Abstract
BackgroundTomato zonate spot virus (TZSV), a new species of genus Tospovirus, caused significant losses in yield and problems in quality of many important vegetables and ornamentals in Southwest China and posed a serious threat to important economic crops for the local farmers. A convenient and reliable method was urgently needed for rapid detection and surveillance of TZSV.MethodsThe nucleocapsid protein (N) of TZSV was expressed in Escherichia coli and purified, and was used as the antigen to immunize BALB/c mice. Three monoclonal antibodies (mAbs) 3A2, 5D2 and 5F7 against TZSV were obtained through the hybridoma technique. The mAb 3A2 was conjugated with colloid gold as detecting reagent; mAb 5D2 was coated on a porous nitrocellulose membrane as the detection line and protein A was coated as the control line respectively. The colloid gold immunochromatographic (GICA) strip was assembled.ResultsThe analysis of Dot-ELISA and Western blot showed that the obtained three independent lines of mAbs 3A2, 5D2 and 5F7 specifically recognized TZSV N. Based on the assembly of GICA strip, the detection of TZSV was achieved by loading the infected sap onto the test strip for visual inspection. The analysis could be completed within 5–10 min. No cross-reaction occurred between TZSV and other tested viruses. The visual detection limit of the test strip for TZSV was 800 fold dilutions of TZSV-infected leaf samples.ConclusionThe mAbs were specific and the colloidal GICA strip developed in this study was convenient, fast and reliable for the detection of TZSV. The method could be applied for the rapid diagnosis and surveillance of TZSV in the field.
Highlights
Tomato zonate spot virus (TZSV), a new species of genus Tospovirus, caused significant losses in yield and problems in quality of many important vegetables and ornamentals in Southwest China and posed a serious threat to important economic crops for the local farmers
Based on monoclonal antibodies (mAbs), we developed a gold immunochromatographic (GICA) strip for the rapid detection of TZSV
One mL of the culture was transferred into 1 L of LB medium containing 50 μg/mL kanamycin and 17 μg/mL chloromycetin at 37 °C to reach the optical density at 600 nm (OD600) of 0.6, 1 mL of 0.1 mM isopropyl beta-D-thiogalactoside (IPTG) was added into the culture, which was incubated at 200 r/min at 20 °C for about 10 h
Summary
Tomato zonate spot virus (TZSV), a new species of genus Tospovirus, caused significant losses in yield and problems in quality of many important vegetables and ornamentals in Southwest China and posed a serious threat to important economic crops for the local farmers. Tospoviruses are the most devastating plant viruses and caused significant losses in many important economic crops throughout the world [1,2,3]. The diseases caused by tospoviruses spreaded rapidly in Yunnan province, Southwest China [4,5,6,7,8,9,10,11,12]. Tomato zonate spot virus (TZSV) was one of main local tospoviruses in Yunnan province and was characterized by Dong et al 2008 [7]. The infection of TZSV is often associated with severe diseases
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