Development of a one-step real-time RT-PCR assay using a minor-groove-binding probe for the detection of duck Tembusu virus.

The design and development of a 3'-conjugated minor-groove-binding (MGB) probe for a real-time RT-PCR assay allowing for the rapid, sensitive, and specific detection of duck Tembusu virus (DTMUV) RNA are described. This assay targeted the 3' terminal non-coding region (NCR) of the TMUV genome and detected 1 × 10¹ copies of RNA per reaction without cross-reaction with other duck pathogens. The linear range of detection was 2 × 10¹-2 × 10⁸ copies/μl. The assay was rapid, requiring just over 2.0 h, including the nucleic acid extraction step. Therefore, this assay is an excellent tool for research routine diagnostic applications, and study of the epidemiology of TMUV infections among duck flocks.