A fast and sensitive method for the simultaneous identification of three important nematode species of the genus Ditylenchus.
BACKGROUND: Nematodes of the genus Ditylenchus are parasites of a wide range of hosts, including higher plants. The most destructive of these species are D. dipsaci and D. destructor, two frequently quarantined pests. No rapid molecular method is available for unambiguous detection and distinguishing of these species from each other or from D. gigas, a pest of Vicia faba, either by multiplex PCR or real-time PCR. RESULTS: By aligning all D. dipsaci, D. destructor and D. gigas rDNA sequences, the authors found a constant-sequence region that could be used as a universal 5′ primer and constant regions in the ITS1 regions of the rDNAs that could be used as species-specific 3′ primers for PCR detection of these nematodes. A standardised protocol was developed for both singleplex- and triplex-mode PCR that yields a single product of distinct length for each of the species. The PCR protocol has also been adapted for real-time PCR. CONCLUSION: The present diagnostic PCR protocol is the only method that can identify all three species with the use of a triplex and/or a singleplex PCR assay. Importantly, the 3′ primer for D. destructor ITS1 rDNA was designed so that it would hybridise all haplotypes.