链延伸反应提高压电基因传感器的灵敏度的实验研究

Sensitivity improvement of piezoelectric gene sensor by strand extension reaction

目的 利用链延伸反应的原理延长引物链以提高压电基因传感器表面的质量负载 ,从而提高传感器的灵敏度。方法 基因传感器阵列表面分别固定上金黄色葡萄球菌mecA及femA两种探针片段 ,待杂交上相应的靶序列片段后 ,加入Klenow酶 ,室温下 (2 0℃左右 )进行链延伸反应 8h。结果 50nmol L单链mecA在杂交反应及链延伸反应的过程中 ,频率的下降值分别为 (99.2± 8.5)Hz、(1 92 .9± 1 3 .3 )Hz。而femA则分别下降了 (1 42 .4± 1 1 .2 )Hz、(2 2 2 .6± 1 6.6)Hz。mecA、femA在链延伸反应前的检测灵敏度为 0 .5nmol L ,但经链延伸反应后检测灵敏度提高到了 0 .0 5nmol L。结论 链延伸反应有效地提高了压电基因传感器的灵敏度 ,可用于微量DNA的检测。

Objective To improve the sensitivity of piezoelectric gene sensor by increasing its surface load on the basis of the principle of strand extension reaction. Methods mecA and femA probes of Staphylococcus aureus were immobilized seperately on the surface of gene sensor array. Klenow large fragment was added to extend the strand for 8 h in room temperature after the probes were hybridized with corresponding target DNA. Results The frequencies of the gene sensor were reduced by (99.2±8.5) Hz and (192.9±13.3) Hz during the hybridization and extension reaction respectively, when 50 nmol/L single strand mecA was added on the surface of the gene sensor array, while (142.4±11.2) Hz and (222.6 ±16.6) Hz for the femA. Before the strand extension reaction, the sensitivity of the gene sensor array was 0.5 nmol/L, and was improved to 0.05 nmol/L after the reaction. Conclusion The sensitivity of piezoelectric gene sensor array is significantly improved by strand extension reaction. This method can be used in the detection of trace DNA and the analysis of homogeneity of nucleic acid.