Gold Immunochromatographic Assay For Rapid Detection Of Papaya Ringspot Virus

Payaya ringspot virus (PRSV), a member of the genus Potyviruses, is the cause of a destructive diseases and a major limiting factor for papaya. So it is very important to detect to PRSV rapidly and accurately. In this paper, the main purpose is to development immunochromatography colloidal gold strip for detection of PRSV in the field.In this paper, through a series of differential hosts and nucleotide sequences and deduced amino acids sequences analysis.The results showed that virus of papaya disease was PRSV-Vb strain. An isolate of PRSV-Vb obtained from leaves of Cucurbita pepo infected by PRSV-Vb was purified, and antiserum to the virus was produced. Virus was purified by 30% sugar-cesium sulfate of density gradient centrifugation after clarification of tissue extracts by chloroform-carbon tetrachloride and concentration of virus particles by polyethylene glycol. The PRSV serums were purified by caprylic acid ammonium sulfate precipitation (CA-AS) and Protein A Columns. The purified immunoglobulin G (IgG) was analyzed by formula, SDS-PAGE and indirect enzyme linked immunosorbent assay (ELISA).The colloidal gold particles were prepared by sodium citrate reduction. The results showed that the 18 nm colloidal gold particles were most suitable for labeling antibody. The optimum concentration was 18 μg/mL and the optimum pH was 7.5 (0.2 M K2CO315 μL/mL) when colloidal gold antibody conjugates. The purified labeled antibody was diluted at ratio of 1:3 and flame plating 15 μL on fiberglass. The best concentration of rabbit anti-PRSV IgG on test line was 1.6 mg/mL and the best concentrations of goat anti-rabbit IgG on control line was 0.5 mg/mL. The Ahlstrom8964 (binder release pad), Millipore 135 (analying membrane), SB06 (sample pad) and SX18 (water absorbing pad) and sm31-25 (plastic back plate) were used to paper the test strip. The PRSV was detectable when juice of leaf of Papaya ringspot virus was diluted 1000 times. The result could be read clearly in 10 minutes.The results showed that the PRSV test strip is time saving, easy to justified, sensitive, and suitable for testing in the field. The date and the techniques will help us research and develop a series of gold immunochromatography assay strip to detect the plant virus.