Objective To investigate the effects of different culture media and pretreatment methods on the results of matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF MS) for the identification of Vibrio parahaemolyticus,and then provide the optimal conditions.Methods Forty strains of Vibrio parahaemolyticus were collected,and subcultured with the blood agar plate,thiosulfate citrate bile salts sucrose(TCBS) agar plate and double wash agar plate,respectively.The pretreatment methods before mass spectrometry included the smear method,extension method and extraction method.The effects of different culture media and pretreatment methods on the results of MALDI-TOF MS for the identification of Vibrio parahaemolyticus were analyzed by the logistics regression model and Chi-square test of SAS software.Results The detection rate of Vibrio parahaemolyticus was the highest in the combination of blood agar plate with the extraction method(genus level:100％;species level:67.5％),followed by the combination of double wash agar plate with the extraction method(genus level:70％;species level:37.5％).For the genus identification level of Vibrio parahaemolyticus,when the pretreatment method was the same,different culture media produced significantly different detection rates(P ＜ 0.01).However,when the culture medium was the same,there was no significant difference in detection rates for different pretreatment methods (P ＞ 0.05).The odd ratios (OR) of the smear method and extention method relative to the extraction method were 0.66 and 0.95,respectively,and there was no significant difference in detection rates for them(P ＞ 0.05),while the ORs of the TCBS agar plate and double wash agar plate relative to blood agar plate were 0.06 and 0.10,respectively,and there was significant difference in detection rates for them(P ＜0.05).The logistic regression equation was Y=2.95-0.41a-0.05b-2.83c-2.83d(a:smear method;b:extension method;c:TCBS agar plate;d:double wash agar plate).For the species identification level of Vibrio parahaemolyticus,when the extension method or the extraction method was used,different culture media produced significantly different detection rates(P ＜ 0.05).When the blood agar plate was used,different pretreatment methods also produced significantly different detection rates(P ＜ 0.01).The ORs of the smear method and extention method relative to the extraction method were 0.32 and 0.55,respectively,and there was significant difference in detection rates for them (P ＜ 0.05),while the ORs of the TCBS agar plate and double wash agar plate relative to blood agar plate were 0.18 and 0.49,respectively,and there was significant difference in detection rates for them(P ＜0.05).The logistic regression equation was Y =0.20-1.15a-0.61b-1.72c-1.72d(a:smear method;b:extension method;c:TCBS agar plate;d:double wash agar plate).Conclusion For improving the detection rate of Vibrio parahaemolyticus by MALDI-TOF MS,the selection of appropriate culture medium is more important than that of the pretreatment method.It is recommended that the extraction method may be as a conventional pretreatment method,and that the optimal medium is the blood agar plate,followed by the double wash agar plate and TCBS agar plate.%目的 评估不同培养基及前处理方法在基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)对副溶血弧菌鉴定中的影响,并寻求最优化条件组合.方法 收集副溶血弧菌40株,分别用血琼脂平板、TCBS琼脂平板和双洗琼脂平板进行培养;用直涂法、扩展法(直涂后裂解)和萃取(裂解、萃取后涂靶)法进行质谱前处理.用SAS软件建立不同培养基及前处理方法对该菌质谱检出率影响的logistic回归分析模型,并进行卡方检验统计分析.结果 “血琼脂平板+萃取法”条件组合检出率最高(100％鉴定到属,67.5％鉴定到种);在不使用血琼脂平板的前提下,“双洗琼脂平板+萃取法”条件组合检出率最高(70％鉴定到属,37.5％鉴定到种).在“属”鉴定水平,前处理方法相同时,不同培养基的检出率差异有统计学意义(P＜0.01);而培养基相同时,不同前处理方法的检出率差异无统计学意义(P＞0.05);直涂法和扩展法相对萃取法的相对危险度(OR)分别为0.66和0.95,检出率差异无统计学意义(P＞0.05);TCBS琼脂平板和双洗琼脂平板相对血琼脂平板的OR分别为0.06和0.10,对检出率差异有统计学意义(P＜0.05),logistic回归方程Y=2.95-0.41a-0.05b-2.83c-2.35d(a:直涂法;b:扩展法;c:TCBS琼脂平板;d:双洗琼脂平板).在“种”鉴定水平,采用扩展法或萃取法,不同培养基的检出率差异有统计学意义(P＜0.05);采用血琼脂平板,不同前处理方法的检出率差异有统计学意义(P＜0.01);直涂法和扩展法相对萃取法的OR分别为0.32和0.55,检出率差异有统计学意义(P＜0.05);TCBS琼脂平板和双洗琼脂平板相对血琼脂平板的OR分别为0.18和0.49,检出率差异有统计学意义(P＜0.05),logistic回归方程y=0.20-1.15a-0.61b-1.72c-0.72d(a:直涂法;b:扩展法;c:TCBS琼脂平板;d:双洗琼脂平板).结论 欲提升副溶血弧菌MALDI-TOF MS鉴定效果,合适培养基的选择优于改善蛋白质提取的前处理操作.推荐萃取法作为常规前处理方法,培养基的选择建议依次为血琼脂平板、双洗琼脂平板和TCBS琼脂平板.
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