Detection and subgrouping of Cucumber mosaic virus isolates by TAS-ELISA and immunocapture RT-PCR.
Eight mouse hybridoma cell lines secreting monoclonal antibodies (MAbs) against Cucumber mosaic virus (CMV) were produced. Analysis of the specificities of the MAbs against CMV isolates by triple antibody sandwich (TAS)-ELISA demonstrated that four MAbs were specific for subgroup I (S-I) isolates and two for subgroup II (S-II) isolates, whereas another two MAbs could detect both S-I and S-II isolates. TAS-ELISA and immunocapture RT-PCR (IC-RT-PCR) methods were then established for reliable and efficient detection and subgrouping of CMV isolates using the produced MAbs. When 197 field samples collected from six provinces in China were tested by TAS-ELISA, 130 samples were found to be infected by CMV. Among them, 121 samples were infected by S-I isolates (93.1%) and another nine samples by S-II isolates (6.9%). In IC-RT-PCR using the MAbs and specific primers in the region of the coat protein (CP) gene, samples shown to contain S-I isolates by TAS-ELISA gave one specific band about 500 nucleotides in length, whereas samples containing S-II isolates produced a single band with the length of approximately 600 nucleotides. The validity and reliability of the results of TAS-ELISA and IC-RT-PCR was confirmed by sequencing and phylogenetic analysis of nearly full-length CP genes of the isolates.
Yu
Cui
C
Institute of Biotechnology, Zhejiang University, Hangzhou 310029, PR China.
Wu
Jianxiang
J
Zhou
Xueping
X
D000818:Animals; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral / Q000276:immunology*; D017799:Cucumovirus / Q000145:classification / Q000276:immunology / Q000302:isolation & purification*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010802:Phylogeny; D010935:Plant Diseases / Q000821:virology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral