To determine the genomic sequence of a duck hepatitis virus type 1 (DHV-1) strain,real-time quantitative polymerase chain reaction (RTQ-PCR) assay based on SYBR Green I technology was developed to target 3D gene of DHV-1. Comparative sequence analysis showed that the genome has a typical picornarivus genetic organization,and strain DHV-1 R genetic organaiztion is 5′ untranslated region (UTR)-VP0-VP3-VP1-2A1-2A2-2B-2C-3A-3B-3C-3D-3′ UTR,DHV-1 R has close relationship with Parechovirus,and has 95.1-99.1% nucleotide sequence identity with other DHV-1 strains. Based on the DHV-1 sequences in GenBank,three pairs of specific primers were designed to amplify DHV-1 using real-time PCR. The results showed that real-time PCR Tm value is 85.6°C and the real-time PCR provides a broad dynamic range,detecting from 102 to 109 copies of DHV-1 cDNA per reaction. No cross-reactions were found in specimens containing DPV,AIV and NDV. It is concluded that DHV-1 belongs to a new group of the family Picornaviridae that may form a separate genus most closely related to the genus Parechovirus. All results showed that the real-time PCR has high sensitivity and specificity to detect DHV-1 using SYBR Green I dissociation curve analysis,isolates can be distinguished by their melting temperature. These methods are rapid,sensitive,and reliable,and can be readily adapted for detection of DHV-1 from other clinical samples.
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