GC夹对三种食源性致病菌的rpoB-PCR-DGGE图谱的影响

doi:10.13560/j.cnki.biotech.bull.1985.2016.05.028

生物技术通报 ›› 2016, Vol. 32 ›› Issue (5): 212-219.doi: 10.13560/j.cnki.biotech.bull.1985.2016.05.028

GC夹对三种食源性致病菌的rpoB-PCR-DGGE图谱的影响

廖超¹，², 张昭寰¹，², 姚鑫¹，², 谢晶¹, 张炜佳¹，²，³, 潘迎捷¹，²，³, 赵勇¹，²，³

1.上海海洋大学食品学院，上海 201306;
2.农业部水产品贮藏保鲜质量安全风险评估实验室，上海 201306;
3.上海水产品加工及贮藏工程技术研究中心，上海 201306

收稿日期:2015-07-22 出版日期:2016-05-25 发布日期:2016-05-27
作者简介:廖超，男，硕士研究生，研究方向：食品微生物风险评估；E-mail：liaochao2499@126.com
基金资助:
国家自然科学基金面上项目（31271870），上海市科委科技创新行动计划项目（14DZ1205100，14320502100），上海市科技兴农重点攻关项目（沪农科攻字2014第3-5号、2005第4-8号），上海市水产品加工及贮藏工程技术研究中心（11DZ2280300）

Effects of GC Clamps on RpoB-PCR-DGGE Fingerprint of Three Types of Food Borne Pathogens

LIAO Chao¹，²,ZHANG Zhao-huan¹，²,YAO Xin¹，²,XIE Jing¹,ZHANG Wei-jia¹，²，³,PAN Ying-jie¹，²，³,ZHAO Yong¹，²，³ ,

1. College of Food Science and Technology，Shanghai Ocean University，Shanghai 201306;
2. Laboratory of Quality ＆ Safety Risk Assessment for Aquatic Production on Storage and Preservation，Ministry of Agriculture，Shanghai 201306;
3. Shanghai Engineering Research Center of Aquatic Product Processing ＆ Preservation，Shanghai 201306

Received:2015-07-22 Published:2016-05-25 Online:2016-05-27

摘要/Abstract

摘要： 旨在探讨不同的GC夹以及GC夹连接引物的不同位置对3种食源性致病菌的DGGE图谱结果的影响，合成6对RNA聚合酶β亚基编码基因rpoB引物（rpoB 1-6），含3种不同GC夹（GC-1，GC-2，GC-3），且每种GC夹分别连接于正反引物5'端。应用rpoB-PCR-DGGE 对副溶血性弧菌（5株），单增李斯特菌（5株）和沙门氏菌（3株）进行分析，并与V3-PCR-DGGE和ERIC-PCR的图谱及聚类分析结果进行比较。结果显示，GC-3夹连接于反引物上的rpoB-PCR-DGGE分辨效果最好，分辨力指数达0.9，与ERIC-PCR相同，高于V3-PCR-DGGE。GC夹序列和连接引物位置均对rpoB-PCR-DGGE图谱结果产生影响。选择或设计无连续鸟嘌呤（G）排列的GC夹序列且将其连接于反引物5'端，均能有效提高rpoB-PCR-DGGE对食源性致病菌检测与分型的效果。

关键词: 副溶血性弧菌, 单增李斯特菌, 沙门氏菌, GC夹, rpoB-PCR-DGGE

Abstract: This work is to investigate the effect of different GC clamps and different positions of primers connected with GC clamps on the DGGE fingerprint of food borne pathogens. We synthesized 6 pairs of primers encoding RNA polymerase β subunit（rpoB 1-6）containing 3 different GC clamps（GC-1，GC-2 and GC-3）that linked to 5'endings of both forward and reverse primers，respectively. Then，rpoB-PCR-DGGE was used to analyze Vibrio parahaemolyticus（5 strains），Listeria monocytogenes（5 strains），and Salmonella spp.（3 strains）. The results of fingerprint analysis were compared with that of V3-PCR-DGGE and ERIC-PCR. GC-3 clamp linked with reverse primer presented the best discriminating effect，and the discriminating index of rpoB-PCR-DGGE reached 0.9 that was equal to ERIC-PCR and greater than V3-PCR-DGGE. In conclusion，both of the different GC clamp sequences and linked positions of primers with GC clamp affected the result of rpoB-PCR-DGGE fingerprint. Choosing or designing GC clamp without continuous guanine（G base）and linked with 5'ending of reverse primer will improve the detecting and typing effect of rpoB-PCR-DGGE for food borne pathogens.

Key words: Vibrio parahemolyticus, Listeria monocytogenes, Salmonella spp., GC clamps, rpoB-PCR-DGGE

廖超, 张昭寰, 姚鑫, 谢晶, 张炜佳, 潘迎捷, 赵勇,. GC夹对三种食源性致病菌的rpoB-PCR-DGGE图谱的影响[J]. 生物技术通报, 2016, 32(5): 212-219.

LIAO Chao,ZHANG Zhao-huan,YAO Xin,XIE Jing,ZHANG Wei-jia,PAN Ying-jie,ZHAO Yong, ,. Effects of GC Clamps on RpoB-PCR-DGGE Fingerprint of Three Types of Food Borne Pathogens[J]. Biotechnology Bulletin, 2016, 32(5): 212-219.

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GC夹对三种食源性致病菌的rpoB-PCR-DGGE图谱的影响

Effects of GC Clamps on RpoB-PCR-DGGE Fingerprint of Three Types of Food Borne Pathogens

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