Abstract
ITS1, the 5.8S rRNA gene and ITS2 of the rDNA region were sequenced from 20
different Pratylenchus species. Additionally, the same region was sequenced
from seven populations of P. penetrans. After purifying, cloning and
sequencing the PCR products, all sequences were aligned in order to find
unique sites suitable for the design of species-specific primers for P.
penetrans. Since ITS regions showed variability between and even within
populations of P. penetrans, only three small DNA sequences were suitable
for the construction of three potentially useful species-specific primers.
New species-specific primers were paired with existing universal ITS primers
and tested in all possible primer combinations. The best performing primer
set, supplemented with a universal 28S rDNA primer set that served as an
internal control, was tested in duplex PCR. The ideal annealing temperature,
Mg2+ concentration and primer ratios were then determined for the most
promising primer set. The optimised duplex PCR was subsequently tested on a
wide range of different Pratylenchus spp. and 25 P. penetrans populations
originating from all over the world. To test the sensitivity, the duplex PCR
was conducted on DNA extracted from a single P. penetrans nematode mixed
with varying amounts of nematodes belonging to another Pratylenchus species.
Results showed that a reliable and sensitive P. penetrans species-specific
duplex PCR was constructed.
Use of species-specific primers and PCR to measure the distributions of planktonic ciliates in coastal waters