• 关键词：腐殖酸  稀释模板  活性污泥  实时荧光定量PCR  抑制效率

• 基金项目：国家自然科学基金（No.51778390）；江苏省自然科学基金（No.BK20171219）；江苏省自然科学研究重大项目（No.14KJA610001）；国家重点研发计划项目（No.2016YFC0401108）

• 倪敏
• 1. 苏州科技大学江苏省环境科学与工程重点实验室, 苏州 215009;2. 苏州科技大学环境科学与工程学院, 苏州 215009

• 刘婷婷
• 1. 苏州科技大学江苏省环境科学与工程重点实验室, 苏州 215009;2. 苏州科技大学环境科学与工程学院, 苏州 215009

• 顾海东
• 1. 苏州科技大学江苏省环境科学与工程重点实验室, 苏州 215009;2. 苏州科技大学环境科学与工程学院, 苏州 215009

• 李祥
• 1. 苏州科技大学江苏省环境科学与工程重点实验室, 苏州 215009;2. 苏州科技大学环境科学与工程学院, 苏州 215009

• 潘杨
• 1. 苏州科技大学江苏省环境科学与工程重点实验室, 苏州 215009;2. 苏州科技大学环境科学与工程学院, 苏州 215009

• 摘要：活性污泥基因组中的腐殖酸会影响实时荧光定量PCR检测的准确性.本研究采用抽提基因组前腐殖酸去除试剂盒、抽提基因组后腐殖酸去除试剂盒、抽提基因组前后共同去除试剂盒3种不同的方法，去除活性污泥基因组中的腐殖酸，经过PCR和实时荧光定量PCR方法的验证找出腐殖酸去除的最适方法；通过DNA酶Ⅰ消化基因组DNA保留杂质腐殖酸，向其中掺入已知浓度的总细菌质粒，5个浓度10倍梯度稀释10 ng·μL^-1的定量模板，探究腐殖酸和梯度稀释模板对活性污泥总细菌实时荧光定量PCR的影响.结果显示，在不同处理组中，通过反复洗脱的方式在基因组抽提前去除活性污泥中的腐殖酸，DNA的得率（162.46 ng·μL^-1）最多，A_260/230值为1.34，A_260/280值为1.80，腐殖酸的残留最少；去除腐殖酸后外加内标质粒组，梯度稀释10 ng·μL^-1模板100倍及以上，腐殖酸的抑制效应显著降低后保持不变（p<0.05），100倍以内变化不显著（p>0.05）；抽提前去除腐殖酸组进行实时荧光定量PCR的抑制效率为6.16%，显著低于对照组的72.68%（p<0.05）.研究表明，抽提前去除活性污泥腐殖酸，抽提后将基因组模板稀释100倍可显著降低活性污泥实时荧光定量PCR的抑制效率，提高定量结果的准确性.

• Abstract：Humic acid in activated sludge affects the accuracy of real-time fluorescence quantitative PCR. This research adopts three different kits such as humic acid removal before, after, and both before and after genomic DNA extraction, to find out the optimal step for humic acid removal in activated sludge by the validation of PCR and real-time fluorescence quantitative PCR. Through eliminating genomic DNA retained impurity humic acid by DNaseⅠ, mixing with known concentrations of total bacterial plasmids to form 10 ng·μL^-1 quantitative template, diluting the template into 5 different concentrations with 10 times gradient dilution to explore the effects of humic acid and gradient diluent on real-time fluorescent quantitative PCR of total bacteria in activated sludge. The results show that, in different groups, the DNA yield is the highest of 162.46 ng·μL^-1, the value of A_260/230 is 1.34, the value of A_260/280 is 1.80, and the humic acid residue is the lowest when the humic acid is removed before activated sludge genomic DNA extraction by means of repeat elution. After removing humic acid, and then mixing with internal standard plasmid group to obtain 10 ng·μL^-1 template, the inhibition effect of humic acid decreased significantly and remains unchanged (p<0.05) by means of 100 times or more gradient dilution on template, while there is no significant change within 100 times gradient dilution (p>0.05). The inhibition efficiency of real-time fluorescence quantitative PCR is 6.16%, which is significantly lower than that of the control group of 72.68% (p<0.05) when humic acid is removed before DNA extraction. humic acid removal in activated sludge before DNA extraction, and 100 times dilution of genomic DNA template, could greatly decrease the inhibition to real-time fluorescent quantitative PCR of activated sludgeand improve the accuracy of quantitative results.

• Key words：humic acid  dilution template  activated sludge  real-time fluorescence quantificative PCR  inhibition efficiency

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