I型鸭肝炎病毒 TaqMan 荧光定量 RT－PCR 方法的建立及初步应用

摘要

Objective To develop a real-time RT-PCR assay ( rRT-PCR) for efficient detection of duck hepatitis virus type 1 ( DHV-I) .Method According to the different gene sequences of DHV-I from different provinces download from NCBI and to find the conserved sequences.One pair of the specific primers and one TaqMan probe were designed. Then reaction parameters were optimized to develop a real-time RT-PCR assay ( rRT-PCR) .Results This developed rRT-PCR assay could detect 20 template copies of RNA, and its sensitivity was higher than that of the conventional RT-PCR. This rRT-PCR assay was found to be specific and able to detect DHV-I, and no positive results were observed when nucleic acid from Muscovy duck parvovirus, goose parvovims, Newcastle disease and avian influenza virus, egg drop syndrome virus, reticuloendotheliosis virus, duck Tembusu virus, poultry intestinal arc virus were used as rRT-PCR templates.The results of this developed rRT-PCR assay used for 100 duck clinical samples showed a positive rate of 92%, indicating that DHV exists in duck group of Jiangsu province in China.Conclusion This rRT-PCR assay can be used as a rapid tool for detection of DHV-I.%目的：建立快速诊断I型鸭肝炎病毒的荧光定量RT－PCR方法。方法根据NCBI下载的20个来自我国不同省份的的I型鸭肝炎病毒的基因序列，找出其保守序列，设计合成一对引物和一条TaqMan探针，进行条件优化，检测其特异性，敏感性，稳定性。结果该方法敏感性达20拷贝，比常规PCR敏感性高。其特异性强，对番鸭细小病毒（MDPV），鹅细小病毒（GPV），新城疫病毒（NDV）和禽流感（AIV），鸭减蛋综合征病毒（EDSV），禽网状内皮组织增生症病毒（REV），鸭坦布苏病毒（DTMUV），禽呼肠弧病毒（ARV）8种病毒的检测均为阴性，I型鸭肝炎病毒检测结果为阳性。用建立的方法检测了江苏徐州采集100份样品，阳性率为92％。说明I型鸭肝炎病毒在江苏徐州地区的鸭群中普遍存在。结论建立了I型鸭肝炎病毒荧光定量PCR方法。