Direct Tissue Blot Immunoassay (DTBIA) for Detection of Citrus Tristeza Virus (CTV)

Abstract

A direct tissue blot immunoassay (DTBIA) procedure was tested for detection of citrus tristeza virus (CTV). Freshly cut stem, petiole or fruit pedicel tissue was carefully pressed to nitrocellulose membranes. The membranes were blocked by incubation in dilute bovine serum albumin and then incubated with unlabeled or biotinylated monoclonal or polyclonal antibodies. Antigen-bound biotinylated antibodies were detected by exposure to a streptavidin-alkaline phosphatase conjugate (APC) and antigen-bound unlabeled antibodies were detected by a goat anti-mouse or goat anti-rabbit IgG-APC. The substrate was NBT-BCIP. Localized areas of the tissue imprints of CTV-infected plants stained intensely and were easily recognized under 10X magnification. Location of CTV in phloem tissues was determined easily without sectioningor other cytologicaltechniques. Nocomparablestaining was observed in imprints of healthy tissue. Assays of 858 healthy and CTV-infected trees in Florida and 560 trees in Spain by ELISA and by DTBIA indicated similar rates of CTV infection. Strain differentiation was accomplished by making duplicate impressions on different test sheets and processing one with the strain-selective monoclonal CTV-MCA13 and the other with polyclonal antibodies, or a mixture of monoclonal antibodies which react to all isolates. DTBIA is rapid, requires little sample preparation, and tissue blots could be stored at room temperature at least 30 days prior to assay. Blotted membranes can be sent safely to another location for testing. DTBIA has been adapted for commercial diagnostic purposes.