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

Abstract

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.