科研之友
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摘要
Background: A number of studies have indicated that the conversion of clopidogrel to its active metabolite is reduced in patients who carry the CYP2C19 *2, *3, *4 or *5 loss-of-function allele, resulting in decreased response of platelet to clopidogrel treatment and worse cardiovascular outcome. The aim of this study was to develop a novel biosensor-based microarray to visually detect CYP2C19 polymorphisms. @@@ Methods: The target DNA was amplified from regions flanking the respective alleles using 5'-biotinylated reverse primer, and plasmids were prepared for the respective alleles. High stringency reversed hybridization, horseradish peroxidase-labeled streptavidin reaction, and color development, with multiple washes in different steps, were carried out and the results were recorded with an optical camera. The gene chips were tested for specificity, detection limit, intra- and inter-batch variations using the constructed plasmids. Finally, 88 clinical samples were assayed with this microarray as well as direct sequencing. @@@ Results: The results could be seen with the naked eye. Concordance tests indicated that for alleles *2, *3, *4, and *5, the. values between this assay and plasmids all reached 1.000. The detection limit was 5 x 10(2) cells/mL. Concordance test between direct sequencing and the microarray assay using 88 clinical samples gave rise to the. value of 0.983, and p < 0.01, indicating very high concordance. @@@ Conclusions: This novel biosensor-based microarray assay can amplify the signal in situ so that it can be detected by simple instruments or even the naked eyes. It is promising for clinical application in hospital laboratories.
