幽门螺杆菌基因组DNA芯片的研制

目的研制幽门螺杆菌(H.pylori)基因组DNA芯片。方法以H.pylori26695和J99作为模板,利用多聚酶链反应(PCR)扩增得到所需要的开放阅读框(open reading frame,ORF)片段。使用Genemachine点样仪进行点样,采用Cy3-dCTP或Cy5-dCTP以及Klenow片段标记H.pylori基因组DNA,并完成芯片杂交和数据读取。数据判断标准是标化后Cy3/Cy5比值(ratio)<0.5则认为不存在这一基因(记作0),若ratio值≥0.5则认为存在这一基因(记作1)。使用芯片重复性、信噪比以及假阳性率和假阴性率评估芯片质量。结果研制的H.pylori基因组DNA芯片共包括1 882个基因片段,相对应于1 636个ORF,其中H.pylori26695为1 549个,H.pyloriJ99为87个。信噪比(S/N)<2的基因点数约为10%。芯片的假阳性率分别为3.4%(H.pylori26695)和7.21%(H.pyloriJ99),假阴性率分别为0.27%(H.pylori26695)和0.24%(H.pyloriJ99)。芯片内点间重复率为98%,芯片间重复率为97%。结论成功制备了H.pylori基因组DNA芯片,所制备的芯片具有较高点一致性、信噪比和统计学重复性,为在全基因组范围内进行H.pylori的基础和应用研究提供了一工具。

Development of the genomic DNA microassay for Helicobacter pylori

The genomic DNA microassay for Helicobacter pylori was developed,in which the open reading frame(ORF) was generated by PCR using specific primers and the genomic DNAs of H.pylori 26695 and J99 were used as templates.DNA fragments on the array were printed by Genemachine printer.The Cy3-dCTP/Cy5-dTCTP and J99 fragments were used to label H.pylori strains and to complete hybridization and data readings.Results were judged on the basis of normalized Cy3/Cy5 ratio,in which a ratio less than 0.5 was considered to be absent for this gene(labeled as 0) whereas a ratio greater than or equal to 0.5 indicated its presence(labeled as 1).In addition,the quality of this microassay was evaluated by means of the reproducibility,signal/noise ratio and false positive or false negative rate.The final microarray included 1882 gene fragments,corresponding to 1636 ORFs of both sequenced strains,of which 1549 strains belonged to H.pylori 26695 and 87 strains belonged to H.pylori J99.It was found that the percentage of the signal/noise ratio <2 was approximately 10%.The false positive and false negative rate for H.pylori 26695 were 3.4% and 0.27% and those for H.pylori J99 were 7.21% and 0.24% respectively.Meanwhile,the repetitive rate between different dots within the same microassay and between gene chips were 98% and 97% respectively.We had successfully prepared the genomic DNA microassay for H.pylori with higher consistent of spots and signal/noise ratio as well as lower false positive and false negative rates,thus providing a useful tool for the studies on H.pylori.