Department of Comparative Biomedical Sciences, University of Teramo, Italy.
Talanta. 2011 Sep 30;85(4):1927-32. doi: 10.1016/j.talanta.2011.07.019. Epub 2011 Jul 18.
A post-PCR nucleic acid work by comparing experimental data, from electrochemical genosensors, and bioinformatics data, derived from the simulation of the secondary structure folding and prediction of hybridisation reaction, was carried out in order to rationalize the selection of ssDNA probes for the detection of two Bonamia species, B. exitiosa and B. ostreae, parasites of Ostrea edulis. Six ssDNA probes (from 11 to 25 bases in length, 2 thiolated and 4 biotinylated) were selected within different regions of B. ostreae and B. exitiosa PCR amplicons (300 and 304 bases, respectively) with the aim to discriminate between these parasite species. ssDNA amplicons and probes were analyzed separately using the "Mfold Web Server" simulating the secondary structure folding behaviour. The hybridisation of amplicon-probe was predicted by means of "Dinamelt Web Server". The results were evaluated considering the number of hydrogen bonds broken and formed in the simulated folding and hybridisation process, variance in gaps for each sequence and number of available bases. In the experimental part, thermally denatured PCR products were captured at the sensor interface via sandwich hybridisation with surface-tethered probes (thiolated probes) and biotinylated signalling probes. A convergence between analytical signals and simulated results was observed, indicating the possibility to use bioinformatic data for ssDNA probes selection to be incorporated in genosensors.
为了合理化选择 ssDNA 探针以检测两种 Bonamia 物种（B. exitiosa 和 B. ostreae），进行了基于实验数据的聚合酶链反应（PCR）后核酸工作，这些实验数据来自电化学基因传感器，以及源自二级结构折叠模拟和杂交反应预测的生物信息学数据。这两种寄生虫是贻贝 Ostrea edulis 的寄生虫。在 B. ostreae 和 B. exitiosa PCR 扩增子（分别为 300 和 304 个碱基）的不同区域内选择了六个 ssDNA 探针（长度为 11 至 25 个碱基，其中 2 个硫醇化和 4 个生物素化），旨在区分这些寄生虫物种。ssDNA 扩增子和探针分别使用“Mfold Web Server”进行分析，模拟二级结构折叠行为。通过“Dinamelt Web Server”预测扩增子-探针的杂交。考虑到模拟折叠和杂交过程中氢键的断裂和形成数量、每个序列的间隙变化以及可用碱基的数量来评估结果。在实验部分，通过与表面固定的探针（硫醇化探针）和生物素化信号探针进行夹心杂交，在传感器界面上捕获热变性的 PCR 产物。观察到分析信号和模拟结果之间的一致性，表明可以使用生物信息学数据来选择 ssDNA 探针以纳入基因传感器。
