Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Highly sensitive detection of gene expression of an intronless gene: amplification of mRNA, but not genomic DNA by nucleic acid sequence based amplification (NASBA).

ABSTRACT: NASBA is an isothermal nucleic acid amplification reaction that amplifies mRNA in a dsDNA background. Although similar to the sensitive reverse transcription/polymerase chain reaction (RT-PCR) in mRNA detection, NASBA is not prone to give false positive results caused by genomic dsDNA. Therefore, NASBA is unique for sensitive detection of transcription of intronless genes, which preclude strategies such as intron spanning primer pairs to control false positive results in RT-PCR. Using NASBA, mRNA of the intronless human interferon-beta gene was demonstrated with a sensitivity of 10 copies, whereas 100 ng genomic DNA gave a negative result.

SUBMITTER: Heim A

PROVIDER: S-EPMC147515 | biostudies-other | 1998 May

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Publications

Highly sensitive detection of gene expression of an intronless gene: amplification of mRNA, but not genomic DNA by nucleic acid sequence based amplification (NASBA).

Heim A A Grumbach I M IM Zeuke S S Top B B

Nucleic acids research 19980501 9

NASBA is an isothermal nucleic acid amplification reaction that amplifies mRNA in a dsDNA background. Although similar to the sensitive reverse transcription/polymerase chain reaction (RT-PCR) in mRNA detection, NASBA is not prone to give false positive results caused by genomic dsDNA. Therefore, NASBA is unique for sensitive detection of transcription of intronless genes, which preclude strategies such as intron spanning primer pairs to control false positive results in RT-PCR. Using NASBA, mRN ...[more]

PMID: 9547289

Similar Datasets

Sensitive isothermal detection of nucleic-acid sequence by primer generation-rolling circle amplification.

Project description:A simple isothermal nucleic-acid amplification reaction, primer generation-rolling circle amplification (PG-RCA), was developed to detect specific nucleic-acid sequences of sample DNA. This amplification method is achievable at a constant temperature (e.g. 60 degrees C) simply by mixing circular single-stranded DNA probe, DNA polymerase and nicking enzyme. Unlike conventional nucleic-acid amplification reactions such as polymerase chain reaction (PCR), this reaction does not require exogenous primers, which often cause primer dimerization or non-specific amplification. Instead, 'primers' are generated and accumulated during the reaction. The circular probe carries only two sequences: (i) a hybridization sequence to the sample DNA and (ii) a recognition sequence of the nicking enzyme. In PG-RCA, the circular probe first hybridizes with the sample DNA, and then a cascade reaction of linear rolling circle amplification and nicking reactions takes place. In contrast with conventional linear rolling circle amplification, the signal amplification is in an exponential mode since many copies of 'primers' are successively produced by multiple nicking reactions. Under the optimized condition, we obtained a remarkable sensitivity of 84.5 ymol (50.7 molecules) of synthetic sample DNA and 0.163 pg (approximately 60 molecules) of genomic DNA from Listeria monocytogenes, indicating strong applicability of PG-RCA to various molecular diagnostic assays.

| S-EPMC2647323 | biostudies-literature

Rapid and Sensitive Isothermal Detection of Nucleic-acid Sequence by Multiple Cross Displacement Amplification.

Project description:We have devised a novel amplification strategy based on isothermal strand-displacement polymerization reaction, which was termed multiple cross displacement amplification (MCDA). The approach employed a set of ten specially designed primers spanning ten distinct regions of target sequence and was preceded at a constant temperature (61-65 °C). At the assay temperature, the double-stranded DNAs were at dynamic reaction environment of primer-template hybrid, thus the high concentration of primers annealed to the template strands without a denaturing step to initiate the synthesis. For the subsequent isothermal amplification step, a series of primer binding and extension events yielded several single-stranded DNAs and single-stranded single stem-loop DNA structures. Then, these DNA products enabled the strand-displacement reaction to enter into the exponential amplification. Three mainstream methods, including colorimetric indicators, agarose gel electrophoresis and real-time turbidity, were selected for monitoring the MCDA reaction. Moreover, the practical application of the MCDA assay was successfully evaluated by detecting the target pathogen nucleic acid in pork samples, which offered advantages on quick results, modest equipment requirements, easiness in operation, and high specificity and sensitivity. Here we expounded the basic MCDA mechanism and also provided details on an alternative (Single-MCDA assay, S-MCDA) to MCDA technique.

| S-EPMC4648395 | biostudies-other

A Sensitive Potentiometric Sensor for Isothermal Amplification-Coupled Detection of Nucleic Acids.

