A highly sensitive fluorescence detection method for tobacco mosaic virus (TMV) RNA based on in situ reaction of disodium 4,4′-diazidostilbene-2,2′-disulfonate tetrahydrate (DES) was developed for the first time. Specifically, cDNA-1 (probe 1) was fixed on carboxylated Fe₃O₄ magnetic beads (MBs), and hybridized with the TMV RNA (tRNA) in the next step. Then, cDNA-2 (probe 2) that was hybridized with tRNA was linked to alkaline phosphatase-conjugated streptavidin (SA-ALP) via the binding of biotin and SA. Next, L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (AAPi) was dephosphorylated and hydrolyzed by alkaline phosphatase (ALP), producing ascorbic acid (AA). The subsequently added DES was reduced by AA, producing reduced DES, which successfully resulted in ‘turn-on’ of the fluorescent signal. Under optimal conditions, a good linear relationship between the fluorescence intensity and the logarithm of the tRNA concentration was obtained in the range from 1 pM to 10 nM, with a detection limit of 0.101 pM. Meanwhile, the method exhibited excellent selectivity and reproducibility. These results showed that the fluorescence detection strategy can be effectively employed for the qualitative and quantitative analysis of tRNA and has a potential application in the actual detection of tRNA.

中文翻译：

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基于 4,4'-二叠氮芪-2,2'-二磺酸二钠原位反应的烟草花叶病毒 RNA 的高灵敏度荧光检测

首次开发了一种基于4,4'-二叠氮芪-2,2'-二磺酸二钠四水合物 (DES)原位反应的烟草花叶病毒 (TMV) RNA 的高灵敏度荧光检测方法。具体而言，将 cDNA-1（探针 1）固定在羧化 Fe ₃ O ₄磁珠 (MB) 上，并在下一步中与 TMV RNA (tRNA) 杂交。然后，与 tRNA 杂交的 cDNA-2（探针 2）通过生物素和 SA 的结合与碱性磷酸酶偶联的链霉亲和素（SA-ALP）连接。接下来，L-抗坏血酸2-磷酸倍半镁盐水合物（AAPi）被碱性磷酸酶（ALP）去磷酸化和水解，产生抗坏血酸（AA）。随后添加的 DES 被 AA 还原，产生还原的 DES，这成功地导致了荧光信号的“开启”。在最佳条件下，荧光强度与tRNA浓度对数在1 pM至10 nM范围内具有良好的线性关系，检测限为0.101 pM。同时，该方法表现出优异的选择性和重现性。这些结果表明荧光检测策略可以有效地用于tRNA的定性和定量分析，在tRNA的实际检测中具有潜在的应用价值。