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.
Sera from patients with connective tissue diseases often contain antibodies against snRNA‐associated proteins. Using one of these sera in an immunological screening of a human lambda gt11 expression vector cDNA library, two cDNA clones for the U1 snRNP‐specific A protein, termed lambda HA‐1 and lambda HA‐2, were isolated. Monospecific antibodies, eluted from the beta‐galactosidase fusion protein of either clone reacted with the U1 snRNP‐specific A antigen. The identity of the clones was confirmed by in vitro translation of hybrid selected mRNA. RNA blot analysis revealed a single polyadenylated transcript of about 1.4 kb in human cells. A cDNA of 1.2 kb, isolated from the same lambda gt11 expression library by cross‐hybridization with a lambda HA‐2 restriction fragment, covered the complete coding sequence of the A protein as demonstrated by in vitro translation of an RNA transcript synthesized from this cDNA. The deduced amino acid sequence contains one very hydrophilic region, and internal sequence duplication and a region highly homologous to the RNP consensus sequence that seems to be common to RNA binding proteins. Sequence comparison with the recently cloned U2 snRNP‐specific B′ protein revealed two extremely homologous regions located in the carboxy‐terminal (homology of 86%) and amino‐terminal part (homology of 77%) of the proteins. This structural relationship indicates that proteins A and B′, although located in different snRNP particles, may have identical functions.
Determination of the number of malaria parasites by routine or even expert microscopy is not always sufficiently sensitive for detailed quantitative studies on the population dynamics of Plasmodium falciparum, such as intervention or vaccine trials. To circumvent this problem, two more sensitive assays, real-time quantitative nucleic acid sequence-based amplification (QT-NASBA) and real-time quantitative PCR (QT-PCR) were compared for quantification of P. falciparum parasites. QT-NASBA was adapted to molecular beacon real-time detection technology, which enables a reduction of the time of analysis and of contamination risk while retaining the specificity and sensitivity of the original assay. Both QT-NASBA and QT-PCR have a sensitivity of 20 parasites/ml of blood, but QT-PCR requires a complicated DNA extraction procedure and the use of 500 l of venous blood to achieve this sensitivity, compared to 50 l of finger prick blood for real-time QT-NASBA. Both techniques show a significant correlation to microscopic parasite counts, and the quantification results of the two real-time assays are significantly correlated for in vitro as well as in vivo samples. However, in comparison to real-time QT-PCR, the results of real-time QT-NASBA can be obtained 12 h earlier, with relatively easy RNA extraction and use of finger prick blood samples. The prospective development of multiplex QT-NASBA for detection of various P. falciparum developmental stages increases the value of QT-NASBA for malaria studies. Therefore, for studies requiring sensitive and accurate detection of P. falciparum parasites in large numbers of samples, the use of real-time QT-NASBA is preferred over that of real-time QT-PCR.Routine clinical diagnosis of malaria is usually based on microscopic detection of Plasmodium parasites in blood smears. However, this technique is relatively laborious when large numbers of samples need to be quantified simultaneously. Furthermore, the detection limit of microscopy, 1 to 20 parasites per l of blood, may not always be sufficiently sensitive. Parasite densities below the detection level of microscopy may play an important role in Plasmodium population dynamics and the epidemiology of the disease; therefore, the availability and use of more-sensitive detection techniques is a prerequisite for many research projects. With the rapid developments in the field of molecular biology, several nucleic acid-based amplification methods, including PCR, reverse transcriptase PCR, and nucleic acid sequence-based amplification (NASBA) (2, 3, 7-9, 11), are now available for detection of Plasmodium parasites. Because many studies require accurate and sensitive quantification of parasites, most of these techniques have been adapted for quantitative analysis (1,3,6,7,11). In preparation for vaccine trials and epidemiological studies directed at evaluation of interventions, the present study was designed to evaluate the newly developed real-time quantitative NASBA (QT-NASBA) and to compare the assay to another quantitative nucleic acidba...
Mycoplasma pneumoniae is a common cause of community-acquired pneumonia and lower-respiratory-tract infections. Diagnosis has traditionally been obtained by serological diagnosis, but increasingly, molecular techniques have been applied. However, the number of studies actually comparing these assays is limited. The development of a novel duplex real-time PCR assay for detection of M. pneumoniae in the presence of an internal control real-time PCR is described. In addition, real-time nucleic acid sequence-based amplification (NASBA) on an iCycler apparatus is evaluated. These assays were compared to serology and a conventional PCR assay for 106 clinical samples from patients with lower-respiratory-tract infection. Of the 106 samples, 12 (11.3%) were positive by all the molecular methods whereas serology with acute sample and convalescent samples detected 6 (5.6%) and 9 (8.5%), respectively. Clinical symptoms of the patients with Mycoplasmapositive results were compared to those of the other patients with lower-respiratory-tract infections, and it was found that the results for mean lower age numbers as well as the presence of chills, increased erythrocyte sedimentation rate, and raised C-reactive protein levels showed significant differences. Molecular methods are superior for diagnosis of M. pneumoniae, providing more timely diagnosis. In addition, using real-time methods involves less hands-on time and affords the ability to monitor the reaction in the same tube.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.