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Results for “Isothermal amplification”

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At least 235 records · Page 13Linked to original sources

PubMed2005

Absence of ribosomal RNA of Mycobacterium tuberculosis complex in sarcoidosis.

OBJECTIVE: To determine whether Mycobacterium tuberculosis ribosomal RNA (rRNA) is present in fresh tissue specimens from patients with sarcoidosis. DESIGN: A prospective study. SETTING: A university-based hospital. Patients Thirty-five patients diagnosed as having sarcoidosis at the University Hospital of Bellvitge, Barcelona, Spain, were included in the study. Fresh tissue samples with granulomatous inflammation were prospectively collected between 1997 and 2001 from all patients. For each sample tested, approximately 1 negative control was included. MAIN OUTCOME MEASURES: Mycobacterium tuberculosis rRNA was detected using an isothermal enzymatic amplification system of target rRNA of M tuberculosis complex via DNA intermediates. Smears for acid-fast staining and mycobacteriological cultures were also obtained. RESULTS: A total of 78 biopsy specimens (57 skin, 10 lymph node, 3 lacrimal gland, 2 spleen, 2 lung, 2 muscle, 1 bone, and 1 nerve) collected from 74 patients (35 patients with sarcoidosis and 39 control patients) were included in the study. Stains for acid-fast bacilli and mycobacterial cultures were negative for organisms in all cases. Mycobacterium tuberculosis rRNA was not detected in the specimens from any patients with sarcoidosis or in those from control patients whose cultures were negative for organisms. Ribosomal RNA was detected in 6 tissue specimens from patients with cultures that were positive for M tuberculosis and that were processed in parallel to the samples included in the study. CONCLUSIONS: Although previous studies have reported that mycobacterial antigens may play a role in granuloma formation in some patients with sarcoidosis, our results suggest that M tuberculosis cannot be considered to be the etiologic agent of the disease.

Case-Control Studies↗

PubMed1999

Mutation identification DNA analysis system (MIDAS) for detection of known mutations.

We introduce a novel experimental strategy for DNA mutation detection named the Mismatch Identification DNA Analysis System (MIDAS) [1, 2], which has an associated isothermal probe amplification step to increase target DNA detection sensitivity to attomole levels. MIDAS exploits DNA glycosylases to remove the sugar moiety on one strand (the probe strand) at a DNA base pair mismatch. The resulting apyrimidinic/ apurinic (AP) site is cleaved by AP endonucleases/lyases either associated with the DNA glycosylase or externally added to the reaction mixture. MIDAS utilizes 32p- or FITC-labeled oligonucleotides as mutation probes. Generally between 20-50 nucleotides in length, the probe hybridizes to the target sequence at the reaction temperature. Mismatch repair enzymes (MREs) then cut the probe at the point of mismatch. Once the probe is cleaved, the fragments become thermally unstable and fall off the target, thereby allowing another full-length probe to hybridize. This oscillating process amplifies the signal (cleaved probe). Cleavage products can be detected by electrophoretic separation followed by autoradiography, or by laser-induced fluorescence-capillary electrophoresis (LIF-CE) of fluorophore-labeled probes in two minutes using a novel CE matrix. In the present experiments, we employed the mesophilic Escherichia coli enzyme deoxyinosine 3'-endonuclease (Endo V), and a novel thermostable T/G DNA glycosylase, TDG mismatch repair enzyme (TDG-MRE). MIDAS differentiated between a clinical sample BRCA 1 wild-type sequence and a BRCA1 185delAG mutation without the need for polymerase chain reaction (PCR). The combination of MIDAS with LIF-CE should make detection of known point mutations, deletions, and insertions a rapid and cost-effective technique well suited for automation.

BRCA1 Protein↗

PubMed2004

DD3PCA3 RNA analysis in urine--a new perspective for detecting prostate cancer.

