Rapid diagnosis of Ditylenchus destructor by loop-mediated isothermal amplification assay based on 28S rRNA sequences

Abstract

Ditylenchus destructor is an important plant pathogenic nematode in agricultural production and is listed as a quarantine pest by many countries and regions. Traditional identification depending on morphological characteristics requires taxonomic expertise and is difficult to apply for rapid identification of plant nematode species. Currently, with the development of molecular biology, many novel tools have been explored for the rapid identification of plant nematodes. In this study, loop-mediated isothermal amplification (LAMP) was used to conduct rapid detection of D. destructor from various origins. First, a set of new LAMP specific primers (DdBIP, DdFIP, DdF3, DdB3 and DdLF) were designed based on 28S rRNA sequences of D. destructor, and LAMP reaction conditions were optimized to use concentrations of 6.0 mmol L−1 Mg2+, 1.0 mmol L−1 dNTPs, and 0.2 U μL−1 Bst 2.0 DNA polymerase, performed at 65 °C for 50 min. Second, the specificity, stability and sensitivity of this method were confirmed using DNA from a single egg and juvenile, male and female nematodes of D. destructor and from D. destructor mixed with various nematode species and soil and tissue samples. The amplification products were detected by checking the ladder-like bands using electrophoresis and visualizing mixture colour changes using SYBR Green I dye. The results showed that the LAMP method could not only detect and identify a single egg and juvenile, female and male nematodes and different populations from different hosts and geographical origins of D. destructor but also directly detect D. destructor from mixed samples of various nematode species, plant tissue and soil. The sensitivity of LAMP amplification was a 1000-fold dilution of the template DNA from an individual female nematode. This report is the first to use LAMP to directly detect D. destructor from samples mixed with various nematode species and with plant tissue and soil.