摘要
目的 探究6种化学试剂型和3种酶型菌体裂解剂在聚合酶链式反应(PCR)扩增体系中的最大允许终浓度和不同浓度IGEPAL CA-630裂解不同种属菌株的差异。方法配制6种化学试剂型(IGEPAL CA-630、CTAB、SDS、EDTA、异硫氰酸胍和NaOH)和3种酶型(溶菌酶、溶葡球菌酶和蛋白酶K)及多个不同稀释度的裂解液,添加于荧光定量PCR体系中,分析比较Ct值差异。用IGEPAL CA-630溶液(浓度为1%和0.1%,TE为溶剂)、TE和去离子水分别裂解嗜热链球菌、金黄色葡萄球菌和大肠埃希菌,经荧光定量PCR扩增,比较Ct值的差异。结果在PCR体系中,IGEPALCA-630、CTAB、SDS、EDTA异硫氰酸胍和NaOH的最大允许终浓度分别为0.800‰、0.256‰、0.012 8‰、0.8 mmol/L、0.012 8 mol/L和0.64 mmol/L,溶菌酶、溶葡球菌酶和蛋白酶K的最大允许浓度分别为0.512、1.28和0.512μg/mL。IGEPAL CA-630裂解嗜热链球菌和金黄色葡萄球菌实验中,TE溶液、0.1%和1%IGEPAL CA-630的Ct值依次减小,TE溶液和去离子水的Ct值没有明显差异;去离子水、TE溶液、0.1%和1%IGEPAL CA-630裂解大肠埃希菌的Ct值均没有明显差异。结论 通过探究,明确了上述9种常见裂解剂不影响PCR扩增的最大浓度,且适当提高IGEPAL CA-630浓度可以增强提取革兰氏阳性菌DNA作用。
Objective To explore the maximum allowable concentration of six chemical reagents and three enzyme lysates in a polymerase chain reaction(PCR)amplification system and the differences of IGEPAL CA-630 cleaving different species.Methods Prepare different kinds of working solutions,including six chemical reagents(IGEPAL CA-630,CTAB,SDS,EDTA,guanidine isothiocyanate,and NaOH)and three enzymes(lysozyme,lysostaphin,and protease K)with different concentrations.Add the working solutions to the fluorescent quantitative PCR system and then analyze the differences in Ct values.Streptococcus thermophilus,Staphylococcus aureus,and Escherichia coli were lysed with IGEPAL CA-630 solutions(1%and 0.1%with TE buffer),TE buffer and deionized water,respectively,and the differences in PCR results were analyzed.Results In the PCR system,the maximum allowable final concentrations of IGEPAL CA-630,CTAB,SDS,EDTA,guanidine isothiocyanate,and NaOH were 0.800‰,0.256‰,0.0128‰,0.8 mmol/L,0.0128 mol/L,and 0.64 mmol/L respectively.The maximum allowable concentrations of lysozyme,lysostaphin,and protease K were 0.512,1.28,and 0.512μg/mL.In the lysing of S.thermophilus and S.aureus with IGEPAL CA-630,the Ct values of TE solution,0.1%and 1%IGEPAL CA-630 decreased successively,with no significant difference between the TE solution and deionized water.There was no significant difference in Ct values of E.coli treated with deionized water,TE solution,0.1%and 1%IGEPAL CA-630.Conclusion This study established the maximum allowable concentration of the above nine components in the PCR amplification solution.Additionally,it was found that increasing the concentration of IGEPAL CA-630 could enhance DNA extraction from gram-positive bacteria.
作者
刘娜
王亚萍
王学硕
崔生辉
LIU Na;WANG Yaping;WANG Xueshuo;CUI Shenghui(National Institute for Food and Drug Control,Beijng 100050,China)
出处
《中国食品卫生杂志》
CSCD
北大核心
2023年第6期843-848,共6页
Chinese Journal of Food Hygiene
基金
中国食品药品检定研究院中青年基金(2020C3)。
关键词
聚合酶链式反应
裂解剂
化学试剂型
酶型
种属
Polymerase chain reaction
lysing reagent
chemical trial dosage form
enzymatic
species