OBJECTIVES: Serum tPSA lacks specificity. The DD3(PCA3) gene is highly specific for prostate cancer and is detectable in prostate cancer cells shed into urine after rectal palpation. A newly developed nucleic acid sequence based amplification assay (uPM3) for detecting DD3(PCA3) RNA in urine samples was evaluated prospectively in patients referred for prostate cancer detection. METHODS: The uPM3 assay simultaneously detects the relative expression of DD3(PCA3) RNA and PSAmRNA as a marker for prostate cells in urine. Urine samples were collected after attentive digital rectal palpation prior to transrectal guided prostate biopsy. Samples were provided as a single void specimen (20-30 ml), stabilized in phosphate buffer and centrifuged. Lysis was performed on cell pellets DD3(PCA3) RNA and PSAmRNA were extracted and amplification was performed using isothermic nucleic acid based amplification (NASBA). The two targets were detected in real-time using specific beacons as probes in a thermostated spectrofluorimeter. Parameters of the amplification curve were defined after a logistic curve fitting routine and a classification tree model was constructed to predict the outcome of patients (i.e. cancer and non-cancer). RESULTS: 201 patients were included in this prospective study. 158/201 analyzed urine samples contained enough prostate cells sufficient for DD3(PCA3) analysis (79% adequacy rate). Prostate cancer was found in 62 (39%) of the evaluable patients. Overall sensitivity, specificity, positive predictive value and negative predictive value for the uPM3 assay at a cut-off 0.5 probability were 82%, 76%, 67% and 87% respectively as compared to 98%, 5%, 40% and 83% respectively for tPSA (at a cutoff of 2.5 ng/ml). In the tPSA categories <4, 4-10 and >10 ng/ml sensitivity was 73%, 84% and 84% and specificity was 61%, 80% and 70%, respectively. The AUC (area under the curve) was 0.87 (CI 0.81-0.92). CONCLUSION: The uPM3 assay showed excellent clinical performances and a specificity far superior to tPSA.

Adult↗

PubMed2000

Quantitative evaluation of simian immunodeficiency virus infection using NASBA technology.

The most commonly used animal model for the study of HIV-1 infection in humans is the infection of non-human primates by simian immunodeficiency virus (SIV). The animal hosts used most frequently are different species of macaques, which are readily infected with SIV, and can therefore be used to study natural infection, pathogenesis, therapy, and vaccine efficacy. The study of HIV-1 infection in humans relies heavily on the quantification of HIV-1 load (i.e. viral RNA) in patient plasma. Given the importance of HIV-1 RNA levels in humans, it follows that SIV RNA levels in animals are also relevant to the study of infection in this model system. This report describes the development of the isothermal amplification-based NASBA technology for the quantification of SIV RNA load in macaque plasma. Evaluation of the assay using model systems demonstrated that the assay is accurate and reproducible over nearly four orders of magnitude. Viral RNA load data were compared to other infection measurements in the macaque system. Further, the assay was used to provide copy number levels of SIV RNA in macaque plasma samples, permitting characterization of viral load during the course of SIV infection.

Animals↗

PubMed2004

Rapid detection of tdh and trh mRNAs of Vibrio parahaemolyticus by the transcription-reverse transcription concerted (TRC) method.

We developed a novel method named the transcription-reverse transcription concerted (TRC) method and an instrument that allowed rapid and completely homogeneous real-time monitoring of RNA isothermal sequence amplification without any post-amplification analysis in our previous study [Ishiguro et al., Anal. Biochem., 314, 77-86 (2003)]. In this study, we newly established rapid and sensitive TRC systems for the detection of the mRNAs transcribed from two major virulence genes of Vibrio parahaemolyticus: the tdh gene encoding the thermostable direct hemolysin (tdh) and the trh gene encoding the thermostable direct hemolysin-related hemolysin. Examination of the standard RNAs prepared in vitro showed that a positive result, increase in the fluorescence intensity to the cut-off value within 25 min, was obtained for as few as 100 copies of RNA. The TRC method specific to the trh mRNA detected the mRNAs transcribed from the trh1 and trh2 genes, two representative trh variants sharing 84% sequence identity. The detection time gave a linear relationship to the logarithm of starting RNA copies ranging from 10(3) to 10(7) copies, showing that quantitative analysis is possible. The detection time for 10(3) copies of the standard RNAs ranged from 11 to 15 min. Examination of the total RNAs extracted from the standard strains of V. parahaemolyticus demonstrated that the new TRC systems are sufficiently sensitive to detect as few as 100 CFUs of the strains carrying the target genes. Total RNA preparations extracted from 24 strains of V. parahaemolyticus, 52 strains belonging to 31 other species of the genus Vibrio and 11 strains belonging to 8 species of non-Vibrio genera were examined. The results of the detection of tdh- and trh-specific mRNAs by the two TRC systems and those of the respective genes by the DNA colony hybridization method agreed. We conclude that the new TRC systems are rapid, highly sensitive, easy to manipulate, and are suitable for routine examination of virulent strains of V. parahaemolyticus in microbiological laboratories.

