Quantification of Paratrichodorus allius in DNA extracted from soil using TaqMan probe and SYBR green real-time PCR assays

Authors: HUANG, DANQIONG - North Dakota State University; YAN, GUIPING - North Dakota State University; Skantar, Andrea

Submitted to: Nematology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/10/2017
Publication Date: 8/1/2017
Citation: Huang, D., Yan, G., Skantar, A.M. 2017. Quantification of Paratrichodorus allius in DNA extracted from soil using TaqMan probe and SYBR green real-time PCR assays. Nematology. 19(8):987-1001. https://doi.org/10.1163/15685411-00003101
DOI: https://doi.org/10.1163/15685411-00003101

Interpretive Summary: Plant-parasitic nematodes are microscopic worms that attack plant roots and cause an estimated ten billion dollars of crop loss each year in the United States and 100 billion dollars globally. The stubby root nematode Paratrichodorus allius may transmit the tobacco rattle virus that is responsible for corky ringspot disease, resulting in significant economic losses to the potato industry. In the present study, scientists from North Dakota State University and an ARS scientist from Beltsville, MD developed highly sensitive molecular diagnostic assays to detect P. allius using DNA extracted directly from soil samples. The results are significant because they demonstrate that nematode numbers detected by two molecular methods were highly correlated with the number of input nematodes manually extracted and counted from artificially inoculated soil. Molecular detection methods were also highly correlated with microscopic counting methods applied to field soil samples. University, state, federal, or commercial diagnostic laboratories, other scientists, action agencies, and extension agencies engaged in nematode research and control will use this research.

Technical Abstract: The ectoparasitic stubby root nematode Paratrichodorus allius transmits Tobacco rattle virus, which causes corky ringspot disease resulting in significant economic losses in the potato industry. This study developed a diagnostic method for direct quantification of P. allius from soil DNA using a TaqMan probe and SYBR Green real-time PCR assays. Specificity of primers/probe designed from the internal transcribed spacer of ribosomal DNA of P. allius was demonstrated by in silico analysis and experimental PCR tests with no cross reactions using non-target nematode species and nematode communities. The SYBR Green method was more sensitive than the TaqMan probe method during detection using serial diluted DNA templates. Standard curves were generated from serial dilutions of DNA extracted from autoclaved soil with artificially inoculated P. allius individuals and were validated by high correlations between the numbers of target nematodes quantified by the assays and added to the soil (R2 > 0.85). Moreover, the numbers of P. allius determined by the real-time PCR assays and estimated by the microscopic method in 17 field soil samples presented correlations (R2) of 0.80 and 0.86, respectively. Although the quantification using TaqMan probe overestimated the target nematodes compared to SYBR Green in eight out of 10 field soil samples that were positive for P. allius, these two methods correlated well (R2 = 0.92). This is the first report of P. allius quantification from soil DNA extracts using real-time PCR, providing a fast and sensitive diagnostic method obviating time-consuming manual nematode extraction from soil and microscopic identification and quantification.