重要肠道病原菌多重PCR-基因芯片检测方法研究  

Development of a Multiplex PCR-Microarray Method for Detection of Important Enteropathogen

作　　者：尤元海[1] 曾浔[1] 过玮[1] 尹焱[1] 张茂俊[1] 张建中[1] 

机构地区：[1]中国疾病预防控制中心传染病预防控制所传染病诊断室,北京102206

出　　处：《中国生物工程杂志》2009年第12期79-84,共6页China Biotechnology

基　　金：科技部社会公益项目(2004DIB2J065)资助项目

摘　　要：目的:建立并初步评价一种针对重要肠道病原菌的多重PCR-基因芯片检测方法。方法:对筛选出的特异引物进行多重PCR优化,将引物分别按种属内混合和种属间混合的方案排查引物间的竞争性抑制现象,再将不同菌属的模板混合,用相对应的混合引物扩增,探寻高效特异的引物组合。分别掺入和不掺入荧光素,验证其对混合PCR反应的影响,并与芯片杂交,探寻多重PCR扩增效率对芯片杂交的影响。分析不同数量引物组合产生的杂交结果,筛选出无交叉反应的最优引物组合。结果:种属内引物混合均得到特异性扩增结果。种属间混合霍乱弧菌和空肠弯曲菌得到部分预期条带,随着混合引物数量的增加,交叉抑制现象也增多。杂交信号强度随多重PCR扩增效率的增加而增强。反应中掺入荧光素的扩增条带产量低于无荧光素的产物。可将35对混合引物拆成3个体系分别标记样品,以避免假阴性结果。结论:PCR反应中掺入荧光素降低扩增效率和杂交效率,但并不影响对杂交结果的判读和数据分析。基因芯片杂交信号强度取决于多重PCR的扩增效率。肠道病原菌多重PCR-基因芯片检测方法具有较高的特异性,混合PCR可以分别按照种属内和种属间的引物组合方案用于多病原的筛检。该基因芯片检测可以采用3个引物体系完成样品标记。Objective： To establish a multiplex PCR-microarray method for detecting important enteropahogens. Methods： Uniplex and multiplex PCR were performed to obtain the best primer sets for identifying the target bacteria at species and multi-species level. Fluorescent dyes were mixed into PCR reaction to determine whether it can affect the efficiency of amplification. To improve the efficiency of microarray, a 35 pairs primer- labeling system was optimized based on the hybridization results to find the best combination to avoid false negative results. Results： Specific PCR products were all obtained using species-specific primer sets. More preferential amplification may happen when more primer pairs were added to the reaction. The hybridization results showed a positive association between the efficiency of multiplex-PCR and signal intensity. Conventional PCR yielded more products than fluorescent dyes labeled PCR. Thirty-five primers were divided into three different combinations to label target respectively, hybridization results showed a high specificity. Conclusion： Mixing fluorescent dyes into PCR may reduce the efficiency of amplification and hybridization, but may have no effect on the analysis of hybridization results. The hybridization efficiency of microarray depends on the amplification efficiency of multiplex PCR. For microarray target labeling, three primer sets could be used to avoid negative hybridization led by preferential amplification of muhiplex-PCR. It indicates that the multiplex PCR- microarray method is an attractive diagnosis tool for the high-throughput identification of enteropathogenic organisms especially for multiple causative agents and epidemiological investigations.

关 键 词：肠道病原菌 多重PCR 基因芯片 

分 类 号：Q819[生物学—生物工程]