Journal Article↗

PubMed2005

Characterization of a thermostable UvrD helicase and its participation in helicase-dependent amplification.

Helicase-dependent amplification (HDA) is an isothermal in vitro DNA amplification method based upon the coordinated actions of helicases to separate double-stranded DNA and DNA polymerases to synthesize DNA. Previously, a mesophilic form of HDA (mHDA) utilizing the Escherichia coli UvrD helicase, DNA polymerase I Klenow fragment, two accessory proteins, MutL and single-stranded DNA-binding protein (SSB), was developed (1). In an effort to improve the specificity and performance of HDA, we have cloned and purified a thermostable UvrD helicase (Tte-UvrD) and the mutL homolog (Tte-MutL) from Thermoanaerobacter tengcongensis. Characterization of the Tte-UvrD helicase shows that it is stable and active from 45 to 65 degrees C. We have found that the Tte-UvrD helicase unwinds blunt-ended DNA duplexes as well as substrates possessing 3'- or 5'-ssDNA tails. Tte-UvrD was used to develop athermophilichelicase-dependent amplification (tHDA) system to selectively amplify target sequences at 60-65 degrees C. The tHDA system is more efficient than mHDA, displaying heightened amplification sensitivity without the need for the MutL and SSB accessory proteins. Amplification independent of MutL corresponds with studies demonstrating that maximal Tte-UvrD helicase activity does not require the mutL homolog. The tHDA system allows for rapid amplification and detection of targets present in genomic DNA. The expeditious nature and simplistic design of the tHDA platform makes the technology ideal for use in diagnostic applications requiring rapid identification of organisms at the point-of-need.

Adenosine Triphosphatases↗

PubMed2004

Multiplex mRNA assay using electrophoretic tags for high-throughput gene expression analysis.

We describe a novel multiplexing technology using a library of small fluorescent molecules, termed eTag molecules, to code and quantify mRNA targets. eTag molecules, which have the same fluorometric property, but distinct charge-to-mass ratios possess pre-defined electrophoretic characteristics and can be resolved using capillary electrophoresis. Coupled with primary Invader mRNA assay, eTag molecules were applied to simultaneously quantify up to 44 mRNA targets. This multiplexing approach was validated by examining a panel of inflammation responsive genes in human umbilical vein endothelial cells stimulated with inflammatory cytokine interleukin 1beta. The laser-induced fluorescence detection and electrokinetic sample injection process in capillary electrophoresis allows sensitive quantification of thousands of copies of mRNA molecules in a reaction. The assay is precise, as evaluated by measuring qualified Z' factor, a dimensionless and simple characteristic for applications in high-throughput screening using mRNA assays. Our data demonstrate the synergy between the multiplexing capability of eTag molecules by sensitive capillary electrophoresis detection and the isothermal linear amplification characteristics of the Invader assay. eTag multiplex mRNA assay presents a unique platform for sensitive, high sample throughput and multiplex gene expression analysis.

Cells, Cultured↗

PubMed2004

Detection and quantification of the red tide dinoflagellate Karenia brevis by real-time nucleic acid sequence-based amplification.

Nucleic acid sequence-based amplification (NASBA) is an isothermal method of RNA amplification that has been previously used in clinical diagnostic testing. A real-time NASBA assay has been developed for the detection of rbcL mRNA from the red tide dinoflagellate Karenia brevis. This assay is sensitive to one K. brevis cell and 1.0 fg of in vitro transcript, with occasional detection of lower concentrations of transcript. The assay did not detect rbcL mRNA from a wide range of nontarget organisms and environmental clones, while 10 strains (all tested) of K. brevis were detected. By the use of standard curves based on time to positivity, concentrations of K. brevis in environmental samples were predicted by NASBA and classified into different levels of blooms per the Florida Fish and Wildlife Conservation Commission (FWC) system. NASBA classification matched FWC classification (based on cell counts) 72% of the time. Those samples that did not match were off by only one class. NASBA is sensitive, rapid, and effective and may be used as an additional or alternative method to detect and quantify K. brevis in the marine environment.

