Development of a Novel Primer-TaqMan Probe Set for Diagnosis and Quantification of Meloidogyne enterolobii in Soil Using qPCR and Droplet Digital PCR Assays

Abstract

Early detection of pathogens before the planting season is valuable to forecast disease occurrence. Therefore, rapid and reliable diagnostic approaches are urgently needed, especially for one of the most aggressive root knot nematodes, Meloidogyne enterolobii. In this study, we developed a novel primer-TaqMan probe set aimed at M. enterolobii. The primer-probe set was successfully applied in the identification and quantification of M. enterolobii via qPCR technology. It was also suitable for improved PCR technology, known as ddPCR analyses, and this work presents the first application of this technology for plant parasitic nematodes. Compared with qPCR, ddPCR exhibited better performance with regard to analytical sensitivity, which can provide a more accurate detection of M. enterolobii concealed in field soil. In addition, we generated standard curves to calculate the number of eggs in soil using the qPCR and ddPCR platforms. Hopefully, the results herein will be helpful for forecasting disease severity of M. enterolobii infection and adopting effective management strategies.

Keywords: Meloidogyne enterolobii; TaqMan probe; ddPCR; identification; qPCR; quantification.

Figure 1

Designed primers and Taqman probe-targeted ITS2 sequence of M. enterolobii in this study. The dots and dashes indicate conserved nucleotides and gaps in the sequences, respectively.

Figure 2

Optimization of annealing temperatures for ddPCR assay to identify M. enterolobii. The total DNA extracted from 0.25 g of soil containing 200 eggs (A) and 5 ng of genomic DNA extracted from eggs (B) was detected under gradient temperature from 50 °C to 70 °C. The droplets achieved at the optimal annealing temperature are indicated in the red box. The blue and gray dots indicate positive and negative droplets. The pink lines represented the horizontal threshold value of 1000.

Figure 3

Specificity tests for primers/Taqman probe set analyzed by qPCR (A) and ddPCR (B) to identify M. enterolobii. ME, M. enterolobii. MI, M. incognita; MJ, M. javanica; MG, M. graminicola; MA, M. arenaria; HG, H. glycines; CE, C. elegans; RR, R. reniformis; NTC, no template control. dRn, normalized fluorescence. The red dashed line indicates the cutoff point in qPCR. The pink line represents the horizontal threshold value of 5000 in ddPCR.

Figure 4

Regression lines for DNA samples of M. enterolobii detected by qPCR and ddPCR. (A) The standard curve built from Ct values obtained from qPCR against the logarithm of gDNA quantities. (B) The standard curve built from number of DNA copies·μL⁻¹ obtained from ddPCR against the gDNA quantities. (C) The standard curves built from Ct values obtained from qPCR against the logarithm of number of eggs inoculated into 0.25 g of soil. The curves were generated using DNA solutions of the handpicked number of eggs added to the soil (black) or twofold dilution series of DNA solutions extracted from soil containing 200 eggs (blue). (D) The standard curve built from the number of DNA copies·μL⁻¹ obtained from ddPCR against the number of eggs inoculated into 0.25 g of soil.

Figure 5

Egg density of M. enterolobii in field soil determined using shallow dish, qPCR, and ddPCR methods, respectively. The value obtained using the shallow dish method was calculated as a function of the number of juveniles hatched from soil, which was assumed as the number of eggs in soil.