Comparison of different methods for extracting nucleic acid from pathogenic microorganisms

CHEN Tao; CHAI Hai-yun; LIU Yan; HUANG Yue-mei; HUANG Wei; ZHANG Zhao; LIN Dong-zi

doi:10.3969/j.issn.1005-4057.2023.06.004

Abstract：Objective To compare and analyze the DNA extraction effects of different extraction reagents for pathogenic microorganisms with simulated sample of alveolar lavage fluid.Methods Candida albicans,staphylococcus aureus,staphylococcus epidermidis,and escherichia coli were selected as representative bacteria.Clinical alveolar samples were prepared by adding a specific number of cells to a mixed bacterial solution of different concentrations based on the cell content of alveolar lavage fluid.Three commercially available nucleic acid extraction kits(Qiagen,Vazyme and Magen)and self-developed and optimized extraction methods(Longsee)were selected for nucleic acid extraction.The extraction performance was compared in terms of the quality of nucleic acid extraction and PCR amplification.Results There was no statistical difference in A260/A280 between Longsee reagent and other three brands(P＞0.05).When the microbial concentration was≥105 CFU/mL,Qiagen had the highest concentration of nucleic acid extracted,followed by Longsee(P＜0.01).Among the four different kinds of samples,the Ct values of 4 kinds of microorganisms treated by 3 brands of reagents were significantly different from those by Longsee reagent(P＜0.01 or 0.05).When the microbial concentration was ≥104 CFU/mL,the Ct corresponding to Longsee reagent was lower than that of the other three reagents(P＜0.01 or 0.05).Conclusion Longsee self-developed nucleic acid extraction method is a cost-effective nucleic acid extraction method for pathogenic microorganisms.