Identification of mycobacteria through conventional microbiological methods is cumbersome and time-consuming. Recently we have developed a novel bacterial identification method to accurately and rapidly identify different mycobacteria directly from water and clinical isolates. The method utilizes the PCR to amplify a portion of the small subunit rRNA from mycobacteria. The 5' PCR primer has a fluorescent label to allow detection of the amplified product. The PCR product is digested with restriction endonucleases, and an automated DNA sequencer is employed to determine the size of the labeled restriction fragments. Since the PCR product is labeled only at the 5' end, the analysis identifies only the restriction fragment proximal to the 5' end. Each mycobacterial species has a unique 5' restriction fragment length for each specific endonuclease. However, frequently the 5' restriction fragments from different species have similar or identical lengths for a given endonuclease. A set of judiciously chosen restriction enzymes produces a unique set of fragments for each species, providing us with an identification signature. Using this method, we produced a library of 5' restriction fragment sizes corresponding to different clinically important mycobacteria. We have characterized mycobacterial isolates which had been previously identified by biochemical test and/or nucleic acid probes. An analysis of these data demonstrates that this protocol is effective in identifying 13 different mycobacterial species accurately. This protocol has the potential of rapidly (less than 36 h) identifying mycobacterial species directly from clinical specimens. In addition, this protocol is accurate, sensitive, and capable of identifying multiple organisms in a single sample.
Analysis of restriction fragment length polymorphism of bacterial small-subunit (SSU) rRNA sequences represents a potential means for characterizing complex bacterial populations such as those found in natural environments. In order to estimate the resolution potential of this approach, we have examined the SSU rRNA sequences in the Ribosomal Database Project bank using a computer algorithm which simulates hybridization between DNA sequences. Simulated hybridizations between a primer or probe sequence and an SSU rRNA sequence yield a value for each potential hybridization. This algorithm has been used to evaluate sites for PCR primers and hybridization probes used for classifying SSU rRNA sequences. Our analysis indicates that length variation in terminal restriction fragments of PCR products from the SSU rRNA sequences can identify a wide spectrum of bacteria. We also observe that the majority of restriction fragment length variation is the result of insertions and deletions rather than restriction site polymorphisms. This approach is also used to evaluate the relative efficiency and specificity of a number of published hybridization probes.
The advent of multiplexed bead assays in recent years has introduced a new dimension of testing for complex diseases such as lupus, which can involve multiple autoantibodies. The ability to rapidly identify multiple autoantibodies, with high sensitivity and specificity in an automated fashion, is highly attractive. The aim of this study was to assess the performance and clinical value of multiplexed bead-based (AtheNA Multi-Lyte ANA-II test system) immunoassays both by comparing the results with those achieved by indirect fluorescent-antibody assay (IFA) or conventional enzyme immunoassays (EIAs) and by independent identification of autoantibodies in well-characterized samples. To achieve this goal, 984 samples were tested for seven analytes ( Detection of antinuclear antibodies (ANAs) has a significant role in diagnosis and prognosis for clinically indicated patients with a variety of autoimmune vascular diseases. Traditionally, the "gold standard" test for detection of ANAs has been the indirect fluorescent-antibody assay (IFA). The advent of using the human HEp-2 cell line for detection of ANAs in the past 20 years has provided sensitivity and brought more standardization and therefore acceptance of this test globally (11). It provided superior resolution for detection of different staining patterns that was not available before. The increased sensitivity also brought forth a more reliable use of titer cutoffs for determining positive results. Though reliable, ANA testing by IFA has had its share of problems and criticism over the years. The test has been deemed "subjective" and highly dependent on the competence of the technician reading the slides (10). IFA testing is also an issue for high-volume laboratories performing ANA screens routinely. To circumvent these problems, researchers have evaluated ANA screening methods using enzyme immunoassays (EIAs) that are usually prepared from HEp-2 cells. A few studies have shown comparable sensitivity and specificity with IFA testing (6, 7). The advocates of EIA-ANA testing herald the objectivity of the results and the ability to automate and run multiple samples reliably (1). Most critics, though, cite issues with sensitivity (either too sensitive, resulting in a high number of false positives, or the opposite, resulting in false negatives) (2), the range of specificity (number of different extractable nuclear antigens [ENAs] detected), and the lack of the ability to detect different patterns available by IFA. There have been suggestions that these shortcomings can be overcome by testing each sample with EIAs specific for testing single ENAs. This idea, although theoretically sound, defeats the purpose of ANA testing by EIA, since it considerably increases cost and time to result, not to mention erroneous diagnosis based on a single test result.Advances in technology have recently provided a new methodology for ANA testing (8,12,14). Fluorescent bead-based flow cytometry, pioneered by Luminex Inc., has allowed different manufacturers to produce kits capable of det...
The detection of anti-telomere antibody appears to be more sensitive and may be as specific as anti-dsDNA (Farr) in SLE. The detection of telomeric repeats may be as accurate as other anti-DNA assay methodologies and more specific for the presence of SLE. The immunogenic potential of telomere biology related to the pathogenesis and/or diagnosis of SLE deserves further investigation.
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.