Project description:A disposable potentiometric sensor was newly developed for the amplification-coupled detection of nucleic acids. The hydrogen-ion is generally released during isothermal amplification of nucleic acids. The surface potential on the oxide-functionalized electrode of the extended gate was directly measured using full electrical circuits with the commercial metal-oxide semiconductor field-effect transistors (MOSFETs) and ring oscillator components, which resulted in cost-effective, portable and scalable real-time nucleic acid analysis. The current-starved ring oscillator changes surface potential to its frequency depending on the square of the variation in pH with a high signal-to-noise ratio during isothermal amplification. The device achieves a conversion rate of 20.5 kHz/mV and a detection resolution of 200 µV for the surface potential. It is demonstrated that the sensor successfully monitors in real-time isothermal amplification of the extracted nucleic acids from Salmonella pathogenic bacteria. The in situ variations in the frequency of the pH-sensitive sensor were compared with the results of both a conventional optical device and pH-meter during isothermal amplification.

| S-EPMC6068556 | biostudies-literature

Highly sensitive and portable mRNA detection platform for early cancer detection.

Project description:Pancreatic cancer, at unresectable advanced stages, presents poor prognoses, which could be prevented by early pancreatic cancer diagnosis methods. Recently, a promising early-stage pancreatic cancer biomarker, extracellular vesicles (EVs) related glypican-1 (GPC1) mRNA, is found to overexpress in pancreatic cancer cells. Current mRNA detection methods usually require expensive machinery, strict preservation environments, and time-consuming processes to guarantee detection sensitivity, specificity, and stability. Herein, we propose a novel two-step amplification method (CHAGE) via the target triggered Catalytic Hairpin Assembly strategy combined with Gold-Enhanced point-of-care-testing (POCT) technology for sensitive visual detection of pancreatic cancer biomarker. First, utilizing the catalyzed hairpin DNA circuit, low expression of the GPC1 mRNA was changed into amplification product 1 (AP1, a DNA duplex) as the next detection targets of the paper strips. Second, the AP1 was loaded onto a lateral flow assay and captured with the gold signal nanoparticles to visualize results. Finally, the detected results can be further enhanced by depositing gold to re-enlarge the sizes of gold nanoparticles in detection zones. As a result, the CHAGE methodology lowers the detection limit of mRNA to 100 fM and provides results within 2 h at 37 °C. Furthermore, we demonstrate the successful application in discriminating pancreatic cancer cells by analyzing EVs' GPC1 mRNA expression levels. Hence, the CHAGE methodology proposed here provides a rapid and convenient POCT platform for sensitive detection of mRNAs through unique probes designs (COVID, HPV, etc.).

| S-EPMC8474757 | biostudies-literature

Multiplex, Rapid, and Sensitive Isothermal Detection of Nucleic-Acid Sequence by Endonuclease Restriction-Mediated Real-Time Multiple Cross Displacement Amplification.

Project description:We have devised a novel isothermal amplification technology, termed endonuclease restriction-mediated real-time multiple cross displacement amplification (ET-MCDA), which facilitated multiplex, rapid, specific and sensitive detection of nucleic-acid sequences at a constant temperature. The ET-MCDA integrated multiple cross displacement amplification strategy, restriction endonuclease cleavage and real-time fluorescence detection technique. In the ET-MCDA system, the functional cross primer E-CP1 or E-CP2 was constructed by adding a short sequence at the 5' end of CP1 or CP2, respectively, and the new E-CP1 or E-CP2 primer was labeled at the 5' end with a fluorophore and in the middle with a dark quencher. The restriction endonuclease Nb.BsrDI specifically recognized the short sequence and digested the newly synthesized double-stranded terminal sequences (5' end short sequences and their complementary sequences), which released the quenching, resulting on a gain of fluorescence signal. Thus, the ET-MCDA allowed real-time detection of single or multiple targets in only a single reaction, and the positive results were observed in as short as 12 min, detecting down to 3.125 fg of genomic DNA per tube. Moreover, the analytical specificity and the practical application of the ET-MCDA were also successfully evaluated in this study. Here, we provided the details on the novel ET-MCDA technique and expounded the basic ET-MCDA amplification mechanism.

| S-EPMC4870240 | biostudies-literature

Highly Sensitive Loop-Mediated Isothermal Amplification for the Detection of Leptospira.

Project description:Leptospirosis is a worldwide zoonosis caused by an infection with the pathogenic species of Leptospira. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the DNA of Leptospira spp. Six sets of primers targeting the gene of the subsurface protein, lipL32, were evaluated for their detection sensitivity. The best primer set detected less than 25 copies of lipL32 per reaction of both plasmid DNA template and purified leptospiral genomic DNA. By combining primers targeting lipL32 with the previously published primer set targeting lipL41, the sensitivity of the assay was improved to 12 copies of L. interrogans. The specificity of the LAMP assay was evaluated by using the genomic DNA from other clinically encountered bacterial species such as different strains of Orientia tsutsugamushi, Rickettsia typhi, Rickettsia conorii, Rickettsia rickettsii, Coxiella burnetii, and Bartonella bacilliformis. These genomic DNA samples were all negative in our LAMP assay. The sensitivity of the LAMP assay was very similar to that of quantitative real time PCR. Several detection methods for the amplified product of LAMP assay were performed to demonstrate the simplicity of the assay. In summary, our results have suggested that this assay is rapid, robust, and easy to perform and has the potential to be used in endemic locations.

| S-EPMC4745460 | biostudies-literature

Detection of dengue viral RNA using a nucleic acid sequence-based amplification assay.

