摘要
本研究旨在建立联合检测胸膜肺炎放线杆菌、猪肺炎支原体和多杀性巴氏杆菌的DNA芯片。用7个从3种病菌基因组中扩增出的不同特异性靶DNA制作基因芯片,并对芯片的靶DNA和探针浓度、杂交温度、重复性、特异性和灵敏度进行了研究。结果表明,检测芯片的特异性强,能与测试的李氏放线杆菌、猪鼻支原体和副猪嗜血杆菌等9种病原区分;灵敏度高,在50μL标记反应体系中,能检测到10~50 pg基组DNA,芯片可重复利用。用芯片对44株目标菌的不同型标准菌株、分离株和疫苗株进行了检测,其信号值≥1 000,信号噪音比（SNR）≥6。用芯片对45头病猪和97头健康猪的临床样品选择培养物进行了检测,其检出率分别为多杀性巴氏杆菌71.1%和49.5%、胸膜肺炎放线杆菌42.2%和26.8%、猪肺炎支原体20%和22.7%,混合感染率分别为42.2%和24.7%。在检测临床样品时,芯片法与PCR的符合率为97.8%~100%,与分离鉴定法的符合率为87.6%~95.6%。研究表明,研制的芯片特异性强、敏感性高、可重复使用,是一种能有效用于胸膜肺炎放线杆菌、猪多杀性巴氏杆菌和猪肺炎支原体鉴定和联合检测的新工具。
The study aimed to establish a DNA microarray to simultaneously detecting Actinobacillus pleuropneumoniae,Mycoplasma hyopneumoniae and Pasteurella multocida.7 different specific target DNA fragments designed and amplified from the genomes of the bacteria were used to construct the DNA microarray.The concentrations of target DNA and probes,hybridization temperature,reproducibility,specificity and sensitivity of the microarray were tested.The microarray could be used repeatedly and possessed high specificity.Microarray hybridization patterns of A.pleuropneumoniae,M.hyopneumoniae and P.multocida could be distinguished from other 9 pathogens species,such as A.lignieresii,Haemophilus parasuis,M.hyorhinitis.The sensitivity of the microarray was determined to 10-50 pg of genome DNA in 50 μL labeling reaction system.44 different serotype reference,isolates and vaccine strains of A.pleuropneumoniae,M.hyopneumoniae and P.multocida were detected with the constructed microarray.Their signal intensities and signal to noise ratio（SNRs） were greater than or equal to 1 000 and 6,respectively.Selective cultures of samples from disease pigs（n=45） and clinical healthy pigs（n=97） were detected by the microarray.Recall ratios of P.multocida,A.pleuropneumoniae,M.hyopneumoniae from disease and healthy pigs were 71.1% and 49.5%,42.2% and 26.8%,20% and 22.7%,respectively.Mixed infection rates were 42.2% and 24.7% in disease and healthy pigs respectively.The coincidence ratios between the microarray and PCR were 97.8%-100%,and the ratios between the microarray and isolation method were 87.6%-95.6%,in detecting clinical samples.Results indicated that the microarray have high specificity and sensitivity,and is a new tool for detection and identification of A.pleuropneumoniae,M.hyopneumoniae and P.multocida.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2010年第1期77-85,共9页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
教育部长江学者和创新团队发展计划基金资助项目(IRT0848)
重庆市科委自然科学基金计划资助项目(CSTC
2009BB5235)
关键词
基因芯片
胸膜肺炎放线杆菌
猪肺炎支原体
多杀性巴氏杆菌
检测
microarray
Actinobacillus pleuropneumoniae
Mycoplasma hyopneumoniae
Pasteurella multocida
detection
