不同病原微生物核酸提取方法的对比

Comparison of different methods for extracting nucleic acid from pathogenic microorganisms

目的通过肺泡灌洗液模拟样本,比较不同提取试剂对病原微生物DNA的提取效果。方法选择白念珠菌、金黄色葡萄球菌、表皮葡萄球菌、大肠埃希菌等代表菌,根据肺泡灌洗液细胞含量,添加特定数量的细胞及不同浓度的混合菌液,模拟临床肺泡样本。采用3种市售核酸提取试剂盒(凯杰、诺唯赞、美基)与自研优化后(南芯自研)的方法进行核酸提取,通过对核酸提取质量及PCR扩增结果对比提取效率。结果南芯自研试剂与3个品牌各样本的A260/A280比较差异均无统计学意义(P>0.05)。当菌液浓度≥105 CFU/mL时凯杰提取核酸浓度最高,南芯自研次之(P<0.01)。4个不同样本中,3家品牌试剂处理的4种微生物的Ct值与南芯自研提取试剂比较,差异有统计学意义(P<0.01或0.05)。当菌液浓度≥104 CFU/mL时,南芯自建提取试剂对应的Ct均低于另外3种提取试剂(P<0.01或0.05)。结论南芯自研核酸提取方法是一种性价比较高的病原微生物核酸提取法。

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 selfdeveloped 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 selfdeveloped nucleic acid extraction method is a cost-effective nucleic acid extraction method for pathogenic microorganisms.