Animals↗

PubMed1998

Evolving reaction-diffusion ecosystems with self-assembling structures in thin films.

Recently, new types of coupled isothermal polynucleotide amplification reactions for the investigation of in vitro evolution have been established that are based on the multi-enzyme 3SR reaction. Microstructured thin-film open bioreactors have been constructed in our laboratory to run these reactions spatially resolved in flow experiments. Artificial DNA/RNA chemistries close to the in vitro biochemistry of these systems have been developed, which we have studied in computer simulations in configurable hardware (NGEN). These artificial chemistries are described on the level of individual polynucleotide molecules, each with a defined sequence, and their complexes. The key feature of spatial pattern formation provides a weak stabilization of cooperative catalytic properties of the evolving molecules. Of great interest is the step to include extended self-assembly processes of flexible structures-allowing the additional stabilization of cooperation through semipermeable, flexible, self-organizing membrane boundaries. We show how programmable matter simulations of experimentally relevant molecular in vitro evolution can be extended to include the influence of self-assembling flexible membranes.

Bioreactors↗

PubMed2004

Invader assay for RNA quantitation.

The Invader assay is a homogeneous, isothermal, signal amplification system for the quantitative detection of nucleic acids. The assay can directly detect either DNA or RNA without target amplification or reverse transcription. It is based on the ability of Cleavase enzymes to recognize as a substrate and cleave a specific nucleic acid structure generated through the hybridization of two oligonucleotides to the target sequence. The combination of sequence-specific oligonucleotide hybridization and structure-specific enzymatic cleavage results in a highly specific assay well suited for discriminating closely related gene sequences. This includes detection of single nucleotide polymorphisms directly from genomic DNA as well as highly homologous mRNAs in closely related gene families. Because Cleavase substrate recognition is structure, and not sequence dependent, cleavage and detection can be applied to virtually any DNA or RNA sequence.

Base Sequence↗

PubMedDate not supplied

The use of dynamic size-sieving capillary electrophoresis and mismatch repair enzymes for mutant DNA analysis.

The detection of base pair mismatches in limiting amounts of DNA is important in the early diagnosis of cancer and other genetic diseases. The specific type and exact location of a bp mismatch in certain genes can yield information on the likelihood of a patient developing a genetic disease, as well as the severity of the disease. We demonstrate two methods of specific DNA point mutation detection and identification that involve the integration of mismatch repair cleavage enzyme analyses with dynamic size-sieving capillary electrophoresis. The mismatch repair cleavage enzymes employ the very mechanism that a cell uses in its own mismatch recognition and repair systems. One analysis employs an isothermal signal amplification process, and the other involves polymerase chain reaction (PCR) (Hoffmann-LaRoche, Nutley, NJ, U.S.A.) for amplification of the DNA. Separation of the DNA fragments using dynamic size-sieving CE yields a cleaved fragment, providing definitive evidence of a bp mismatch. The specificity and sensitivity of the assay are facilitated by the detection of fluorescently labeled DNA fragments using laser-induced fluorescence detection; picogram quantities of a target DNA can be analyzed reproducibly.

Base Pair Mismatch↗

PubMed2003

[Comparative evaluation of the isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN) and Roche Amplicor PCR and culture for detecting Mycobacterium tuberculosis complex in sputum samples].

We compared the ability of the newly developed ICAN MTB Detection Kit (TaKaRa Bio Inc.), which uses the Isothermal and Chimeric primer-initiated Amplification of Nucleic acid (ICAN), with that of COBAS Amplicor PCR System (Roche Diagnostics) to directly detect Mycobacterium tuberculosis complex (MTB) in sputum samples. A total of 142 sputum samples from 120 patients were examined in this study. The results were compared with those of acid-fast staining and MGIT liquid culture system (BD) following identification by the probe test (DDH Mycobacteria Kit). A total of 68 specimens were MGIT positive for MTB. In addition, 62 specimens were positive by the combination of staining and MGIT assay for MTB. When compared with that for MGIT, the sensitivity of each assay system was 88.2% for ICAN and 92.6% for COBAS Amplicor, respectively. The specificity of each assay system was 65.7% for ICAN and 62.7% for COBAS Amplicor, respectively. Coincidence between ICAN and COBAS Amplicor assay results was 96.3% (130 of 135 samples). No significant difference was observed between the results of the two assay methods. It is concluded that although both nucleic acid amplification methods are sensitive and specific for the detection of MTB in the respiratory specimens, ICAN system appeared to be more rapid (within 3.5 h from the specimen collection) than Amplicor system. The ICAN system will be useful in clinical laboratories for the rapid detection of MTB without specially programmed thermo-cycler.

