PCR结合变性高效液相色谱法与荧光定量PCR法在检测禽白血病中的比较与应用

摘要

Compared the new molecular method for rapid detection of avian leukemia virus by using denaturing high performance liquid chromatography(DHPLC) combined with nucleic acid amplification and Real-time PCR in this study. According to the sequence of pol gene of ALV, one pair of primers and the TaqMan probe were designed by using Primer Premier 5. 0. The PCR fragment which was amplified by the primers were analysised by DHPLC and the results of Real-time PCR by the primers and the TaqMan probe. They all compared to normal chicken embryo allantoic fluid, duck plague virus(DPV),infectious bronchitis virus(IBV) ,goose parvovirus(GPV) ,avian influenza virus(H5Nl AIV),Newcastle disease virus(NDV), infectious bursal disease virus (IBDV) ,EDSV. There were tested to confirm the specificity of the PCR-DHPLC assay and no positive absorption peaks occurred. The detection limit of ALV AV228 by PCR-DHPLC was 3 pg,10 fold iower than the ordinary Realtime PCR. The results of detcting organ samples from the chickens were tested by PCR-DHPLC and Real-time PCR,showing 100% agreement.%本研究旨在比较PCR结合变性高效液相色谱技术(PCR-DHPLC)与荧光定量PCR (Real-time PCR)两种方法在检测禽白血病中的应用.根据禽白血病pol基因序列,设计1对引物和1条探针,利用引物进行禽白血病模板的RT-PCR扩增,产物经变性高效液相色谱上样处理；利用引物及探针进行荧光定量PCR扩增,结果与PCR-DHPLC进行比对.两种方法同时用正常鸡胚尿囊液、鸭瘟病毒、传染性支气管炎病毒、鹅细小病毒、H5N1亚型禽流感病毒、新城疫病毒、传染性法氏囊病毒、减蛋综合症病毒做特异性检测;以稀释成不同梯度的AV228毒株核酸做敏感性检测.试验结果表明PCR-DHPLC方法只对禽白血病病原有阳性扩增的吸收峰,Real-time PCR也只对禽白血病病原有阳性扩增,两法均对其他禽源病毒核酸无特异性扩增；PCR-DHPLC与Real-time PCR法相比,灵敏度虽低于Real-time PCR 1个数量级,但检测限仍可达到3 pg核酸模板.两种方法均能检测到感染鸡的泄殖腔拭子所采集的病毒量.采集120份蛋鸡棉拭子同时进行临床检测,两种方法检测ALV的符合率为100％(15/120).研究表明两种方法均可用于诊断禽白血病病毒.