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 (Pratylenchus dakotaensis) 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 P.dakotaensis. The specificity of the primers was evaluated in silico analysis and laboratory PCR experiments. Results showed that only P.dakotaensis 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 P.dakotaensis. To validate the assays to distinguish P.dakotaensis from other Pratylenchus spp. commonly detected in North Dakota soybean fields, 20 soil samples collected from seven counties were tested. The PCR assays amplified the DNA of P.dakotaensis and discriminated it from other Pratylenchus 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 P.dakotaensis.

Keywords: DNA; ITS rDNA; Pratylenchus dakotaensis; conventional PCR; detection; endomigratory; identification; in silico analysis; real-time PCR; root-lesion nematode.

Figure 1

Melting curve profiles of P. dakotaensis. Amplicons with a single peak at a melting temperature of 81.5 °C were observed for populations of P. dakotaensis.

Figure 2

Amplification sensitivity of the IC-ITS1F/IC-ITS1R primer set in conventional polymerase chain reaction (PCR) compared to real-time PCR. The quantification cycle (Cq) value is presented as the mean ± standard deviation of three technical replicates of one of the biological replicates used for standard curve development. DNA was extracted from 4 P. dakotaensis individuals and sequential two-fold serial dilutions (2, 1, 1/2, 1/4, 1/8, 1/16, 1/32, and 1/64) were conducted, but the last dilution level 1:256 (1/64) was not included in this figure.

Figure 3

Standard curve of the real-time PCR assay for the new species of Pratylenchus detected in a soybean field of North Dakota: quantification cycle number (Cq) plotted against the log number of individuals of P. dakotaensis by sequential two-fold dilutions (4, 2, 1, 1/2, 1/4, 1/8, 1/16, 1/32, and 1/64). Each red dot represents an independent reaction run in triplicate for three biological replicates at each dilution level. The efficiency (E) of amplification was calculated as E = 1^1/-m − 1, where m indicates the slope.