NASBA isothermal enzymatic in vitro nucleic acid amplification optimized for the diagnosis of HIV-1 infection.
Isothermal nucleic acid amplification of target RNA or DNA sequences is accomplished by the simultaneous enzymatic activity of AMV reverse transcriptase, T7 RNA polymerase and RNase H. Amplification factors of the nucleic acid sequence based amplification (NASBA) method range from 2 x 10(6) to 5 x 10(7) after 2.5 h incubation at 41 degrees C. During NASBA there is a major accumulation of specific single stranded RNA. RNA:DNA hybrid and double stranded DNA are also synthesized, although to a minor extent. The system is optimized for the detection of HIV-1 sequences in in vitro infected cells, blood and plasma. Detection levels are 10 molecules of HIV-1 in a model system with in vitro generated HIV-1 RNA as input and 5 infected cells on a background of 5 x 10(4) non-infected cells. Blood and plasma can also be used as the source of nucleic acid for detection of HIV-1 sequences using a specifically developed sample preparation method. Using NASBA it is possible to amplify specifically RNA or DNA from a pool of total nucleic acid, which permits the investigation of the expression of specific genes involved in pathogenesis of infectious agents. The combination of NASBA with a rapid and user-friendly nucleic acid extraction method makes the whole procedure suitable for large scale diagnosis of infectious agents (e.g. HIV-1).
Kievits
T
T
Organon Teknika, Boxtel, The Netherlands.
van Gemen
B
B
van Strijp
D
D
Schukkink
R
R
Dircks
M
M
Adriaanse
H
H
Malek
L
L
Sooknanan
R
R
Lens
P
P
D001483:Base Sequence; D002478:Cells, Cultured; D012321:DNA-Directed RNA Polymerases; D015750:Genes, gag; D015751:Genes, pol; D015658:HIV Infections / Q000175:diagnosis* / Q000235:genetics; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D015347:RNA Probes; D012367:RNA, Viral / Q000302:isolation & purification*; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H; D012680:Sensitivity and Specificity; D013601:T-Lymphocytes; D014764:Viral Proteins
D015347:RNA Probes; D012367:RNA, Viral; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D012321:DNA-Directed RNA Polymerases; D016914:Ribonuclease H