The Plant Pathology Journal

The Korean Society of Plant Pathology (한국식물병리학회)
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Development and Evaluation of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Tylenchulus semipenetrans Using DNA Extracted from Soil

  • Song, Zhi-Qiang (College of Plant Protection, Hunan Agricultural University) ;
  • Cheng, Ju-E (Institute of Plant Protection, Hunan Academy of Agricultural Sciences) ;
  • Cheng, Fei-Xue (Institute of Plant Protection, Hunan Academy of Agricultural Sciences) ;
  • Zhang, De-Yong (College of Plant Protection, Hunan Agricultural University) ;
  • Liu, Yong (College of Plant Protection, Hunan Agricultural University)
  • Received : 2016.10.20
  • Accepted : 2016.12.29
  • Published : 2017.04.01
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Abstract

Tylenchulus semipenetrans is an important and widespread plant-parasitic nematode of citrus worldwide and can cause citrus slow decline disease leading to significant reduction in tree growth and yield. Rapid and accurate detection of T. semipenetrans in soil is important for the disease forecasting and management. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed to detect T. semipenetrans using DNA extracted from soil. A set of five primers was designed from the internal transcribed spacer region (ITS1) of rDNA, and was highly specific to T. semipenetrans. The LAMP reaction was performed at $63^{\circ}C$ for 60 min. The LAMP product was visualized directly in one reaction tube by adding SYBR Green I. The detection limit of the LAMP assay was $10^{-2}J2/0.5g$ of soil, which was 10 times more sensitive than conventional PCR ($10^{-1}J2/0.5g$ of soil). Examination of 24 field soil samples revealed that the LAMP assay was applicable to a range of soils infested naturally with T. semipenetrans, and the total assay time was less than 2.5 h. These results indicated that the developed LAMP assay is a simple, rapid, sensitive, specific and accurate technique for detection of T. semipenetrans in field soil, and contributes to the effective management of citrus slow decline disease.

Keywords

References

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