土壤固碳微生物的绝对定量检测实验设计  

Experimental Design of Absolute Quantitative Detection for Soil CO2-fixing Bacteria

作　　者：付小花[1,2] 陈皓[1,2] 张华[1,2] 周磊[1,2] 唐贤春[1] FU Xiaohua;CHEN Hao;ZHANG Hua;ZHOU Lei;TANG Xianchun(School of Environmental Science and Engineering,Tongji University,Shanghai 200092,China;State Key Laboratory of Pollution Control and Resource Reuse,Tongji University,Shanghai 200092,China)

机构地区：[1]同济大学环境科学与工程学院,上海200092 [2]同济大学污染控制与资源化研究国家重点实验室,上海200092

出　　处：《实验室研究与探索》2024年第4期18-22,共5页Research and Exploration In Laboratory

基　　金：同济大学第十七期实验教学改革项目(0400104179)。

摘　　要：为了定量检测土壤中的固碳微生物,设计以功能基因cbbL为靶标的固碳微生物微滴数字聚合酶链式反应(ddPCR)检测方法。选择合适的引物探针,从退火温度、探针浓度以及引物浓度进行反应条件的优化,分析ddPCR检测方法的线性范围、敏感性、重复性和特异性。结果显示,当退火温度为55.8℃、探针与引物浓度分别为350、750 nmol/L时,建立的cbbL-ddPCR扩增反应效率最高,阴阳性微滴分布界限最明显,平均拷贝数较高;检测的线性范围为2.3×10^(0)~2.3×10^(5)copies/μL-DNA,曲线方程y=0.1077x-95.562,相关系数R^(2)为0.9997,检出限为0.5 copy/μL-DNA,21个重复的变异系数仅为3.92%,与其他4种非固碳微生物DNA未发生交叉反应。所建立的cbbL-ddPCR方法可用于土壤微生物固碳潜能测定。To quantitatively detect CO_(2)-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 2.3×100~2.3×10^(5)copies/μL-DNA,with the linear equation is y=0.1077x-95.562,the correlation coefficient R^(2) is 0.9997.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.

关 键 词：微滴数字聚合酶链式反应 固碳微生物 cbbL基因 反应条件优化 

分 类 号：X832[环境科学与工程—环境工程] G642[文化科学—高等教育学]