Highly sensitive qualitative and quantitative detection of reverse transcriptase activity: optimization, validation, and comparative analysis with other detection systems.
An ultra-sensitive assay for reverse transcriptase (RT) activity called Amp-RT has been developed. An in vitro transcribed heteropolymeric RNA sequence was used as a template, and polymerase chain reaction (PCR) amplification with Southern-blot hybridization served as a detection system for the cDNA product of the reaction. Titration of Mg2+ and Mn2+ concentrations using the human immunodeficiency virus type 1 (HIV-1) and the human T lymphotropic virus type 1 (HTLV-I), respectively, showed optimal assay reactivity for both viruses at 2-20 mM of Mg2+. Analysis of density banded HIV-1 showed that the peak RT activity of the assay was associated with the fractions consistent with retrovirus particles. The sensitivity of Amp-RT was also compared with that of three conventional RT assays by using seven different retroviruses including HIV-1, simian immunodeficiency virus (SIV), caprine arthritis-encephalitis virus (CAEV), HTLV-I and HTLV-II, simian retrovirus type 2 (SRV-2), and gibbon ape leukemia virus (GALV). HTLV-I, HTLV-II, and GALV could not be detected by the three conventional RT assays. Amp-RT was able to detect all these viruses in 10(1)-10(3)-fold dilutions. Similarly, Amp-RT was found to be 10(3)-10(6)-fold more sensitive than the other RT assays in detecting HIV-1, SIV< or CAEV. Culture supernatants from uninfected cell lines were all Amp-RT negative. A quantitative Amp-RT assay was also developed by using recombinant HIV-1 RT and signal quantitation. The assay was found to have a 5 log linear range, and therefore, provides a useful tool for quantitating RT and retroviruses. Amp-RT offers a sensitive generic tool for the qualitative and quantitative detection of known and unknown retroviruses.
Yamamoto
S
S
Retrovirus Diseases Branch, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA.
Folks
T M
TM
Heneine
W
W
D000818:Animals; D015139:Blotting, Southern / Q000379:methods*; D002499:Centrifugation, Density Gradient; D004797:Enzyme-Linked Immunosorbent Assay; D054303:HIV Reverse Transcriptase / Q000235:genetics; D015497:HIV-1 / Q000201:enzymology; D006801:Humans; D008274:Magnesium; D008345:Manganese; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis*; D012194:RNA-Directed DNA Polymerase / Q000235:genetics*; D012190:Retroviridae / Q000302:isolation & purification*; D012191:Retroviridae Proteins / Q000235:genetics*; D012680:Sensitivity and Specificity; D013395:Sucrose; D014407:Tumor Cells, Cultured
D012367:RNA, Viral; D012191:Retroviridae Proteins; D008345:Manganese; D013395:Sucrose; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D008274:Magnesium