Development of real-time PCR technique for the estimation of population density of Pythium intermedium in forest soils.
Pythium intermedium is known to play an important role in the carbon cycling of cool-temperate forest soils. In this study, a fast, precise and effective real-time PCR technique for estimating the population densities of P. intermedium from soils was developed using species-specific primers. Specificity was confirmed both with conventional PCR and real-time PCR. The detection limit (sensitivity) was determined and amplification standard curves were generated using SYBR Green II fluorescent dye. A rapid and accurate assay for quantification of P. intermedium in Takayama forest soils of Japan was developed using a combination of a new DNA extraction method and PCR primers were developed for real-time PCR. And the distribution of P. intermedium in forest soil was investigated with both soil plating method and the developed real-time PCR technique. This new technique will be a useful tool and can be applied to practical use for studying the role of Pythium species in forest and agricultural ecosystems.
Li
Mingzhu
M
The United Graduate School of Agriculture Science, Gifu University, Gifu 501-1193, Japan. mingzhu_li@green.gifu-u.ac.jp
Senda
Masako
M
Komatsu
Tsutomu
T
Suga
Haruhisa
H
Kageyama
Koji
K
D015169:Colony Count, Microbial; D017931:DNA Primers / Q000235:genetics; D007564:Japan; D009172:Mycology / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011775:Pythium / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D012988:Soil Microbiology; D014197:Trees
D017931:DNA Primers