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Singleplex, mixed, and multiplex amplicon detection

YS Yajing Song
PG Peter Gyarmati
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To prepare the spiked DNA (sDNA), 106 copies of each gDNA was added into 200 µL of human serum, and sDNA was extracted using the Qiagen Blood DNA Mini kit following the manufacturer’s instructions. Both singleplex and multiplex PCR assays in 1× Kapa PCR readymix were used to prepare the detection targets on a Techne primer thermal cycler. The detection with the mixed targets was processed by mixing two or three of singleplex PCR products. The multiplex detection was processed with the PCR products from duplex or triplex PCR assay. Five hundred nanograms of target DNA bound with 10 µL of magnetic beads was used for each detection. The average intensity of each specific signal was compared to that of NTC on the same paper. Each set of conditions was completed in triplicate.

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