Effect of ethanol in nucleic acid extraction on quantitative real-time PCR
10.13200/j.cnki.cjb.004405
- VernacularTitle:核酸提取产物中乙醇对实时荧光定量PCR的影响
- Author:
GONG Xianglian
- Publication Type:Journal Article
- Keywords:
Ethanol;
Quantitative real-time PCR(qPCR);
Freeze-dried;
PCR reagent;
Nucleic acid extraction;
Residue
- From:
Chinese Journal of Biologicals
2025;38(01):102-108+114
- CountryChina
- Language:Chinese
-
Abstract:
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%.
