Rapid Diagnosis Of DTMUV Based On Molecular Techniques And The Variability Analysis Of DTMUV E Gene

Since June2010, a severe outbreak of egg-laying and breeder duck infectious disease, with egg drop, loss of appetite and ovary-oviduct disease, has spread around the major duck-producing regions in China. A novel Flavivirus, named Duck Tembusu virus (DTMUV), was isolated from sick duck. The DTMUV genome was positive-sense, single-strand RNA,10,990nucleotides in length. In order to carry out the epidemiological investigation of the disease and to improve accuracy of disease diagnostic, the study established the DTMUV RT-PCR and quantitative RT-PCR detection methods, and from different sources DTMUV the E protein gene variability analysis. These findings will lay a foundation for disease prevention and control technology research.1. The detection of one step RT-PCR assay of DTMUVA pair of primers was designed based on the analysis of TMUV sequences. The One-step RT-PCR method had been successfully developed for detection of DTMUV through optimization of viral RNA extraction and RT-PCR assay. The results showed that1.82TCID50of virus could be detected in two hours. The rate of positivity in106clinically suspected tissues and organs of diseased duck subjected to detection by RT-PCR was46.2%. The results indicated that DTMUV is high prevalent in Zhejiang and its neighboring provinces.2. The detection of one step quantitative RT-PCR assay of DTMUVThe design and development of a3’-conjugated minor-groove-binding (MGB) probe for a real-time RT-PCR assay allowing for the rapid, sensitive, and specific detection of duck Tembusuvirus (DTMUV) RNA are described. This assay targeted the3’terminal non-codingregion (NCR) of the TMUV genome and detected1×101copies of RNA perreaction without cross-reaction with other duck pathogens. The linear range of detection was2×101～2×108copies/μL.The assay was rapid, requiring just over2.Oh, including the nucleic acid extraction step. Therefore, this assay is an excellent tool for research routine diagnostic applications, and study of the epidemiology of DTMUV infections among duck flocks. 3. The variability analysis of E gene of DTMUVAccording to35strains of DTMUV E gene nucleotide sequence and amino acid sequence alignment, found that nucleic acid sequence (1503bp) and amino acid sequence (501AA) homology were97%～100%and97.4%～100%, respectively. There was the main nucleotide difference at the positon191, resulted amino acid difference at the position64.The resulting showed that the E gene of DTMUV was not obvious genetic variations.