Infrared Fluorescence Detection Of Nucleic Acid Molecules With Rolling Circle Amplification On Nylon Membrane

Positively-charged nylon membrane is a general solid-phase support for nucleic acid detection due to its convinient immobilization of detected nucleic acid materials by direct electrostatic adherence and simple UV crosslinking. Rolling circle amplification (RCA) is a widely used isothermal DNA amplification technique for nucleic acid detection. Near-infrared fluorescence (NIRF) is a new fluorescence technique with high sensitivity due to low background. This study thus developped a new facile method for detecting various nucleic acid molecules by combining the advantages of nylon membrane, RCA, and NIRF, named NIRF-based solid phase RCA on nylon membrane (M-NIRF-sRCA).The detection system of this method only need two kinds of nucleic acid molecules:specific probes with a RCA primer (P) at its 3' end and a rolling circle (RC). The detection procedure consists of four steps: (1) immobilization:immobilize detected nucleic acids on nylon membrane by UV crosslinking; (2) hybridization:hybridize nylon membrane with specific probes and RC; (3) amplification:amplify target DNA by a RCA reaction containing ?29 DNA polymerase, RC, dNTP, and biotin-dUTP; (4) imaging: incubate sRCA products with NIRF-labeled streptavidin (streptavidin-800CW) and imaged with a Odysssey near-infrared fluorescence imager system.The feasibility and reliability of M-NIRF-sRCA were demonstrated by the detection of oligonucleotides, plasmids, and bacteria genomic DNAs. The results showed that M-NIRF-sRCA could quantitatively detect target oligonucleotide in the range of 0.17 fmol?0.35 pmol with the sensitivity as few as 0.17 fmols. M-NIRF-sRCA could quantitatively detect HPV plasmids including HPV16, HPV18, and HPV33 in the range of 0.25 fmol?4 fmol with the sensitivity as few as 0.25 fmols. Moreover, the specificity of M-NIRF-sRCA detection was demonstrated by detecting 6 kinds of HPVs, including high-risk types of HPV16, HPV18, HPV33, HPV35, and low-risk types of HPV6 and HPV11. Finally, with some improvements, M-NIRF-sRCA system could be also used to quantitatively detect DH5? genomic DNAs in the range of 3.09 ng?49.4 ng with the sensitivity as few as 3.09 ng (corresponding to about 3000 bacteria).In conclusion, this study developped a new facile method for detecting nucleic acid molecules based on nylon membrane and NIRF-sRCA that has high sensitivty. This method provides a new biomedical tool for nucleic acid molecule detection.