title: Development and Evaluation of Loop-Mediated Isothermal Amplification Assay for Rapid Detection ofTylenchulus semipenetransUsing DNA Extracted from Soil.
abstract: Tylenchulus semipenetransis 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 ofT. semipenetransin soil is important for the disease forecasting and management. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed to detectT. semipenetransusing DNA extracted from soil. A set of five primers was designed from the internal transcribed spacer region (ITS1) of rDNA, and was highly specific toT. semipenetrans. The LAMP reaction was performed at 63°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 10-2J2/0.5 g of soil, which was 10 times more sensitive than conventional PCR (10-1J2/0.5 g of soil). Examination of 24 field soil samples revealed that the LAMP assay was applicable to a range of soils infested naturally withT. 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 ofT. semipenetransin field soil, and contributes to the effective management of citrus slow decline disease.
authors.affiliation: College of Plant Protection, Hunan Agricultural University, Changsha 410128, P. R. China; Institute of Plant Protection, Hunan Academy of Agricultural Sciences, Changsha 410125, P. R. China.
authors.forename: Zhi-Qiang
authors.initials: ZQ
authors.lastname: Song
authors.affiliation: Institute of Plant Protection, Hunan Academy of Agricultural Sciences, Changsha 410125, P. R. China.
authors.forename: Ju-E
authors.initials: JE
authors.lastname: Cheng
authors.affiliation: Institute of Plant Protection, Hunan Academy of Agricultural Sciences, Changsha 410125, P. R. China.
authors.forename: Fei-Xue
authors.initials: FX
authors.lastname: Cheng
authors.affiliation: College of Plant Protection, Hunan Agricultural University, Changsha 410128, P. R. China; Institute of Plant Protection, Hunan Academy of Agricultural Sciences, Changsha 410125, P. R. China.
authors.forename: De-Yong
authors.initials: DY
authors.lastname: Zhang
authors.affiliation: College of Plant Protection, Hunan Agricultural University, Changsha 410128, P. R. China; Institute of Plant Protection, Hunan Academy of Agricultural Sciences, Changsha 410125, P. R. China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Liu
keywords: DNA extraction; Tylenchulus semipenetrans; citrus slow decline disease; loop-mediated isothermal amplification; nematode detection