膜微阵列技术的建立及检测细菌条件的优化

Establishment of membrane microarray technique and optimizing the conditions for detecting bacteria

目的 探讨膜微阵列技术检测细菌的可行性。方法 在细菌的 1 6SrRNA基因内设计了通用引物和特异性探针 ,将探针点在膜上制备微阵列 ,再与地高辛标记的DNA杂交 ,比较了随机引物标记法和PCR掺入标记法的灵敏度 ,探讨了硝酸纤维素膜和尼龙膜作基片的可行性 ,并比较了不同的固定条件和杂交条件下的杂交结果 ,以标准菌株DNA为样本 ,建立起微阵列技术。结果 随机引物标记法和PCR掺入标记法的灵敏度均为 0 .1pg ;尼龙膜较之硝酸纤维素膜可更好的结合寡核苷酸 ,经紫外线交联 3min ,与杂交液 3(5×SSC ,5g/LSDS ,1 %封闭剂 )在 5 5℃杂交结果最好。标准菌株除结核杆菌、变形杆菌外 ,均能与其对应的特异探针杂交。结论 利用膜微阵列技术检测细菌有快速、敏感、特异。

Objective To explore the feasibility of membrane microarray technique for detection of bacteria.Methods A set of universal primers and two specific probes for each bacterium were designed according to conserved regions and variable regions of 16S rRNA gene respectively.Each probe was spotted on the membrane to make up microarray,and then hybridized with digoxin labeling DNA.The sensitivity of labeling methods of random primer and PCR incorporation was compared.The feasibility of nitri cacid cellulose membrane and nylon membrane as a substrate was researched,and hybridized result of different conditions for fixing and hybridizing were analysed.The technique was established by hybridization of DNA from standard strain after amplification.Results The sensitivity of both the methods of random primer and PCR incorporation was 0.1pg.The nylon membrane immobilized oligonucleotide better than nitric acid cellulose membrane did.The best result was obtained by ultraviolet cross linkage for 3 minutes and then hybridizition at 55℃ with hybridizing fluid 3.It was shown that all standard strains except tuberculosis and proteus bacillus were hybridized with corresponding probes.Conclusion Membrane microarray is fast, sensitive, specific technique without radioactive contamination.