长、短链探针以链延伸反应提高基因传感器检测MRSA灵敏度的实验研究

Sensitivity Improvement of Detecting MRSA Using Gene Sensor by Strand Extension Reaction with Long and Short Chain Probes

目的 利用链延伸反应的原理延长引物链以提高石英谐振式基因传感器表面的质量负载 ,从而提高传感器检测 MRSA的灵敏度。方法 以不对称 PCR的反应产物做为长链探针 ,人工合成的寡核苷酸片段为短链探针 ,在基因传感器阵列表面分别固定上金黄色葡萄球菌 mec A及 fem A的长链及短链探针共 4种探针片段 ,与相应的靶序列杂交后 ,加入 Klenow酶 ,37℃进行链延伸反应 1h。结果 长、短链探针均有效地固定在了传感器表面。mec A、fem A短链探针在链延伸反应前的检测灵敏度为 0 .5 nm ol/L,但经链延伸反应后检测灵敏度提高到了0 .0 5 nmol/L;而长链探针却无法与相应的基因组靶序列发生杂交。结论 短链探针链延伸反应有效地提高了石英谐振式基因传感器的灵敏度 ,但该法不适用于长链探针。

OBJECTIVE To improve the sensitivity of detecting MRSA using quartz oscillation gene sensor(QOGS) by increasing its surface load on the basis of the principle of strand extension reaction.METHODS Product of asymmetric PCR was used as long chain probe and man made oligonucleotide used as short chain probe respectively. Four kinds of mecA and femA probes of Staphylococcus aureus were immobilized on the surface of QOGS. Klenow large fragment was added to extend the strand for one hour at 37℃ after the probes were hybridized with corresponding target DNA. RESULTS Long and short probes were all immobilized on the surface of QOGS effectively. As to short chain probe, the sensitivity of the QOGS was 0.5 nmol/L before the strand extension reaction, and it improved to 0.05 nmol/L after strand extension reaction. However, long chain probes could not hybridize with genomic target sequence.CONCLUSIONS Sensitivity of QOGS was significantly improved by strand extension reaction with short chain probe, while it was not suitable for long chain probes.