摘要
为建立同时检测皮毛中携带的布鲁氏菌、大肠杆菌O157、金黄色葡萄球菌、乙型溶血性链球菌、丹毒杆菌、铜绿假单胞杆菌6种致病菌的基因芯片检测方法,本研究根据NCBI中细菌的16S rDNA和gyrB基因序列,分别设计通用引物和特异性寡核苷酸探针。点样制备检测基因芯片,核酸杂交后,优化并建立同时检测6种致病菌的基因芯片方法。结果表明:使用47%甲酰胺杂交液,42℃摇转杂交4 h为最佳杂交条件。建立的基因芯片方法在多种致病菌之间无交叉反应,检测敏感性可达10拷贝。制备的基因芯片稳定,有效保存期为6个月。该基因芯片对临床样品的检测结果与PCR平行检测结果的符合率为100%。本研究建立的基因芯片检测皮毛中6种致病菌的方法具有高通量、灵敏和特异的特点,为临床皮毛中致病菌的检测和监控提供了新的检测方法。
To establish a DNA chip method for detection of Brucella, E.coli O157, Staphylococcus aureus, f3-Streptococcus, Erysipelothrix rhusiopathiae and Pseudomonas aeruginosa in animal fur simultaneously, two pair of universal primers and the specific oligonucleotide probes of the 6 kinds of bacteria were designed and synthesized based on the 16S rDNA and gyrB gene sequences, respectively. Then DNA chips were prepared by spotting the probes on amino-modified glass slides and followed by DNA hybridization with the DNA products amplified f^om the bacteria by asymmetric PCR. Under the optimized hybridization conditions including incubation for rotating 4 hours in 47% formamide at 42 ~C, the DNA chip method was no cross-reactivity with other bacteria and the sensitivity was approximate 100 copies. In addition, the coincidence rate of the DNA chip and the PCR for clinical fur sample was 100%. The prepared DNA chip was validated for 6 months. Our data indicated the DNA chip assay was a high throughput, sensitive and specific for rapid quarantine of the 6 bacteria in animal fur.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2013年第5期379-383,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
中国检科院基本科研业务费专项(2009JK010)
关键词
皮毛
6种致病菌
基因芯片
检测
animal fur
six kinds of bacteria
DNA chip
detection
