Development of a loop-mediated isothermal amplification assay for detection of Phytophthora sojae.
Phytophthora sojae is a devastating pathogen that causes soybean Phytophthora root rot. This study reports the development of a loop-mediated isothermal amplification (LAMP) assay targeting the A3aPro element for visual detection of P. sojae. The A3aPro-LAMP assay efficiently amplified the target element in < 80 min at 64 °C and was evaluated for specificity and sensitivity. The specificity was evaluated against P. sojae, Phytophthora spp., Pythium spp., and true fungi isolates. Magnesium pyrophosphate resulting from the LAMP of P. sojae could be detected by real-time measurement of turbidity. Phytophthora sojae DNA products were visualized as a ladder-like banding pattern on 2% gel electrophoresis. A positive colour (sky blue) was only observed in the presence of P. sojae with the addition of hydroxynaphthol blue prior to amplification, whereas none of other isolates showed a colour change. The detection limit of the A3aPro-specific LAMP assay for P. sojae was 10 pg μL(-1) of genomic DNA per reaction. The assay also detected P. sojae from diseased soybean tissues and residues. These results suggest that the A3aPro-LAMP assay reported here can be used for the visual detection of P. sojae in plants and production fields.
Department of Plant Pathology, Nanjing Agricultural University, China.
Ting-Ting
TT
Dai
Chen-Chen
CC
Lu
Jing
J
Lu
SuoMeng
S
Dong
WenWu
W
Ye
YuanChao
Y
Wang
XiaoBo
X
Zheng
D017931:DNA Primers / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010838:Phytophthora / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D010935:Plant Diseases / Q000382:microbiology*; D013025:Glycine max / Q000382:microbiology*
D017931:DNA Primers