Liu Zongliang, Fu Yuguang, Ji Yanhong, Wei Jianzhong, Cai Xuepeng, Zhu Qiyun
State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 1 Xujiaping, Chengguan District, Lanzhou 730046, PR China.
Avian Dis. 2013 Sep;57(3):595-601. doi: 10.1637/10484-010713-Reg.1.
Duck Tembusu virus (DTMUV) is a single-stranded positive-sense RNA virus that causes disease to emerge in duck flocks and results in huge economic losses to the duck industry. However, no vaccines and control measures are available in China to date. Development of reliable and fast detection methods is necessary to prevent and control this disease. Therefore, a one-step SYBR Green real-time reverse transcription polymerase chain reaction (RT-PCR) method is established here for DTMUV detection. The results show that the method can specifically detect DTMUV without cross-reactions with selected avian pathogens. The sensitivity of the assay was 1000 times greater than that of a conventional RT-PCR and able to test as few as 20 copies from RNA standard samples. The coefficients of variations of inter- and intra-assay values ranged from 0.09% to 0.36% and 0.1% to 0.23%, respectively. Testing 168 field samples and 96 experimentally infected samples by conventional RT-PCR and the one-step SYBR Green real-time RT-PCR, the positive rates were 35.1% and 73.8% from field samples and 30.2% and 64.6% from infected samples. The one-step SYBR Green real-time RT-PCR developed in this study was shown to be a sensitive, specific, high-throughput, cost-effective, and simple diagnostic tool for the rapid detection and epidemiological surveillance of the emerging DTMUV infection.
鸭坦布苏病毒（DTMUV）是一种单链正链RNA病毒，可导致鸭群发病，给养鸭业造成巨大经济损失。然而，迄今为止中国尚无可用的疫苗和防控措施。开发可靠且快速的检测方法对于预防和控制这种疾病至关重要。因此，本文建立了一种用于检测DTMUV的一步法SYBR Green实时逆转录聚合酶链反应（RT-PCR）方法。结果表明，该方法能够特异性检测DTMUV，与所选禽病原体无交叉反应。该检测方法的灵敏度比传统RT-PCR高1000倍，能够检测低至20个RNA标准样品拷贝。批内和批间检测值的变异系数分别为0.09%至0.36%和0.1%至0.23%。通过传统RT-PCR和一步法SYBR Green实时RT-PCR检测168份田间样品和96份实验感染样品，田间样品的阳性率分别为35.1%和73.8%，感染样品的阳性率分别为30.2%和64.6%。本研究开发的一步法SYBR Green实时RT-PCR是一种灵敏、特异、高通量、经济高效且简单的诊断工具，可用于新兴DTMUV感染的快速检测和流行病学监测。
