摘要
目的探讨并明确量子点核酸传感器检测微生物核酸样本时探针长度对杂交效率和检测结果的影响。方法首先合成金黄色葡萄球菌16S rDNA探针,然后在其5′端添加不同长度的重复碱基序列,分别合成5条长度不同的以胸腺嘧啶结尾的探针;采用巯基法固定金黄色葡萄球菌16S rDNA探针于石英晶体微阵列上,并用石英晶体微天平(QCM)检测样品频率漂移量以判断不同核酸长度时的杂交效率,并用荧光显微镜观察形成不同双链复合物的荧光强度和荧光效率,以此判断不同碱基序列长度探针对荧光效率和杂交效率的影响,并运用SPSS软件对数据进行统计分析。结果对于普通的短链核酸杂交反应,随着探针长度增加,杂交效率呈现依次递增的趋势,当离子强度为40mmol/L时,杂交效率和荧光强度增加最快,其后再增加探针长度,杂交效率增加速度变缓。结论探针长度是影响杂交效率的主要原因之一,通过增加探针长度可以提高杂交效率和荧光强度,但同时也会增加二聚体产生概率,故应根据实际需要设计长度适合的探针。
OBJECTIVE To discuss and identify the influence of probe length on the hybridization effect and fluorescence intensity during the detection of microorganism biosensor. METHODS Hydrosulfide group was adopted to immobilize the 16S rDNA probes of Staphylococcus aureus on the quartz crystal biosensor array. Firstly we synthesized series of probes with different length. Then we detected the frequency shifts induced by nucleic acid hybridization. Then we detected the total fluorescence intensity with fluorescence microscopy. All of the data were analyzed by the SPSS software. RESULTS The hybridization efficiency increased gradually with the increase in probe length. The hybridization efficiency firstly climbed quickly then slow down when the probe length reached 40 bp. Similarly, the peak fluorescence intensities produced were the same when the probe length was 40 bp and 60 bp. CONCLUSIONS Probe length is the important factor influencing the results of quantum dot microorganism biosensor. And the long probe can increase hybridization effect and fluorescence intensity. However, multiple parameters should be considered when determining the optimal length in practice.
出处
《中华医院感染学杂志》
CAS
CSCD
北大核心
2010年第11期1531-1533,共3页
Chinese Journal of Nosocomiology
基金
国家"863"项目(2007AA02Z416)
国家自然科学基金(30900348)
全军科技成果推广扩试项目(08JKS01)
军队科技攻关(08G089)
重庆市科技攻关项目(CSTC
2008AC0001)
重庆市自然科学基金(CSTC
2007BB5067)
第三军医大学军事医学专项(2008XG24)
福建省医疗器械和医药技术重点实验室开放基金(09001)
关键词
探针长度
微生物传感器
核酸杂交
量子点
Probe length
Microorganism biosensor
Nucleic acid hybridization
Quantum dots
