On-site detection of Citrus tristeza virus (CTV) by lateral flow immunoassay using polyclonal antisera derived from virions produced by a recombinant CTV

Maintenance of virus-free citrus in nurseries and orchards is essential to control the spread of aphid-transmitted Citrus tristeza virus (CTV). To this end, a lateral flow immunoassay (LFIA) was developed to detect CTV that was user-friendly and field-deployable. Polyclonal antisera was generated in rabbit against purified virions from Nicotiana benthamiana inoculated with an infectious recombinant cDNA clone of CTV (rCTV). Affinity purified rCTV IgG (1 mg/ml) and Mouse IgG (0.5 mg/ml) were conjugated with gold nanoparticles and coated onto a glass fiber membrane conjugate pad. Affinity purified rCTV IgG and anti-mouse IgG were used as test line and control line, respectively on the test strip. The LFIA detected CTV within 10 min and was sensitive up to a 1:80 dilution of crude plant sap extract. The LFIA was validated using CTV strains T30, T36, VT and RB from greenhouse and field sources and results were 100% in agreement with Enzyme-Linked Immunosorbent Assay and Reverse Transcription quantitative PCR. The immunostrip readily detected CTV after six months in storage in a sealed bag with silica gel at room temperature indicating good stability. The LFIA test strips offered a cost-effective tool to detect CTV in the field or nursery by non-skilled personnel without laboratory equipment.