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作 者 ：廖超, 张昭寰, 姚鑫, 谢晶, 张炜佳, 潘迎捷, 赵勇,

期 刊 ：生物技术通报 2016年 32卷 5期 页码：212-219

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

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

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。

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.

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