B - Fast ELISA: application of a new ELISA technique for the rapid detection of Pepino mosaic virus

摘要

In recent decades, many plant diseases have spread worldwide, favored by the steady increase of global trade and transport of plant products and germplasm. Pepino mosaic virus (PepMV) is an emerging virus with a high economic impact on tomato production in Europe, originating geographically from South America. In recent years, a number of new detection methods have been developed, but most are of limited suitability for routine testing. Enzyme-linked immunosorbent assay (ELISA) is still the most widely used method for large-scale testing of plant viruses, despite being limited by the many separate operating steps and long incubation times required. In order to overcome these shortcomings, the B-Fast ELISA technique has been developed, combining therobustness of an ELISA with the speed of PCR. This test allows reliable results to be obtained within only 2 h. Following the simultaneous incubation of a mixture of capture and detection antibody (conjugate) together with the test sample for 1 h, the ELISA wells are washed and substrate is added without any further operating steps. This new ELISA is fully compatible with standard ELISA buffers and equipment. B-Fast ELISA was comprehensively validated and compared with a standard double antibody sandwich (DAS)-ELISA for the detection of PepMV, demonstrating its suitability for sensitive and specific detection of all relevant PepMV strains in tomato. In addition, 15 of 27 randomly selected tomato samples offered in German supermarkets tested positive for the presence of PepMV by both B-Fast and standard DAS-ELISA. Infections were confirmed by RT-PCR, and subsequent sequencing of amplification products demonstrated that Chile 2 was the predominant strain. The successful development of the B-Fast assay shows that it provides a very rapid and convenient screening alternative to standard ELISA.