Peter Franklin Lens

NASBATM 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).

A one-tube quantitative HIV-1 RNA NASBA nucleic acid amplification assay using electrochemiluminescent (ECL) labelled probes

Quantification of HIV-1 viral RNA based on co-amplification of an internal standard Q-RNA dilution series requires a number of NASBA nucleic acid amplification reactions. For each internal standard Q-RNA concentration a separate NASBA amplification has to be performed. The development of an electrochemiluminescent (ECL) detection system with a dynamic signal detection range over 5 orders of magnitude enabled simplification of the Q-NASBA protocol. Three distinguishable Q-RNAs (QA, QB and QC) are mixed with the wild-type sample at different amounts (i.e. 10(4) QA, 10(3) QB and 10(2) QC molecules) and co-amplified with the wild-type RNA in one tube. Using ECL-labelled oligonucleotides the wild-type, QA, QB and QC amplificates are separately detected with a semi-automated ECL detection instrument and the ratio of the signals determined. The amount of initial wild-type RNA can be calculated from the ratio of wild-type signal to QA, QB and QC signals. This one-tube Q-NASBA protocol was compared to the earlier described protocol with six amplifications per quantification using model systems and HIV-1 RNA isolated from plasma of HIV-1-infected individuals. In all cases the quantification results of HIV-1 RNA were comparable between the two methods tested. Due to the use of only one amplification per quantification in the one-tube Q-NASBA protocol the QA, QB and QC internal standard RNA molecules can be added to the sample before nucleic acid isolation. The ratio of QA:QB:QC:WT RNAs, from which the initial input of WT-RNA is calculated, will remain constant independent of any loss that might occur during the nucleic acid isolation.

Quantification of HIV-1 RNA in plasma using NASBAtm during HIV-1 primary infection

Quantification of HIV-1 viral RNA in plasma was achieved by competitive co-amplification of a dilution series of in vitro generated RNA using the nucleic acid sequence based amplification (NASBA) technology. This 1.5 kilobase in vitro RNA, comprising the gag and part of the pol region, differs only by sequence-randomization of a 20 nt fragment from the wild-type RNA, ensuring equal efficiency of amplification. In model systems the accuracy of this method is within one log. Application of the Q-NASBA to plasma samples of a patient with a primary HIV-1 infection shows good concordance of the HIV-1 RNA profile with the p24 antigen profile. However, the HIV-1 RNA determination is more sensitive than the p24 antigen determination. Peak values of HIV-1 RNA are around 10(8) RNA molecules per ml plasma at the moment of seroconversion. Quantitative nucleic acid detection methods, like Q-NASBA, allow the monitoring of HIV-1 RNA during the course of infection which might have predictive value for disease development.

Qualitative and Quantitative Detection of Nucleic Acids of Infectious Agents by NASBA

Since the advent of the polymerase chain reaction (PCR; Saki 1988; Mullis & Faloona 1987), a number of other nucleic acid amplification techniques have been developed. One of the most important and well developed of these new technologies is the Nucleic Acid Sequence Based Amplification (NASBATM method (Kievits et al 1991). NASBATM utilizes the coordinated activities of AMV reverse transcriptase (RT), RNase H, and T7 RNA polymerase to amplify a specific nucleic acid target. The specificity of the reaction is determined by a pair of oligonucleotide primers, which are specific for the sequence of interest. One of these primers (designated P1) is synthesized so as to include the promoter for T7 RNA polymerase as a 5’ overhang. The reaction is conducted at constant temperature (41°C) and produces a single stranded RNA product which represents a 106-109 amplification of the original target sequence (figure 1). Although capable of amplifying both DNA and RNA target sequences, NASBATM is most suitable for the amplification of RNA. Thus, NASBATM has become an extremely powerful technique for the detection and quantification of retroviruses (particularly HIV-1).