Fluorometric determination of Simian virus 40 based on strand displacement amplification and triplex DNA using a molecular beacon probe with a guanine-rich fragment of the stem region

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• 作者：Yubin Li ; Ruimin Li ; Li Zou ; Manjun Zhang ; Liansheng Ling
• 关键词：Tumor virus ; Triplex DNA ; Molecular beacon ; Hairpin structure ; G ; quadruplex ; Fluorescence ; SV40 infection
• 刊名：Microchimica Acta
• 出版年：2017
• 出版时间：February 2017
• 年：2017
• 卷：184
• 期：2
• 页码：557-562
• 全文大小：
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Nanochemistry; Nanotechnology; Characterization and Evaluation of Materials; Analytical Chemistry; Microengineering;
• 出版者：Springer Vienna
• ISSN：1436-5073
• 卷排序：184

文摘

The authors describe a method for sensitive quantitation of the tumor inducing DNA virus Simian virus 40. It is based on strand displacement amplification using triplex DNA. The molecular beacon (MB) probe with a guanine-rich fragment of the stem region is locked in its G-quadruplex structure and inactive. However, on addition of target dsDNA (oligo-1/oligo-2), which is the homopyrimidine/homopurine sequence located in the T antigen gene of SV40 DNA, it will hybridize with the hairpin structure. The resulting G-rich fragment is liberated and forms a G-quadruplex structure that binds the added fluorophore N-methyl mesoporphyrin IX that has a red fluorescence. The released 3′-end forms a shorter stem, which can trigger another strand displacement amplification in the presence of deoxy-ribonucleoside triphosphates (dNTPs) and Klenow Fragment (exo−) polymerase. Under the optimum conditions, fluorescence intensity (peaking at 614 nm) is proportional to the concentration of target dsDNA in the 1 pM to 250 pM concentration range, with a detection limit of 0.4 pM. The method is selective and does not recognize other dsDNAs.