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.

► Duck Tembusu virus is a newly emerged flavivirus.
► A minor groove binder assay was developed for real-time RT-PCR detection of DTMUV.
► The assay was quantitative and highly sensitive for detection of clinical samples.
► The assay was a reliable real-time method for epidemiological study of DTMUV infection.
► The assay will also be useful for early diagnosis of TMUV and management of TMUV.