Adult↗

PubMed2004

Strand displacement amplification: a versatile tool for molecular diagnostics.

Strand displacement amplification is an isothermal process that permits 10(10)-fold amplification of a DNA target sequence in as little as 15 min. In the form of the BD ProbeTec ET System, strand displacement amplification was the first nucleic acid amplification technology to be coupled with real-time homogeneous fluorescence-based detection for routine application in the clinical laboratory. The isothermal nature of the reaction process offers distinct advantages with regard to the cost and simplicity of instrumentation, while a universal detection format permits the use of the same fluorescent detector probes across multiple analytes. This has important potential in the field of genetic analysis, in which disease predisposition and therapeutic efficacy are frequently determined by multiple nucleic acid markers.

Base Sequence↗

PubMed2004

Multi-analyte single-membrane biosensor for the serotype-specific detection of Dengue virus.

A multi-analyte biosensor based on nucleic acid hybridization and liposome signal amplification was developed for the rapid serotype-specific detection of Dengue virus. After RNA amplification, detection of Dengue virus specific serotypes can be accomplished using a single analysis within 25 min. The multi-analyte biosensor is based on single-analyte assays (see Baeumner et al (2002) Anal Chem 74:1442-1448) developed earlier in which four analyses were required for specific serotype identification of Dengue virus samples. The multi-analyte biosensor employs generic and serotype-specific DNA probes, which hybridize with Dengue RNA that is amplified by the isothermal nucleic acid sequence based amplification (NASBA) reaction. The generic probe (reporter probe) is coupled to dye-entrapping liposomes and can hybridize to all four Dengue serotypes, while the serotype-specific probes (capture probes) are immobilized through biotin-streptavidin interaction on the surface of a polyethersulfone membrane strip in separate locations. A mixture of amplified Dengue virus RNA sequences and liposomes is applied to the membrane and allowed to migrate up along the test strip. After the liposome-target sequence complexes hybridize to the specific probes immobilized in the capture zones of the membrane strip, the Dengue serotype present in the sample can be determined. The amount of liposomes immobilized in the various capture zones directly correlates to the amount of viral RNA in the sample and can be quantified by a portable reflectometer. The specific arrangement of the capture zones and the use of unlabeled oligonucleotides (cold probes) enabled us to dramatically reduce the cross-reactivity of Dengue virus serotypes. Therefore, a single biosensor can be used to detect the exact Dengue serotype present in the sample. In addition, the biosensor can simultaneously detect two serotypes and so it is useful for the identification of possible concurrent infections found in clinical samples. The various biosensor components have been optimized with respect to specificity and sensitivity, and the system has been ultimately tested using blind coded samples. The biosensor demonstrated 92% reliability in Dengue serotype determination. Following isothermal amplification of the target sequences, the biosensor had a detection limit of 50 RNA molecules for serotype 2, 500 RNA molecules for serotypes 3 and 4, and 50,000 molecules for serotype 1. The multi-analyte biosensor is portable, inexpensive, and very easy to use and represents an alternative to current detection methods coupled with nucleic acid amplification reactions such as electrochemiluminescence, or those based on more expensive and time consuming methods such as ELISA or tissue culture.

Biosensing Techniques↗

PubMed1995

Modelling viral evolution in vitro using exo- Klenow polymerase: continuous selection of strand displacement amplified DNA that binds an oligodeoxynucleotide to form a triple-helix.

