Image-based detection of oligonucleotides--a low cost alternative to spectrophotometric or fluorometric methods.
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| 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.
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| Authors | Rajesh Ahirwar, Swati Tanwar, Shahila Parween, Ashok Kumar, Pradip Nahar |
| Journal | The Analyst (Analyst) Vol. 139 Issue 9 Pg. 2186-92 (May 07 2014) ISSN: 1364-5528 [Electronic] England |
| PMID | 24653995 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't) |
| Chemical References |
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