High sensitive one-step RT-PCR using MMLV reverse transcriptase, DNA polymerase with reverse transcriptase activity, and DNA/RNA helicase

•We devised a one-step RT-PCR with a thermostable MMLV RT and other two enzymes.•One is a Thermotoga petrophila family A DNA polymerase variant, K4polL329A.•The other is a Thermococcus kodakarensis DNA/RNA helicase, Tk-EshA.•The concentrations of enzymes, MgCl2, and Mn(OCOCH3)2 were optimized.•As a result, 30 copies/μl of target RNA in 10 μl reaction volumes were detected.