Target-specific in vitro transcribed (IVT) RNA was synthesized in order to determine the analytical sensitivity of each multiplex RT-qPCR assay as previously described [45 (link),46 ,47 (link)]. Inserts containing the target regions of each assay (flanked by PstI and HindIII restriction enzymes) were chemically synthesized and cloned into the multiple cloning site of the pGEM®-3Z vector (Promega, Madison, WI, USA).
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