Development of a serological procedure for sensitive and rapid detection of Thladiantha dubia mosaic virus

Thladiantha dubia mosaic virus (ThDMV) from the wild Manchurian tuber gourd ( Thladiantha dubia Bunge, Cucurbitaceae), is a novel tentative specie of the genus Potyvirus within the family Potyviridae . It may pose a potential risk to both cultivated crops and plants in the natural ecosystem. In the present study, we developed an ELISA procedure for detection of ThDMV. Coat protein (CP) of ThDMV was successfully expressed in Escherichia coli , and used to immunize rabbit for antiserum after being purified by using Ni-NTA affinity column. Results of indirect ELISA showed that the optimum dilutions of the coating antigen and antiserum were 1:160 (1 μg per well), and 1:2000 respectively. Western blot indicated that antibodies in the antiserum could specifically recognize and bind CP of ThDMV. An ELISA method was developed with the antiserum to detect ThDMV in plant extracts. Samples collected from potato fields in Northeast China and Northwest China were analyzed by ELISA and RT-PCR and both gave consistent results. The ELISA assay developed here has potential to be used as a sensitive, reliable and cost-effective tool for ThDMV detection in epidemiological studies.