Scientia Agricultura Sinica ›› 2013, Vol. 46 ›› Issue (3): 534-544.doi: 10.3864/j.issn.0578-1752.2013.03.010

• PLANT PROTECTION • Previous Articles     Next Articles

Loop-Mediated Isothermal Amplification Assay for Rapid Diagnosis of Meloidogyne enterolobii Directly from Infected Plants

 HE  Xu-Feng, PENG  Huan, DING  Zhong, HE  Wen-Ting, HUANG  Wen-Kun, PENG  De-Liang   

  1. 1.State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193
    2.College of Biosafety Science and Technology, Hunan Agricultural University, Changsha 410128
  • Received:2012-08-13 Online:2013-02-01 Published:2012-11-07

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Abstract: 【Objective】 The objective of this study is to establish a rapid diagnostic method for Meloidogyne enterolobii from the infected plants based on the loop-mediated isothermal amplification (LAMP), and to provide technical supports for monitoring and prevention of M. enterolobii. 【Method】 The LAMP specific primers were designed according to the distinction of ITS (internal transcribed spacer) sequences between M. enterolobii and other Meloidogyne spp.. The reaction conditions of LAMP, including the concentrations of Mg2+, dNTPs, betain and reaction times were optimized, and the specificity and sensitivity of the LAMP were testified. A rapid method for M. enterolobii from infected plants by LAMP was established on this basis. 【Result】 The optimum conditions for LAMP reaction could be carried out under the concentrations of 5.0 mmol•L-1 Mg2+ and 2.4 mmol•L-1 dNTPs, without betain and 45 minutes of reaction. The method developed in this paper was able to specifically detect M. enterolobii from 11 species of plant nematodes and to sensitivity detect DNA as low as 1/200000 nematode DNA. The sensitivity of LAMP reaction was 100 times higher than the conventional PCR method, which was capable of detecting M. enterolobii directly from the infected roots with 100% accuracy.【Conclusion】The rapid detection of M. enterolobii by LAMP assay targeted the ITS region of ribosomal DNA was built. The method is high specificity, sensitivity, and economic value, which makes it possible for quick and accurately detecting M. enterolobii from the infected plant tissue, and has higher actual application value.

Key words: Meloidogyne enterolobii , LAMP , specificity , sensitivity , diagonosis

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