NASBA: a novel, isothermal detection technology for qualitative and quantitative HIV-1 RNA measurements.

Although immunoassays have long served as the standard in the field of diagnostics, the advent of nucleic acid amplification technologies allows for a new array of diagnostic applications. NASBA, nucleic acid sequence-based amplification, is one such technology that is highly suited for the amplification of RNA. As such, NASBA is applied readily as a diagnostic tool for infectious diseases, particularly for RNA viruses, such as retroviruses. The development and application of NASBA technology as a qualitative and quantitative diagnostic system for HIV-1 are described in this article.

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.

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.

Qualitative and quantitative detection of HIV-1 RNA by nucleic acid sequence-based amplification.

AIM: To develop a method to detect HIV-1 viral RNA by amplifying a specific nucleic acid sequence. METHOD: The 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. VALIDATION: An in vitro cultured HIV-1 stock solution was used to validate the NASBA method and determine the variation in RNA measurement. CONCLUSION: Although 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.

Activation of the mas oncogene involves coupling to human alphoid sequences.

We have shown previously that mouse NIH3T3 cells transfected with DNA from a human ovarian carcinoma were rendered tumourigenic by an activated mas oncogene in four independent transfection experiments. In all cases the 5'-noncoding region was rearranged in comparison to the original ovarian tumour DNA. We now report that in all four transfectants the newly acquired sequences consist of human centromeric alpha satellite repeat DNA. In at least three transfectants the alphoid DNA originates from the centromere of chromosome three. Analysis of the sequences of the recombination site in one transfectant revealed that a homologous sequence of five base pairs (CAGCA) is present in both parental strands, and might thus have contributed to the recombinational event. To establish a conclusive role for alphoid DNA in the activation of mas, we performed a co-transfection experiment in NIH3T3 cells with cloned alphoid DNA and the mas coding sequence. We show that the transfectants expressing a transformed phenotype contain amplified mas linked to alphoid DNA. NIH3T3 cells transfected with plasmids that contained alphoid sequences cloned directly upstream of the mas coding sequence, and injected into nude mice, gave rise to tumours with amplified mas sequences (7/7). In six of these tumours the alphoid sequences were amplified as well. Our data suggest a novel mechanism of oncogene activation: recombination with normal alphoid repeat DNA resulting in amplification of the oncogene.

Quantification of HIV-1 RNA in plasma using NASBA 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.

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).

Frequent somatic imbalance of marker alleles for chromosome 1 in human primary breast carcinoma.

Loss of heterozygosity at particular chromosomal loci in the tumor cell, as evidenced by restriction fragment length polymorphism analysis, has been taken as a hallmark of the presence of tumor suppressor genes. Recent studies of breast carcinoma have suggested that such genes might be located on the short as well as on the long arm of chromosome 1. We report here that comparison of constitutional and tumor genotypes of 84 breast tumors at 7 polymorphic chromosome 1 loci indicates a frequent imbalance of alleles on both 1p (12 of 61 informative patients) and 1q (37 of 71 informative patients). In about one-half of these cases, however, this imbalance was consistent with a gain in copy number of one allele in tumor DNA relative to normal DNA, rather than loss of the other. In 10 tumors we performed chromosome 1 enumeration in the interphase nucleus using in situ hybridization with a probe detecting the heterochromatin region at 1q12. These experiments confirmed the supernumerary presence of region 1q12 in those tumors showing an allelic copy number gain of 1q. We suggest that there are several genes on chromosome 1 serving as targets for these changes, some of them associated with breast cancer development through their deletion and others through an increase in copy number.

Direct nonradioactive in situ hybridization of somatic cell hybrid DNA to human lymphocyte chromosomes.

Biotinylated DNA from various human-rodent hybrids was hybridized to human lymphocyte spreads after preannealing of the repeated sequences with sonicated total human DNA. Fluorescent labeling was achieved by successive treatments with fluorescein-labeled avidin and biotinylated antiavidin antibody. The use of labeled total DNA from hybrids with known chromosome composition permits the fluorescent staining-("painting") of specific chromosomes, or parts thereof, in human lymphocyte metaphases. Alternatively, the human chromosome content of cell hybrids with unknown chromosome composition is directly assessed from the labeling pattern of human lymphocyte spreads using the total hybrid DNA as probe.

Isolation and characterization of cell hybrids containing human Xp-chromosome fragments.

We have subjected C12D, a Chinese hamster hybrid containing only the human X chromosome, to 6-thioguanine selection. The majority of the derivative clones retained rearranged Xp-fragments, which were characterized by using a combination of enzyme markers, DNA probes, and in situ hybridization. Two of these, TG2 and TG5sc9.1, contained only an Xpter----p21 fragment and should be an ideal resource for directed cloning from this region. A possible mechanism for the specific retention of Xp-fragments is discussed.

