摘要
本研究基于IS1081基因建立了鉴定犬结核的SYBRGreen荧光定量PCR方法。在最佳反应条件下反应效率为1．09，获得的标准曲线相关系数R值达0．9995，标准曲线的线性范围达到1～1×10^7拷贝8个数量级，最低可检测到约10个目的拷贝，即31．4fg DNA。根据溶解曲线分析，扩增产物中无引物二聚体的影响，说明所设计引物特异，退火温度合适。这表明本研究建立的SYBR Green荧光定量方法灵敏度高、定量范围广。利用该方法对临床样品进行检测，结果发现：与结核杆菌分离培养方法比较，符合率为85％。240份犬猫鼻拭子中150份被检测为阳性，这说明犬猫鼻腔中分枝杆菌携带率很高。
A SYBR Green Real-time quantitative PCR assay was developed based on insertion sequence 1081 （IS1081） DNA of Mycobacterium tuberculosis to detect tuberculosis in dogs and cats. The Real-time PCR assay was highly specific and had a broad linear detecting range （1～1×10^7 copies/μL, R= 0. 999 5） with 1.09 PCR amplification efficiency. The sensitivity of this method approached up to 10 copies （31. 4 fg DNA）. The melting curve also showed that primers were specific and annealing temperature was suitable. This method had good agreement （85%） with bacterial isolation when known positive clinical samples were tested. The Real-time PCR assay was used to detect IS1081 DNA in dogs and cats nasal swabs. Results showed that 150 out of 240 samples were positive, which indicated a high mycobacterium carrier rate in nasal cavities of these animals.
出处
《中国动物传染病学报》
CAS
2009年第2期55-59,共5页
Chinese Journal of Animal Infectious Diseases
基金
上海市科技兴农重点项目(沪农科攻字(2006)第5-4号)
