Reverse transcription loop-mediated isothermal amplification (RT-LAMP).
Step 1: At the 3′-end of the viral RNA, reverse transcriptase
and BIP primer initiate conversion of RNA to cDNA. Step 2: At the
same end, DNA polymerase and B3 primer continue to generate the second
cDNA strand to displace and release the first cDNA strand. Step 3:
The FIP primer binds to the released cDNA strand and DNA polymerase
generates the complementary strand. Step 4: F3 primer binds to the
3′ end, and DNA polymerase then generates a new strand while
displacing the old strand. LAMP cycling produces various sized double-stranded
looped DNA structures containing alternately inverted repeats of the
target sequence as detected by a DNA indicator dye. Reagents*: Primers
and master mix containing reverse transcriptase, DNA polymerase with
strand displacing activity, dNTPs, and buffers.