椰毒假单胞菌酵米面亚种荧光标记扩增片段长度多态性分型方法的建立  

Establishment of the typing method by fluorescent amplification fragment length polymorphism for Pseudomonas cocovenenans subsp. farinofermentans

作　　者：王岗 郭云昌 杜春明 付萍 李薇薇 赵悦[2] 刘秀梅 杨大进 裴晓燕 

机构地区：[1]国家食品安全风险评估中心,北京100021 [2]中国疾病预防控制中心营养与食品安全所,北京100050

出　　处：《中国食品卫生杂志》2013年第2期125-131,共7页Chinese Journal of Food Hygiene

基　　金：国家自然科学基金青年基金(30901200)

摘　　要：目的建立椰毒假单胞菌酵米面亚种荧光标记扩增片段长度多态性(FAFLP)分型方法,并对4株模式菌株和19株分离菌株进行分型。方法选用4种低频限制性内切酶(ApaⅠ、EcoRⅠ、HindⅢ和PstⅠ)和2种高频限制性内切酶(MseⅠ和TaqⅠ)进行组合,对FAFLP预扩增和选择性扩增体系进行优化,用BioNumerics软件对分离株的指纹图谱进行聚类分析,并与VITEK 2 GN生化结果和16S rDNA序列分析进行比较。结果 ApaⅠ-G/TaqⅠ-G组合将19株椰毒假单胞菌酵米面亚种分为7个群,菌株间最低相似度为80.85%,最高相似度为98.77%。PstⅠ-T/TaqⅠ-G组合将19株椰毒假单胞菌酵米面亚种分为10个群,菌株间最低相似度为48.68%,最高相似度为99.67%。两种FAFLP组合均能将菌株进行完全区分,而VITEK 2 GN和16S rDNA序列分析无法将菌株进行完全区分。结论 ApaⅠ-G/TaqⅠ-G组合与PstⅠ-T/TaqⅠ-G组合FAFLP对椰毒假单胞菌酵米面亚种具有足够的分辨率,本研究建立的FAFLP分型方法可应用于椰毒假单胞菌酵米面亚种的分子分型和溯源。Objective To establish a genotyping method for Pseudomonas cocovenenans subsp, farinofermentans with fluorescent amplification fragment length polymorphism （FAFLP）. Methods Four low-frequency-cleavage restriction endonucleases （Apa [ , EcoR [ , Hind lII, Pst [ ） and two high-frequency-cleavage restriction endonuc]eases （MseI, Taq I ） were tested to optimize the combination for FAFLP. Pre-selective and selective PCR reactions were then optimized. The selective PCR products was separated on LI-COR 4300 DNA Analyzer and digital imagines were analyzed by BioNumerics software. In addition, the FAFLP method was compared with other two typing methods （VITEK 2 and 16s rDNA）. Results 19 isolates were clustered into 7 groups with 80. 85% as the minimum similarity and 98.77% the maximum among groups using the combination of Apa I -G/Taq I -G. The isolates were clustered into 10 groups with 48.68% as the minimum similarity and 99.67% the maximum among groups using the combination of Pst I -T/Taq I -G. The two combinations of FAFLP could deseriminate all the strains in the study, but not the other two methods （ VITEK 2 and 16s rDNA）. Conclusion The two combinations of FAFLP both had a good discrimination and could be applied for tracing Pseudomonas cocovenenans subsp, farinofermentans in the investigation of foodborne diseases.

关 键 词：椰毒假单胞菌酵米面亚种 荧光标记扩增片段长度多态性(FAFLP) 分子分型 食源性致病菌 食品安全 

分 类 号：R155.3[医药卫生—营养与食品卫生学]