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 × 10? 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.     

Immunosorbent analysis of toxin contamination in milk and ground beef using IgY-based ELISA

Analytical methodology to detect ricin and Shiga toxins (Stx) in food matrices is important for food safety and biosecurity. Monoclonal antibodies (mAbs) that bind each toxin were used for capture in sandwich enzyme-linked immunosorbent assay, and IgY polyclonal antibodies were prepared as detection antibodies. The ricin assay systems, using colorimetric or chemiluminescent substrates, detected native, but not heat-denatured ricin. The lower limit of detection (LOD) was 0.13?ng?mL^?1 in milk and 0.8?ng?g^?1 in ground beef. The Stx2 assay systems detected native Stx2, but not heat-denatured Stx2 or Stx1. The LOD was 0.13?ng?mL^?1 in milk and 0.7?ng?g^?1 in ground beef. Using a standard 96-well-plate format, the assays can detect less than 1?×?10^?4 of an estimated lethal oral dose of either toxin in a serving of milk. The IgY detection antibodies for ricin were more heat-stable than mouse polyclonal anti-ricin at 65°C.