In April 2010, a severe viral disease spread out in most duck-farming regions in China including Zhejiang, Jiangsu, Hebei and Shandong provinces. The disease affected both meat ducks and laying ducks. The affected layer ducks showed clinical symptom of heavy egg-laying decrease ranging from 20% to 60%, even 90% .During the course of disease, some ducks developed neurological signs including unsteady standing, falling and quivering to death. In some young-duck farms, the disease developed as early as in 10-day-old ducklings, with a peak in 20-40 days old ducklings. The main symptoms were unable to stand steadily and falling. The death rate was normally within 10-30%, and could be as high as 80%, causing huge economic losses in duck-farming. We isolated some apparently new flavivirus from duck incubated in 10-day-old SPF chicken embryos and duck embryos, and found that the isolates belonged to the genus flavivirus based on sequence analysis. The virus had lower nucleotide homology with other genus of flavivirus.
In consideration of biological characters and clinical symptoms, we suggested Duck Encephalitis virus (DEV) was named for this newly virus. DEV infection can cause duck encephalitis with severe central nervous system disorders and egg laying decrease. Diagnosis of DEV infection is mainly based on viral culture and molecular approaches . Virus isolation is a definitive diagnosis for DEV infection, however, this assay is usually unfeasible owing to less sensitive and time-consuming. RT-PCR assays for detection of specific genomic sequence of DEV have shown high sensitivity and specificity. However, these assays are time-consuming and require expensive and sophisticated equipments . DEV often occur in rural areas where routine RT-PCR diagnostic facilities are limited. Therefore, it is necessary for us to develop a rapid, simple, sensitive, and specific diagnostic method for DEV infection and surveillance.
LAMP is a novel PCR method and its most important advantage is amplification of nucleic acids under isothermal conditions at a temperature range between 60 and 65°C within 1 h [4–6]. Another advantage of the method is that the amplification can lead to the accumulation of large amounts of products of various lengths, making detection of amplified nucleic acids much easier. Recently, LAMP technology has been successfully applied for rapid detection of various pathogens [8–12]. A RT-LAMP assay for detecting JEV was firstly reported by Toriniwa and Komiya and the sensitivity was similar to conventional RT-PCR . Later, Parida used the application of RT-LAMP assay to detect JEV in the cerebrospinal fluid samples from patients with clinical diagnosis of acute encephalitis .
So far, there has been no report on using the RT-LAMP to rapidly diagnose and identify DEV. In this study, we established the RT-LAMP method to rapidly diagnose and identify DEV. The assay can be a new standard for the virus identification.