Experimental Design of Absolute Quantitative Detection for Soil CO2-fixing Bacteria
To quantitatively detect CO2-fixing bacteria in soil,an assay of droplet digital PCR(ddPCR)was developed,which was based on the functional gene cbbL.We selected suitable probe and a pair of primers for ddPCR,then optimized the reaction conditions in terms of annealing temperature,probe concentration and primer concentration,and evaluated the specificity,sensitivity,and repeatability of this method.When the annealing temperature is 55.8 ℃,and the concentrations of primers and probes are 750 and 350 nmol/L,the established cbbL-ddPCR amplification reaction efficiency is the highest,the distribution boundary of the positive and negative droplets is the most obvious,and the average copy number is higher.The cbbL-ddPCR shows a good linear relationship between 2.3 × 100～2.3 × 105 copies/μL-DNA,with the linear equation is y=0.107 7x-95.562,the correlation coefficient R2 is 0.999 7.The sensitivity is 0.5 copy/μL-DNA.The coefficient of variation of 21 replicates is 3.92％.There is no cross reaction with DNA of other four non-carbon-fixing bacteria.Therefore,the cbbL-ddPCR assay established in this paper can be used to determine the carbon sequestration potential of cbbL-containing soil bacteria in a rapid,accurate,sensitive and specific way,thus supports the assessment of soil carbon sequestration capacity.
droplet digital polymerase chain reaction(ddPCR)CO2-fixing bacteriacbbL geneoptimization of reaction conditions
NETL
NSTL
万方数据
维普
