title: Image-based detection of oligonucleotides--a low cost alternative to spectrophotometric or fluorometric methods.
abstract: Herein, we report a sensitive and low cost image-based (photocolorimetric) method for the detection of oligonucleotides on an activated polypropylene microtest plate (APPμTP). The assay was developed on the APPμTP by covalently immobilising 20-mer amino-modified oligonucleotides. Biotin-tagged complementary target sequences were then hybridised with the immobilised oligonucleotides. Colour was developed by streptavidin-HRP conjugate and the image of the coloured assay solution was taken by a desktop scanner and analysed using colour saturation. The developed method was analysed for its detection limit, accuracy, sensitivity and interference. The linearity range was found to be 1.7-170 ng mL(-1) while the lower limit of detection and limit of quantification were 1.7 and 5.6 ng mL(-1) respectively. The method showed comparable sensitivity to fluorometric methods, and was found to be correlated to fluorescence (R(2) = 0.8081, p-value < 0.0001) and absorbance (R(2) = 0.9394, p-value < 0.0001)-based quantification. It discriminates mismatched base sequences from perfectly matched sequences efficiently. Validation of the method was carried out by detecting por A DNA of Neisseria meningitidis in bacterial meningitis samples. The por A-specific probe having a 6-carbon spacer at its 5'-NH2 terminus was immobilised covalently to the APPμTP and hybridised with different samples of biotinylated single-stranded por A DNA.
authors.affiliation: Department of Chemical and Systems Biology, CSIR-Institute of Genomics and Integrative Biology, Mall Road, Delhi, 110007, India. pnahar@igib.res.in.
authors.forename: Rajesh
authors.initials: R
authors.lastname: Ahirwar
authors.forename: Swati
authors.initials: S
authors.lastname: Tanwar
authors.forename: Shahila
authors.initials: S
authors.lastname: Parween
authors.forename: Ashok
authors.initials: A
authors.lastname: Kumar
authors.forename: Pradip
authors.initials: P
authors.lastname: Nahar
mesh_terms: D001483:Base Sequence; D057230:Limit of Detection; D009841:Oligonucleotides / Q000032:analysis*; D013050:Spectrometry, Fluorescence / Q000191:economics / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet / Q000191:economics / Q000379:methods*
chemical_list: D009841:Oligonucleotides