Evolution comprises cycles of amplification, mutagenesis and selection. To study evolutionary phenomena, isothermal strand displacement amplification (SDA) of double-stranded DNA as an in vitro model for rolling-circle replication of viruses has been coupled to a positive selection procedure. First, two subsequent amplification reactions utilizing exo- Klenow polymerase were performed under direct observation using the fluorescent dye thiazole orange. Under the chosen conditions, the mutation rate was 1.5 x 10(-3) and 0.4 x 10(-3) for base substitutions and deletions, respectively. Then, a 16mer oligodeoxynucleotide with an acridine moiety coupled to its 5' end was used to select for double strands that retained their ability to form a triple-helix with the oligodeoxynucleotide. Conditions for triple-helix formation were chosen such that only 10 to 40% of the SDA products were allowed to bind the third strand. Non-denaturing polyacrylamide gel electrophoresis was used to separate triple-helices from unmodified double strands, and only triplex strands were used to initiate a new round of error-prone amplification and selection. Nine such rounds with about 270 molecular generations were performed. The final mutant spectrum was characterized and compared with those of amplification reactions without additional selection pressure. While without selection pressure base substitutions and deletions throughout the initial wild-type rapidly produce a diverse mutant distribution, the consensus after nine selection rounds clearly shows two mutational hotspot positions. Using gel shift assays and a newly developed non-radioactive DNase I footprinting technique, it could be shown that both the initial wild-type and the final consensus do not differ significantly in their triplex formation ability. As opposed to this, they do show different amplification efficiencies. The final consensus sequence is amplified with the highest rate in the exponential reaction phase, while the most abundant clone, which is characterized by two additional point deletions, is the sequence with the highest amplification rate in the linear growth phase.

Base Sequence↗

PubMed2004

Real-time nucleic acid sequence-based amplification in nanoliter volumes.

Real-time nucleic acid sequence-based amplification (NASBA) is an isothermal method specifically designed for amplification of RNA. Fluorescent molecular beacon probes enable real-time monitoring of the amplification process. Successful identification, utilizing the real-time NASBA technology, was performed on a microchip with oligonucleotides at a concentration of 1.0 and 0.1 microM, in 10- and 50-nL reaction chambers, respectively. The microchip was developed in a silicon-glass structure. An instrument providing thermal control and an optical detection system was built for amplification readout. Experimental results demonstrate distinct amplification processes. Miniaturized real-time NASBA in microchips makes high-throughput diagnostics of bacteria, viruses, and cancer markers possible, at reduced cost and without contamination.

Humans↗

PubMed2002

Multiplexed protein profiling on microarrays by rolling-circle amplification.

Fluorescent-sandwich immunoassays on microarrays hold appeal for proteomics studies, because equipment and antibodies are readily available, and assays are simple, scalable, and reproducible. The achievement of adequate sensitivity and specificity, however, requires a general method of immunoassay amplification. We describe coupling of isothermal rolling-circle amplification (RCA) to universal antibodies for this purpose. A total of 75 cytokines were measured simultaneously on glass arrays with signal amplification by RCA with high specificity, femtomolar sensitivity, 3 log quantitative range, and economy of sample consumption. A 51-feature RCA cytokine glass array was used to measure secretion from human dendritic cells (DCs) induced by lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-alpha). As expected, LPS induced rapid secretion of inflammatory cytokines such as macrophage inflammatory protein (MIP)-1beta, interleukin (IL)-8, and interferon-inducible protein (IP)-10. We found that eotaxin-2 and I-309 were induced by LPS; in addition, macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC), soluble interleukin 6 receptor (sIL-6R), and soluble tumor necrosis factor receptor I (sTNF-RI) were induced by TNF-alpha treatment. Because microarrays can accommodate approximately 1,000 sandwich immunoassays of this type, a relatively small number of RCA microarrays seem to offer a tractable approach for proteomic surveys.

Antibodies↗

PubMed2002

Characteristics and applications of nucleic acid sequence-based amplification (NASBA).

Nucleic acid sequence-based amplification (NASBA) is a sensitive, isothermal, transcription-based amplification system specifically designed for the detection of RNA targets. In some NASBA systems, DNA is also amplified though very inefficiently and only in the absence of the corresponding RNA target or in case of an excess (>1,000-fold) of target DNA over RNA. As NASBA is primer-dependent and amplicon detection is based on probe binding, primer and probe design rules are included. An overview of various target nucleic acids that have been amplified successfully using NASBA is presented. For the isolation of nucleic acids prior to NASBA, the "Boom" method, based on the denaturing properties of guanidine isothiocyanate and binding of nucleic acid to silica particles, is preferred. Currently, electro-chemiluminescence (ECL) is recommended for the detection of the amplicon at the end of amplification. In the near future, molecular beacons will be introduced enabling "real-time detection," i.e., amplicon detection during amplification. Quantitative HIV-1 NASBA and detection of up to 48 samples can then be performed in only 90 min.

DNA↗