Real-time quantitative PCR assay on bacterial DNA: In a model soil system and environmental samples.
Real-time quantitative PCR (RTQ-PCR) was used to quantify the bacterial target DNA extracted by three commonly used DNA extraction protocols (bead mill homogenization, grinding in presence of liquid nitrogen and hot detergent SDS based enzymatic lysis). For the purpose of our study, pure culture of Bacillus cereus (model organism), sterilized soil seeded with a known amount of B. cereus (model soil system) and samples from woodland and grassland (environmental samples) were chosen to extract DNA by three different protocols. The extracted DNA was then quantified by RTQ-PCR using 16S rDNA specific universal bacterial primers. The standard curve used for the quantification by RTQ-PCR was linear and revealed a strong linear relationship (r(2)=0.9968) with a higher amplification efficiency, e5=1.02. High resolution gel electrophoresis was also carried out to observe the effect of these extraction methods on diversity analysis. For the model soil system, the liquid nitrogen method showed the highest target DNA copy number (1.3 x 10(9) copies/microl). However, for both the environmental samples, the bead beating method was found to be suitable on the basis of the high target DNA copy numbers (5.38 x 10(9) and 4.01 x 10(8) copies/ml for woodland and grassland respectively), high yield (6.4 microg/g and 1.76 microg/g of soil for woodland and grassland respectively) and different band patterns on high resolution gel electrophoresis suggesting an overall high extraction efficiency. This difference in the extraction efficiency between the model soil system and environmental samples may be attributed to different affinity of seeded and native DNA to soil particles.
Kabir
Shaila
S
Graduate School of Environment and Information Sciences, Yokohama National University, Yokohama 240-8501, Japan. kabir_shaila@hotmail.com
Rajendran
Narasimmalu
N
Amemiya
Takashi
T
Itoh
Kiminori
K
D001409:Bacillus cereus / Q000235:genetics* / Q000302:isolation & purification; D004269:DNA, Bacterial / Q000032:analysis*; D004587:Electrophoresis, Agar Gel; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics; D015203:Reproducibility of Results; D012988:Soil Microbiology
D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S