We have developed a novel strategy for the colorimetric detection of PCR products by utilizing a target-specific primer modified at the 5′-end with an anti-DNAzyme sequence. A single-stranded DNAzyme sequence folds into a G-quadruplex structure with hemin and shows strong peroxidase activity. When the complementary strand binds to the DNAzyme sequence, it blocks the formation of the G-quadraduplex structure and loses its peroxidase activity. In the presence of the target gene, PCR amplification proceeds, and anti-DNAzyme sequence modified primers present in the reaction mixture form a double strand through primer extension. Therefore, it does not block the DNAzyme sequence. Further, a colorimetric signal is generated by the addition of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and H₂O₂ at the end of the reaction. We have successfully detected a single copy of the HIV type 1 gag gene in buffer and 10 copies in human serum. The strategy developed could be used to detect DNA and RNA in complex biological samples by simple primer designing that includes DNAzyme and a DNA extended primer.

中文翻译：

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使用DNAzyme和互补的DNA延伸引物 对HIV gag基因进行比色分子诊断†

我们已经开发了一种新的策略，用于通过比色法检测PCR产物，方法是利用在5'末端修饰了抗DNAzyme序列的靶标特异性引物。单链DNAzyme序列折叠成带有血红素的G-四链体结构，并显示出强大的过氧化物酶活性。当互补链与DNAzyme序列结合时，它将阻止G-四链体结构的形成，并失去其过氧化物酶活性。在靶基因的存在下，进行PCR扩增，并且反应混合物中存在的抗DNA酶序列修饰的引物通过引物延伸形成双链。因此，它不阻断DNAzyme序列。此外，通过添加2,2'-叠氮基双（3-乙基苯并噻唑啉-6-磺酸盐）（ABTS）和H ₂ O生成比色信号₂在反应结束时。我们已经成功地在缓冲液中检测到1型HIV gag基因的单个拷贝，在人血清中检测到10个拷贝。通过简单的引物设计（包括DNAzyme和DNA延伸引物），可以将开发的策略用于检测复杂生物样品中的DNA和RNA。