核酸提取产物中乙醇对实时荧光定量PCR的影响  

Effect of ethanol in nucleic acid extraction on quantitative real-time PCR

作　　者：龚向莲 黄玉麟 林伟春[1] 杨连威 胡祐 邵珊 张东旭[1] GONG Xianglian;HUANG Yulin;LIN Weichun;YANG Lianwei;HU You;SHAO Shan;ZHANG Dongxu(State Key Laboratory of Vaccines for Infectious Diseases,Xiamen University,Xiamen 361104,Fujian Province,China)

机构地区：[1]厦门大学国家传染病诊断试剂与疫苗工程技术研究中心,福建厦门361104 [2]厦门万泰凯瑞生物技术有限公司,福建厦门361022

出　　处：《中国生物制品学杂志》2025年第1期102-108,114,共8页Chinese Journal of Biologicals

基　　金：国家自然科学基金青年项目(62003284)。

摘　　要：目的研究核酸提取产物中乙醇对实时荧光定量PCR(quantitative real-time PCR,qPCR)的影响,为更高灵敏度核酸检测提供乙醇浓度界限。方法建立重铬酸钾氧化法检测两种商品化磁珠法核酸提取试剂盒提取产物的乙醇残留,检测其是否对qPCR产生影响,再将不同浓度乙醇溶液混合核酸模拟提取产物溶解冷冻干燥处理的qPCR试剂(以下简称冻干试剂)并进行扩增,观察其灵敏度变化,并对Ct值、荧光增益强度和扩增效率进行统计学分析。结果两种试剂盒的提取产物中残留3%~9%乙醇,对扩增均造成影响,有可能会导致灵敏度下降。对于人巨细胞病毒(human cytomegalovirus,HCMV),乙醇浓度达到4.38%时,荧光增益强度差异有统计学意义(P<0.001),扩增效率出现明显差异;对于呼吸道合胞病毒(respiratory syncytial virus,RSV),乙醇浓度达到2.63%时,荧光增益强度差异有统计学意义(P=0.004);乙醇浓度达到3.50%时,Ct值差异有统计学意义(P=0.004),扩增效率出现明显差异。结论乙醇会影响qPCR灵敏度、Ct值、荧光增益和扩增效率。为进行更高灵敏度的核酸检测,建议乙醇在提取产物中的残留不超过3.50%。Objective To study the impact of ethanol in nucleic acid extraction products on quantitative real-time PCR(qPCR),and to provide ethanol concentration limits for the extraction quality of more sensitive nucleic acid detection methods.Methods A potassium dichromate oxidation method was developed to detect the ethanol residues in the products extracted by two commercialized magnetic bead nucleic acid extraction kits,testing whether the ethanol residues impact qPCR.Then different concentrations of ethanol solutions were mixed with qPCR reagents that had been dissolved in simulated extraction products and freeze-dried(hereinafter referred to as freeze-dried reagents)for amplification.The sensitivity changes were observed,and the Ct values,fluorescence gain intensity and amplification efficiency were statistically analyzed.Results The extraction products of the two types of reagent kits contained 3%-9%of residual ethanol,both of which caused an impact on the amplification,potentially leading to a decrease in sensitivity.For human cytomegalovirus(HCMV),when ethanol concentration reached 4.38%,the fluorescence gain intensity showed significant difference(P<0.001),and the amplification efficiency showed significant difference.For respiratory syncytial virus(RSV),there was a significant difference in fluorescence gain intensity when ethanol concentration reached 2.63%(P=0.004).When the concentration of ethanol reached 3.50%,the difference of Ct values was significant(P=0.004),and the amplification efficiency was significantly different.Conclusion Ethanol can affect the sensitivity,Ct values,fluorescence gain,and the amplification efficiency of qPCR.In order to detect nucleic acid with higher sensitivity,it is suggested that the residue of ethanol in the extracted products should not exceed 3.50%.

关 键 词：乙醇 实时荧光定量PCR 冷冻干燥 PCR试剂 核酸提取 残留 

分 类 号：R446.5[医药卫生—诊断学]