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Aim: The 961delT, C1494T and A1555G mutations in the mitochondrial 12S rRNA are the hotspot mutations associated with aminoglycoside antibiotics induced deafness (AAID).The objective of this study was to apply the "on/off" switch consisting of 3 phosphorothioate-modified allele specific primers and Exo+ polymerase in single base discrimination of the three hotspot point mutations rapidly.