副溶血性弧菌细胞猝灭及代谢物组提取方法的优化

Comparison of Quenching and Extraction Methods for Metabolome Analysis of Vibrio Parahaemolyticus

以气相色谱-质谱联用(GC-MS)为分析方法,比较了液氮和75%甲醇(-80℃)两种溶液对副溶血性弧菌细胞的猝灭效果,以及氯仿、75%冰甲醇、水、甲醇-氯仿-水(10∶3∶1,V/V)、甲醇-氯仿-水(3∶1∶1,V/V)、甲醇-氯仿(1∶1,V/V)6种提取剂对副溶血性弧菌代谢物组的提取效果。结果表明,用75%甲醇(-80℃)猝灭副溶血性弧菌时,出现了代谢物泄漏现象,而液氮猝灭则不存在这个现象;检索发现,采用75%冰甲醇提取获得了47种代谢物,峰面积标准偏差为8.02%,其它5种提取剂获得代谢物种类少于40种,且重现性差。比较色谱峰数量、面积和重现性后发现,液氮猝灭、75%冰甲醇提取适于副溶血性弧菌代谢物组提取。

A reliable cell quenching and metabolite extraction method was crucial for microbial metabolism analysis. For metabolism analysis of Vibrio parahaemolyticus, liquid nitrogen and 75% methanol （-80℃） were compared for their efficiency of cell quenching, and six different extraction solvents based on （i） chloroform, （ii） 75% cold methanol, （iii） water, （iv） methanol：chloroform： water=10：3：l （V/V）, （v） methanol：chloroform：water--3：1 ： 1 （V/V） and （vi） methanol ： chloroform=l：l （V/V） were compared for their efficiency of metabolites extracting. The metabolites were detected by （/C-MS. The results showed that leakage of metabolite occurred when quenched with 75% methanol （-80℃）; however this phenomenon did not occur with liquid nitrogen. 47 metabolites were found when extracted with 75 G cold methanol, and the relative standard deviation of peak area was 8.0 %. In contrast, 40 or less metabolites were detected with other extraction methods, and the reproducibilities of those methods were unsatisfactory. For global analysis, quenching with liquid nitrogen and extraction with 75 % cold methanol was very suitable for metabolomics studies of Vibrio parahaemolytieus.