RNA pathogen detection with one-step reverse transcription PCR and strand-displacement based signal amplification.
A novel detection method for RNA pathogens based on one-step reverse transcription PCR is introduced here. This method utilized the reverse transcriptase activity and the 5'-nuclease activity of TaqM1 DNA polymerase to transform target RNA into cDNA. The following PCR process released a fragment from the 5' end as a specific probe. Afterwards this fragment triggered a strand-displacement based signal amplification to release large amounts of G-quadruplex DNAzymes. All the probes applied in our method were unmodified DNA oligonucleotides. The detection results could be reported without sophisticated instruments either in the colorimetric way through oxidizing ABTS or in the fluorometric way by using tyramine as substrate. This approach could successfully detect HIV-1 in a blood sample and it has a linear concentration range of 6 fM to 60 pM.
Natural Products Research Center, Chengdu Institution of Biology, Chinese Academy of Science, Chengdu 610041, P. R. China.
Feng
F
Du
Frank
F
Streckenbach
Haodong
H
Chen
Xin
X
Huang
Zhuo
Z
Tang
Andreas
A
Marx
D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D005470:Fluorometry / Q000379:methods; D015658:HIV Infections / Q000097:blood / Q000175:diagnosis*; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D012367:RNA, Viral / Q000097:blood / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D019914:Taq Polymerase / Q000378:metabolism
D018076:DNA, Complementary; D012367:RNA, Viral; D019914:Taq Polymerase