单增李斯特菌检测基因芯片的研制与评价  

Development and evaluation of a DNA microarray for Listeria monocytogenes detection

作　　者：何冬梅[1] 王洪敏[2] 柯昌文[1] 邓小玲[1] 杨杏芬[1] 赖蔚苓[1] 柯碧霞[1] 李柏生[1] 谭海玲[1] 

机构地区：[1]广东省疾病预防控制中心病原微生物检验所,广州510300 [2]解放军第458医院全军肝病中心

出　　处：《中华微生物学和免疫学杂志》2011年第10期916-921,共6页Chinese Journal of Microbiology and Immunology

基　　金：广东省科技计划项目（2005833701013）

摘　　要：目的研制快速、特异、灵敏的检测单增李斯特菌的基因芯片。方法选择gyrB、ISR、16SrRNA、23SrRNA、hlyA、lap和pffA作为单增李斯特菌的检测靶基因，研制一种Oligo探针基因芯片，对18个不同种属来源的已知参考生物样品进行检测和鉴定，并且采用对比试验、重复性试验、灵敏度试验和特异性试验对该j芷：片进行验证评估。结果通过对比发现IDT合成的70merOligoj告片探针在芯片打印与芯片检测两个方面较优。比较10、40和80txmol／L3个Oligo探针点样浓度，结果显示10txmol／L的探针点样浓度已能获得很好的芯片检测结果。单增李斯特菌检测芯片具有较好的重复性；样品检测绝对量下限为0．9ngDNA左右。结论Oligo基因芯片可以快速准确地检测单增李斯特菌。Objective To develop a rapid and sensitive DNA microarray for Listeria monocytogenes detection. Methods A DNA microarray was developed using gyrB, 1SR, 16S rRNA, 23S rRNA, hlyA, iap and pgCA as the target genes and tested against 18 different species of known reference for repeatability, sensitivity, and specificity to verify the effectiveness of the chip. Results After testing of samples by the LM array, results show that the 70 mer Oligos synthesized by IDT are superior to the Oligos synthesized by Sagon with respect to both probe spotting or samples detection. The comparison of 3 spotting probe concentrations of 10 μmol/L, 40 μmol/L and 80 μmol/L demonstrated that the 10 μmol/L probes result in good detection signals equivalent to the 40 μmol/L and 80 μmol/L probes. The repeatability and sensitivity evaluated by sample testing on the LM array revealed that the chips developed in this study have good repeatability and the lower limit of sample detection is 0.9 ng DNA. The LM array can distinguish clearly and definitively between Listeria and non-Listeria bacteria in the sample. Conclusion The microarray is able to rapidly detect and identify Listeria monocytogenes.

关 键 词：单增李斯特菌 基因芯片 寡核苷酸探针 

分 类 号：R4[医药卫生—临床医学]