Project description:Faster techniques are needed for the early diagnosis of dengue fever and dengue hemorrhagic fever during the acute viremic phase of infection. An isothermal nucleic acid sequence-based amplification (NASBA) assay was optimized to amplify viral RNA of all four dengue virus serotypes by a set of universal primers and to type the amplified products by serotype-specific capture probes. The NASBA assay involved the use of silica to extract viral nucleic acid, which was amplified without thermocycling. The amplified product was detected by a probe-hybridization method that utilized electrochemiluminescence. Using normal human plasma spiked with dengue viruses, the NASBA assay had a detection threshold of 1 to 10 PFU/ml. The sensitivity and specificity of the assay were determined by testing 67 dengue virus-positive and 21 dengue virus-negative human serum or plasma samples. The "gold standard" used for comparison and evaluation was the mosquito C6/36 cell culture assay followed by an immunofluorescent assay. Viral infectivity titers in test samples were also determined by a direct plaque assay in Vero cells. The NASBA assay was able to detect dengue viral RNA in the clinical samples at plaque titers below 25 PFU/ml (the detection limit of the plaque assay). Of the 67 samples found positive by the C6/36 assay, 66 were found positive by the NASBA assay, for a sensitivity of 98.5%. The NASBA assay had a specificity of 100% based on the negative test results for the 21 normal human serum or plasma samples. These results indicate that the NASBA assay is a promising assay for the early diagnosis of dengue infections.

| S-EPMC88241 | biostudies-literature

Nucleoside analogs to manage sequence divergence in nucleic acid amplification and SNP detection.

Project description:Described here are the synthesis, enzymology and some applications of a purine nucleoside analog (H) designed to have two tautomeric forms, one complementary to thymidine (T), the other complementary to cytidine (C). The performance of H is compared by various metrics to performances of other 'biversal' analogs that similarly rely on tautomerism to complement both pyrimidines. These include (i) the thermodynamic stability of duplexes that pair these biversals with various standard nucleotides, (ii) the ability of the biversals to support polymerase chain reaction (PCR), (iii) the ability of primers containing biversals to equally amplify targets having polymorphisms in the primer binding site, and (iv) the ability of ligation-based assays to exploit the biversals to detect medically relevant single nucleotide polymorphisms (SNPs) in sequences flanked by medically irrelevant polymorphisms. One advantage of H over the widely used inosine 'universal base' and 'mixed sequence' probes is seen in ligation-based assays to detect SNPs. The need to detect medically relevant SNPs within ambiguous sequences is especially important when probing RNA viruses, which rapidly mutate to create drug resistance, but also suffer neutral drift, the second obstructing simple methods to detect the first. Thus, H is being developed to detect variants of viruses that are rapidly mutating.

| S-EPMC6159519 | biostudies-literature

Rapid and sensitive detection of norovirus genomes in oysters by a two-step isothermal amplification assay system combining nucleic acid sequence-based amplification and reverse transcription-loop-mediated isothermal amplification assays.

Project description:We developed a two-step isothermal amplification assay system, which achieved the detection of norovirus (NoV) genomes in oysters with a sensitivity similar to that of reverse transcription-seminested PCR. The time taken for the amplification of NoV genomes from RNA extracts was shortened to about 3 h.

| S-EPMC2446545 | biostudies-literature

Highly sensitive disposable nucleic acid biosensors for direct bioelectronic detection in raw biological samples.

Project description:The development of rapid, low-cost and reliable diagnostic methods is crucial for the identification and treatment of many diseases. Screen-printed gold electrodes (Au/SPEs), coated with a ternary monolayer interface, involving hexanedithiol (HDT), a specific thiolated capture probe (SHCP), and 6-mercapto-1 hexanol (MCH) (SHCP/HDT/MCH) are shown here to offer direct and sensitive detection of nucleic acid hybridization events in untreated raw biological samples (serum, urine and crude bacterial lysate solutions). The composition of the ternary monolayer was modified and tailored to the surface of the Au/SPE. The resulting SHCP/HDT/MCH monolayer has demonstrated to be extremely useful for enhancing the performance of disposable nucleic acid sensors based on screen-printed electrodes. Compared to common SHCP/MCH binary interfaces, the new ternary self-assembled monolayer (SAM) resulted in a 10-fold improvement in the signal (S)-to-noise (N) ratio (S/N) for 1 nM target DNA. The SHCP/HDT/MCH-modified Au/SPEs allowed the direct quantification of the target DNA down to 25 pM (0.25 fmol) and 100 pM (1 fmol) in undiluted/untreated serum and urine samples, respectively, and of 16S rRNA Escherichia coli (E. coli) corresponding to 3000 CFU ?L(-1) in raw cell lysate samples. The new SAM-coated screen-printed electrodes also displayed favorable non-fouling properties after a 24h exposure to raw human serum and urine samples, offering great promise as cost-effective nucleic acid sensors for a wide range of decentralized genetic tests.

| S-EPMC4386838 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data