Development of Real-Time and Conventional PCR Assays for Identifying a Newly Named Species of Root-Lesion Nematode (Pratylenchus dakotaensis) on Soybean

Abstract

A rapid and accurate PCR-based method was developed in this study for detecting and identifying a new species of root-lesion nematode (<i>Pratylenchus</i>&nbsp;<i>dakotaensis</i>) recently discovered in a soybean field in North Dakota, USA. Species-specific primers, targeting the internal transcribed spacer region of ribosomal DNA, were designed to be used in both conventional and quantitative real-time PCR assays for identification of <i>P.</i><i>dakotaensis</i>. The specificity of the primers was evaluated in silico analysis and laboratory PCR experiments. Results showed that only <i>P.</i><i>dakotaensis</i> DNA was exclusively amplified in conventional and real-time PCR assays but none of the DNA from other control species were amplified. Detection sensitivity analysis revealed that the conventional PCR was able to detect an equivalent to 1/8 of the DNA of a single nematode whereas real-time PCR detected an equivalent to 1/32 of the DNA of a single nematode. According to the generated standard curve the amplification efficiency of the primers in real-time PCR was 94% with a R² value of 0.95 between quantification cycle number and log number of <i>P.</i><i>dakotaensis</i>. To validate the assays to distinguish <i>P.</i><i>dakotaensis</i> from other <i>Pratylenchus</i> spp. commonly detected in North Dakota soybean fields, 20 soil samples collected from seven counties were tested. The PCR assays amplified the DNA of <i>P.</i><i>dakotaensis</i> and discriminated it from other <i>Pratylenchus</i> spp. present in North Dakota soybean fields. This is the first report of a species-specific and rapid PCR detection method suitable for use in diagnostic and research laboratories for the detection of <i>P.</i><i>dakotaensis</i>.