Visual and Highly Sensitive Serological Detection Technology for Pepper mild mottle virus Based on Monoclonal Antibody
LIU Nai-Ru, ZHANG Xi, ZHANG Cui, DONG Jie, MAO Wei-Jia, WANG Ya-Qin, ZHOU Xue-Ping, FU Shuai, WU Jian-Xiang*
State Key Laboratory of Rice Biology & Breeding/Zhejiang Key Laboratory of Biology and Ecological Regulation of Crop Pathogens and In‐sects, Institute of Biotechnology, Zhejiang University, Hangzhou 310058, China
Abstract：Pepper mild mottle virus (PMMoV) seriously damages the pepper (Capsicum annuum) industry worldwide. The establishment of sensitive and practical virus detection technology is the key to the prevention and control of this viral disease. In this study, using purified PMMoV virions as the immunogen, six monoclo‐ nal antibodies (i. e. 15A3, 15G7, 17D1, 11E1, 6A10 and 3B2) that specifically and sensitively recognize PM‐MoV were finally obtained through the immunization of BALB/c mice (Mus musculus), cell fusion, screeningof hybridoma cells, antibody selection, continuous cell subcloning and preparation of monoclonal antibody as‐ cites. Two serological techniques, dot enzyme-linked immunosorbent assay (Dot-ELISA) and colloidal gold immunochromatographic strip (CGICS), were established to detect PMMoV using the produced monoclonal antibodies as the detection antibodies. Both Dot-ELISA and CGICS could specifically and broadly detect dif‐ ferent isolates of PMMoV, and showed negative reactions when detecting Tobacco mosaic virus (TMV), Cucum- ber green mottle mosaic virus (CGMMV), Tomato brown rugose fruit virus (ToBRFV), Tomato mosaic virus (ToMV), Cucumber mosaic virus (CMV) and healthy pepper plants. Moreover, the sensitivities of Dot-ELISA and CGICS for detecting diseased leaf homogenates were separately up to 1∶25 600 and 1∶204 800 times dilu‐ tion (W/V, g/mL), which was 2 times and 16 times more sensitive than RT-PCR, respectively. Additionally, the monitoring results of field samples by the two serological detection techniques were 100% consistent with those from RT-PCR monitoring, indicating that the two established serological techniques can be used to accu‐ rately, broadly, sensitively and quickly detect PMMoV. This study provides technical support for the detection and diagnosis of PMMoV, and its scientific prevention and control.
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