鸭副黏病毒和鸭圆环病毒二重荧光定量PCR检测方法的建立

摘要

根据鸭副黏病毒(DPMV)和鸭圆环病毒(DuCV)保守基因序列,设计了2对针对鸭副黏病毒和鸭圆环病毒的特异性引物和2条不同荧光基团标记的TaqMan探针,建立了鸭副黏病毒和鸭圆环病毒的二重荧光定量PCR检测方法.该方法敏感性好,对鸭副黏病毒和鸭圆环病毒的检测敏感性分别达到1 60和140个拷贝数；该方法特异性强,对鸭肝炎病毒、番鸭细小病毒、鸭瘟病毒和H9型禽流感病毒等病原体的检测全为阴性；应用该方法对118份临床病料进行检测,结果检出鸭副黏病毒和鸭圆环病毒阳性感染率分别为0.85％和8.47％,无混合感染.本试验建立的二重荧光定量PCR具有快速、特异、敏感和重复性好等优点,适用于鸭副黏病毒和鸭圆环病毒的快速诊断和监测.%A duplex Real-time polymerase chain reaction(drRT-PCR) assay was developed and optimized to simultaneously detect duck paramyxovirus and duck circovirus in one reaction. Two sets of specific primers for duck paramyxovirus and duck circovirus, along with two TaqMan probes specific for each virus were used in the assay. This drRT-PCR assay was found to be specific and be able to detect and differentiate duck paramyxovirus and duck circovirus, and no positive results were observed, when nucleic acid from duck hepatitis virus, Muscovy duck parvovirus, duck plague virus and H9 subtype avian influenza virus and so on were used as drRT-PCR templates. The sensitivity of this drRT-PCR assay was 160 and 140 copies for duck paramyxovirus and duck circovirus, respectivly. The drRT-PCR assay was applied to 118 clinical samples detection. Among these samples, duck paramyxovirus positive rate was 0. 85%, duck circovirus positive tate was 8. 47%, no co-infection. This drRT-PCR assay was a quick, sensitive and specific test for detecting duck paramyxovirus and duck circovirus,and would be useful for the control of these viruses in ducks.