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ARS Home » Pacific West Area » Parlier, California » San Joaquin Valley Agricultural Sciences Center » Crop Diseases, Pests and Genetics Research » Research » Publications at this Location » Publication #326931

Title: Lateral flow immunoassay for the rapid detection of citrus tristeza virus

item MAHESHWARI, YOGITA - Foreign Agricultural Service (FAS, USDA)
item SELVARAJ, VIJAY ANAND RAJ - Foreign Agricultural Service (FAS, USDA)
item RAMADUGU, CHANDRIKA - University Of California
item Keremane, Manjunath
item HAJERI, SUBHAS - Central California Tristeza Eradication Agency
item Yokomi, Raymond - Ray

Submitted to: Conference of International Organization of Citrus Virologists
Publication Type: Abstract Only
Publication Acceptance Date: 3/22/2016
Publication Date: 4/11/2016
Citation: Maheshwari, Y., Selvaraj, V., Ramadugu, C., Keremane, M.L., Hajeri, S., Yokomi, R.K. 2016. Lateral flow immunoassay for the rapid detection of citrus tristeza virus. Conference of International Organization of Citrus Virologists. p.57.

Interpretive Summary:

Technical Abstract: A lateral flow methodology was developed using gold nanoparticles for rapid detection of Citrus tristeza virus (CTV). The test strip was based on a sandwich immunoassay and could be accomplished within 10 minutes. A sample was considered negative for CTV when only the control line appeared; whereas, a sample was considered positive when both control and test lines appeared. A single test required 0.1-0.2 ml of plant extract prepared using coating buffer (pH 9.6). Colloidal gold was prepared by a reduction method using tetrachloroauric acid with trisodium citrate. Purified polyclonal antibodies were developed using concentrated preparation of CTV virions generated in Nicotiana benthamiana plants inoculated with infectious cDNA clone of CTV. The minimum quantity of antibodies that avoided aggregation of colloidal gold solution by NaCl was chosen for gold conjugation. Affinity purified IgG to CTV and Mouse IgG were conjugated with gold nanoparticles and coated onto a glass fiber membrane to serve as a conjugate pad. The nitrocellulose membrane was coated using CTV IgG (1mg/ml) and anti-mouse IgG (1mg/ml) as test line and control line, respectively. The sensitivity of the lateral flow test was up to 1:160 dilution of clarified plant extract. The methodology detected different strains of CTV such as T30, T36, VT and RB. The lateral flow strip provides a rapid and cost effective instant field CTV detection method usable by non-skilled personnel.