禽流感H9亚型一步法RT-PCR快速检测试剂盒的研制及应用

为建立一种简单、快速、灵敏、准确的禽流感H9检测方法,本研究根据GenBank中收录的禽流感H9病毒高度保守的基因序列,设计1对H9的特异性引物,建立了一步法快速检测禽流感的PCR,对反应条件进行优化后,组装成快速检测试剂盒,并对临床收集的疑似禽流感病料进行检测。结果显示,试验成功建立了禽流感H9病毒一步法RT-PCR检测方法,在此基础上完成了试剂盒的组装,对30份疑似禽流感病料的检出率高达98%以上,检测灵敏度达10^1.5ELD₅₀,稳定性良好, 冷冻保存期达12个月。经证实该试剂盒具有操作简便、经济、快速、灵敏度高、特异性强、重复性好、稳定性高等优点,值得推广应用。

Development and Application of Rapid RT-PCR Detection Kit for AIV H9 Subtype

In order to establish a simple, sensitive, accurate and rapid PCR detection method and develop a one-step RT-PCR detection kit for AIV H9 subtype,one pair of specific primers was designed and synthesized according to the conserved gene of AIV H9 subtype in GenBank, and then PCR method of rapid detection of AIV H9 subtype was formed, and through optimizing the reaction conditions, a rapid detection kit was assembled, and lots of samples from suspected AIV H9 subtype were to be detected. The results showed that AIV H9 subtype one-step RT-PCR detection method was successfully established, and then developed a kit, in addition, the rate of detection to samples suspected AIV H9 subtype was up to 98%, the detection sensitivity was 10^1.5 ELD₅₀, and the stability was good, in the cryopreservation case, it could survive for at least 12 months. Moreover, the operation was easy, fast, economic, effective. Detection of clinical samples confirmed that the kit was simple, rapid, high sensitivity, specificity, repeatability and stability, it was worthy to be promoted and applied.