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Optimization of real-time ERA assay

Following successful primer selection, the optimization of thereal-time ERA assay reaction mixture was conducted. This stepemployed the fluorescent nucleic acid amplifcation kit (KS103; GenDx Biotechnology Co., Ltd) and was performed using a CFX96 Real-Time PCR Detection System (Bio-Rad LaboratoriesInc.). Each 19.2 uL reaction mixture contained 2 uL of DNA tem-plate,8 uL of rehydration buffer, primers optimized from 200 to 700 nmol L-1, probes optimized from 100 to 350 nmol L¹ andddH₂O. The primers and probes were maintained at a fxed ratioof 2:1. The activator (0.8 uL) was added to the lid, followed by cen-trifugation for thorough mixing. The reactions were incubated at 37℃ for 20 min with fuorescence measurements taken every 30s. Visual detection was achieved by imaging the tubes under LED blue light illuminator ,with images captured using a smartphone.

Doi: 10.7506/spkx1002-6630-20201016-146