荧光定量RT-PCR检测禽白血病病毒方法的建立及应用

gag基因是禽白血病病毒(ALV)的群特异性基因,根据GenBank中登录的gag基因的保守序列(E46390),设计合成了1对引物,建立了基于SYBR GreenI模式的检测禽白血病病毒的实时荧光定量RT-PCR方法。该方法只对目的基因有扩增,对其它无关病毒核酸无特异性扩增;扩增效率为94.6%;在8.2×102～8.2×107拷贝的范围内有很好的线性关系(Y=3.46X+31.8,r=0.9995);最低可检测82个拷贝的病毒核酸,与普通PCR方法相比,灵敏度高出1000倍。组内CV为0.28%～1.45%;组间CV为2.13%～3.84%;对151例临床疑似样本的检出率为54.9%(83/151)。随机选择15份阳性样本PCR产物克隆测序,结果证明扩增片断为ALV的特异序列,与ALVgag基因核苷酸同源性为97.8～98.9%;对重庆14个县的14个健康商品蛋鸡群的134份粪便棉拭子进行检测,ALV的检出率为38.1%,鸡群的阳性率为14/14。同时用实时RT-PCR方法和ELISA方法对从种鸡场随机采集的30份100日龄蛋鸡的血清进行检测,实时RT-PCR检测率为5/30,ELISA方法检测率为4/30。本方法的建立为禽白血病的快速诊断、大规模检疫、监测、流行病学调查以及药物筛选等提供了新的检测方法。

Development and Application of Real-Time RT-PCRfor Detection of Avian Leukosis Virus

Gag gene is a group-specific gene in avian leukosis virus (ALV). A SYBR Green I-based real-time PCR was developed for detection of ALV by using a pair of primers that was designed to target conservative region of gag gene(E46390). The results demonstrated that the real time RT-PCR established in this study showed several advantages: (1) High specificity. Positive amplification could be observed only from ALV DNA; (2) Good repeatabil- ity. The intra-assay and inter-assay CVwere 0.28%~1.45% and 2.13%~3.84% respectively; (3) Sensitive and rapid. The detection limit of the assay was 82 copies, 103 times higher than traditional PCR. The RRT-PCR also showed a broad linear range (8.2×102~8.2×107 copies, r=0.999) of quantification and high efficiency (94.6%). 151 suspected clinical samples were detected by the real time RT-PCR. And the detection rate is 83/151. Sequencing results of PCR products from 15 positive samples showed 97.8~98.9% nucleotide homology between sequences determined in this study and in GenBank, suggesting that this assay have good specificity for ALV. Total 134 fecal swabs were collected from 14 healthy egg flocks in 14 counties of Chongqing and detected by the real-time RT-PCR. 38.1% of samples were positive for ALV. The positive rate of ALV was 100 % (14/14) in virus-carrying flocks. 30 serum samples from the layers of 100 days were also detected using both real-time PCR and ELISA. The positive ratio were 5/30 and 4/30, suggesting a little higher detection rate of real-time PCR compared to ELISA. The real-time PCR assay developed in this study will be useful for rapid laboratory diagnosis, large-scale inspection,drug screen- ing and epidemiology investigation for the avian leukosis .