficult to distinguish from clinically.To establish a duplex Taq Man real-time PCR method for the differential diagnosis of these diseases,primers and probes were designed according to the conserved specific regions of the genomes of Mycoplasma mycoides subsp.mycoides(Gen Bank:u61140.1,from nt826 to nt1742),and Mycoplasma bovi(Gen Bank:af130119.1,from nt189 to nt618).By optimizing the reaction system and conditions,a duplex Taq Man real-time PCR was established to detect these pathogens simultaneously.The specificity test results showed that the method was specific fo Mycoplasma mycoides subsp.mycoides and Mycoplasma bovis without cross-reaction with other related pathogens,including Pasteurella,infectious bovine rhinotracheitis virus,Mycoplasma agalactia,Mycoplasma capricolum subsp.capripneumoniae Mycoplasma mycoides subsp.capri,and Mycoplasma leachii strain,indicating strong specificity.The sensitivity test results showed that the detection limit of this method for Mycoplasma mycoides subsp.mycoides and Mycoplasma bovis recombinant plasmid standards both were 1.0×10~1copies/μL,representing a high sensitivity.The repeatability test showed that the coefficient o variation in intra-and inter-batch was less than 2.5%,suggesting excellent repeatability.This method detected 112 clinical sample in our laboratory,which were identified as negative by routine PCR for Mycoplasma mycoides subsp.mcoides and Mycoplasm bovis.The results showed that the detection results for Mycoplasma mycoides subsp.mycoides were negative.However Mycoplasma bovis was detected in 4 positive samples,which were confirmed to be authentic positive samples by sequencing.Thi study established a duplex Taq Man real-time PCR to detect Mycoplasma mycoides subsp.mycoides,and Mycoplasma bovi simultaneously.The method showed strong specificity,high sensitivity,and excellent repeatability.It can detect various cli
作者
马芷忻
武琪
刘桐
辛九庆
徐青元
MA Zhi-xin;WU Qi;LIU Tong;XIN Jiu-qing;XU Qing-yuan(State Key Laboratory for Animal Disease Control and Prevention,Harbin Veterinary Research Institute,Chinese Academy of Agriculture Science,Harbin 150069 China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2024年第3期262-268,共7页
Chinese Journal of Preventive Veterinary Medicine
基金
重要新发与外来动物疫病口岸检疫新型检测技术研究(2021YFD1800503)
中国农业科学院科技创新工程项目。
