Tim Kievits

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 HIV-1 RNA by nucleic acid sequence-based amplification.

AimTo develop a method to detect HIV-1 viral RNA by amplifying a specific nucleic acid sequence. MethodThe nucleic acid sequence-based amplification (NASBA) method uses the simultaneous activity of avian myeloblastosis virus reverse transcriptase, T7 RNA polymerase and RNase H to amplify a specific nucleic acid target sequence. ValidationAn in vitro cultured HIV-1 stock solution was used to validate the NASBA method and determine the variation in RNA measurement. ConclusionAlthough NASBA is theoretically capable of specific amplification of RNA or DNA, it is most suitable for amplification of RNA, and therefore for detection of HIV-1 viral RNA.

Detection of HIV-1 RNA in plasma and serum samples using the NASBA amplification system compared to RNA-PCR

The presence of HIV-1 RNA in the plasma and serum of European and African patients was monitored using RNA-polymerase chain reaction (RNA-PCR) and the new isothermal NASBA nucleic acid amplification system encompassing a gel-based detection assay (ELGA). Identical RNA extraction procedures, provided by the NASBA amplification system, were used for both methods. The detection limit for HIV-1 RNA, measured on a 10-fold dilution series of spiked HIVIIIB in negative plasma, was about 0.05 CCID50 per test for both methods. Both NASBA and RNA-PCR were more sensitive than a p24 assay for the detection of circulating HIV-1 virus in blood: 17 of the 34 (50%) p24 antigen-tested seropositives were p24-positive while 32 (94%) were positive by NASBA and 30 (88%) by RNA-PCR. Among the 45 seropositives, 34 of which were tested for p24 antigen, 43 (96%) were positive by NASBA and 41 (91%) by RNA-PCR. Almost all seropositives had a detectable viral load in 100 microliters plasma. Lower viral loads were only encountered in some healthy seropositives with a higher CD4 count. There was no cross-reactivity with HIV-2 or HIV-I with both the RNA-PCR and NASBA. The extraction method used permitted the detection of HIV-1 RNA equally well in serum and in plasma with heparin or EDTA.

Detection of HIV-1 RNA in plasma by isothermal amplification (NASBA) irrespective of the stage of HIV-1 infection

Using an experimental assay for isothermal amplification of HIV RNA in plasma (NASBA, Organon Teknika, Boxtel, The Netherlands), 70 samples from 59 HIV-1-infected persons and 29 samples from 28 uninfected volunteer blood donors were tested for the presence of HIV-1 RNA. The HIV-1-positive plasma samples were drawn from patients at various stages of infection: two samples from persons with signs of acute HIV infection (CDC stage I); 29 samples from 25 symptom-free persons (CDC stage II) and 39 samples from 32 persons with AIDS-related symptoms (CDC stage IV). All samples from HIV-1-infected persons had HIV-1 RNA, irrespective of the stage of infection (100% sensitivity). Testing the donor samples in duplicate, initially 54/58 samples (93%) tested negative for HIV-1 RNA. Repeated testing of the 4 samples with inconsistent test results showed all samples to be negative (specificity 100%). Detection of HIV-1 RNA in plasma by isothermal amplification (NASBA) promises to be a valuable alternative to the detection of HIV-1 RNA or DNA by the polymerase chain reaction.

Abstract 4322: Kinase activity profiles distinguish papillary thyroid cancers with and without BRAF V600E mutations

Background: Most differentiated non medullary thyroid cancers (DTC) are curatively treated by surgery and radio-active iodine ablation therapy. A subset of patients shows recurrence due to a loss of iodine transport. Two main subgroups of recurrent DTC are seen: papillary thyroid cancers (PTC) with somatic BRAF mutations (V600E) and oncocytic follicular cancer. Recurrent DTC are clinically treated by multi-kinase inhibitors such as sorafenib, with a low affinity for BRAF V600E. Vemurafenib and dabrafenib were specifically designed against this mutant. The aim of this study was twofold: - Can benign and malign DTC be classified based on kinase activity profiles? - Do sorafenib (and regorafenib) show different inhibition profiles than dabrafenib? Methods: Tissue cryosections from fresh frozen thyroid tumors were lysed. All tumor specimens were analyzed for BRAF mutations. Serine/threonine kinase (STK) activity profiles of the lysates (0.5 μg protein per array) were generated on PamChip® peptide microarrays, comprising peptide sequences from known human phosphorylation sites. The ex vivo effect of BRAF inhibitors sorafenib, regorafenib and dabrafenib on kinase activity profiles of 14 PTC9s was determined as well. Data were analysed with Bionavigator software. Results: A classifier built on the STK kinase activity profiles of 57 thyroid cancer samples was able to classify malignant and benign tumors with a limited error rate. Leave One Out Cross Validation classified 26/35 of malignant and 17/22 of benign samples correctly. Kinase inhibition profiles of PTC9s with sorafenib and regorafenib did not discrimate V600E mutants from wild type tumors whereas with dabrafenib 34/144 peptides were identified that potentially differentiated the groups. Conclusions: Serine/threonine kinase activity profiling appears to be able to differentiate benign and malignant thyroid tumors. Ex vivo spiking in of kinase inhibitors shows differential inhibition in tumors with a somatic BRAF mutation. Potentially, an industrial prediction platform can be envisioned for testing of novel drugs in tumor tissue. Whether individual patient responses against registered kinase inhibitors can be predicted must be investigated. Citation Format: Maria H. Hilhorst, Adrienne van den Berg, Tom van Wezel, Tim Kievits, Piet J. Boender, Rik de Wijn, Rob Ruijtenbeek, Wim Corver, Hans Morreau. Kinase activity profiles distinguish papillary thyroid cancers with and without BRAF V600E mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4322. doi:10.1158/1538-7445.AM2015-4322

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).