Rapid subchromosomal localization of cosmids by nonradioactive in situ hybridization.

A rapid method for localizing large numbers of complete cosmids by nonradioactive in situ hybridization is described. The cosmids are nick translated in the presence of biotin-16-dUTP, incubated with an excess of sonicated human DNA, and used as a probe for in situ hybridization. Sites of hybridization are detected by successive treatments with FITC-labeled avidin and biotinylated anti-avidin antibody. Fifty-two cosmids were localized on chromosome 16 in 5 d relative to translocation breakpoints contained in two cell lines. Rapid identification of chromosome 16 was achieved by cohybridization with a chromosome 16-specific centromeric repeat probe.

Rapid detection of chromosome 16 inversion in acute nonlymphocytic leukemia, subtype M4: regional localization of the breakpoint in 16p.

The pericentric inversion of chromosome 16 characteristic for acute nonlymphocytic leukemia, subtype M4, was detected in five patients by means of nonradioactive in situ hybridization of complete cosmids. First, five cosmids situated along the short arm of chromosome 16 were used to map the breakpoint of the inversion distal to the rare folate-sensitive fragile site FRA16A. Then, the use of two cosmids on either side of the breakpoint, combined with a probe specific for the centromeric region of chromosome 16, readily detected the inversion, even in poor metaphase spreads.

der(1)t(1;9): a specific chromosome abnormality in polycythemia vera? Cytogenetic and in situ hybridization studies.

Two patients with polycythemia vera and an extra der(1)t(1;9) chromosome are reported. In one patient this was found as the sole abnormality. The other patient originally presented with trisomy 9 but later developed an extra der(1) during the further course of the disease with disapperance of the extra chromosome 9. In situ hybridization studies on this latter patient proved that the centromere of chromosome 1 was involved in the formation of the derivative chromosome.

Detection of chromosome aneuploidy in interphase nuclei from human primary breast tumors using chromosome-specific repetitive DNA probes.

We have used in situ hybridization with chromosome specific repetitive DNA sequences as a probe to reveal particular chromosomes as distinct spots or clusters of signal within interphase nuclei. Using karyotypically defined cells and cell lines, we show that the number of signals obtained per nucleus correlates with the number of particular chromosomes present in that nucleus. Further, admixtures of karyotypically different cell lines could be detected. In situ hybridization of nuclei and metaphase spreads derived from the breast cancer cell line MCF-7 shows that a deviant number of spots/nucleus indicates a numerical and/or structural chromosomal aberration. In seven primary breast tumors studied, we detected numerical aberrations of the target sites of chromosomes 1 and/or 18. Although all had a single peak in DNA flow measurements, six of the cases appeared to be heterogeneous with respect to their spots/nucleus content.

Chromosome-specific alpha satellite DNA: isolation and mapping of a polymorphic alphoid repeat from human chromosome 10.

Distinct subsets of the human alpha satellite repetitive DNA family can be found in the centromeric region of each chromosome. Here we described the isolation and mapping of an alpha satellite repeat unit specific for human chromosome 10, using a somatic cell hybrid in which the only human centromere derives from chromosome 10. A hierarchical higher-order repeat unit, consisting of eight tandem approximately 171-bp alphoid monomer units, is defined by six restriction endonucleases. Under high-stringency conditions, a cloned representative of this 8-mer repeat family hybridizes to chromosome 10 only, both by Southern blot analysis of a somatic cell hybrid panel and by in situ hybridization. The probe furthermore detects a polymorphic restriction pattern of the alpha satellite array on chromosome 10. These features will make this probe a valuable genetic marker for studies of the centromeric region of chromosome 10.

A deletion hot spot in the Duchenne muscular dystrophy gene.

We have made a detailed study of a deletion hot spot in the distal half of the Duchenne muscular dystrophy (DMD) gene, using intragenic probe P20 (DXS269), isolated by a hybrid cell-mediated cloning procedure. P20 detects 16% deletions in patients suffering from either DMD or Becker muscular dystrophy (BMD), in sharp contrast to the adjacent intragenic markers JBir (7%) and J66 (less than 1%), mapping respectively 200-320 kb proximal and 380-500 kb distal to P20. Of the P20 deletions, 30% start within a region of 25-40 kb, the majority extending distally. P20 was confirmed to map internal to a distal intron of the DMD gene. This region was recently shown by both cDNA analysis (M. Koenig et al., 1987; Cell 50: 509-517), and field inversion electrophoresis studies (J.T. Den Dunnen et al., 1987, Nature (London) 329: 640-642) to be specifically prone to deletions. In addition, P20 detects MspI and EcoRV RFLPs, informative in 48% of the carrier females. Together, these properties make P20 useful for carrier detection, prenatal diagnosis, and the study of deletion induction in both DMD and BMD.