Simultaneous Detection of Multiple Nucleic Acid Targets in a Homogeneous Format

Abstract

The acridinium ester 4-(2-succinimidyloxycarbonylethyl)phenyl-10-methylacridinium 9-carboxylate trifluoromethane sulfonate (AE), which reacts rapidly with alkaline hydrogen peroxide to produce light, has been used as a detection label in a number of assay procedures, including nucleic acid probe-based systems [Nelson et al. (1995) in Nonisotopic Probing, Blotting and Sequencing (Kricka, L. J., Ed.) pp 391-428, Academic Press, Inc., San Diego, CA]. We have synthesized a number of derivatives of this AE and characterized their chemiluminescent properties. These derivatives display significant differences in the kinetics of the chemiluminescence reaction as well as optimal pH for light production. These differences allow two or more derivatives to be simultaneously detected and quantitated in a single reaction vessel. Several of these derivatives have been covalently linked to nucleic acid probe molecules and have been further characterized in regard to chemiluminescence properties as well as hydrolysis of the ester bond in both single- and double-stranded conformations. On the basis of these properties, homogeneous assay formats utilizing DNA probes labeled with various AE derivatives were developed. Simultaneous detection and quantitation of Chlamydia trachomatis and Neisseria gonorrhoeae, the gag and pol regions of HIV, and wild-type and mutant HIV sequences was achieved with high sensitivity and discrimination.

Sensitivity of the Procleix HIV-1/HCV Assay for Detection of Human Immunodeficiency Virus Type 1 and Hepatitis C Virus RNA in a High-Risk Population

“…Accordingly, to estimate the Procleix assay clinical sensitivity, the assay was used to test prospectively collected plasma samples from a population at high risk for HIV-1 and HCV infection. The principal risk factors in this population, including intravenous drug use, male homosexual activity, multiple sexual partners, and sex with high-risk partners, were comparable to those described previously in studies of the epidemiology of HIV-1 and HCV infection (4,5,6,7,10). A total of 72.6% of the study subjects were intravenous drug users.…”

Significant Closure of the Human Immunodeficiency Virus Type 1 and Hepatitis C Virus Preseroconversion Detection Windows with a Transcription-Mediated-Amplification-Driven Assay

“…The IC signal in each reaction can be discriminated from the HIV-1 and/or HCV signal by the differential kinetics of light emission from probes with different acridinium ester labels (12). The IC amplicon is detected by using a probe with rapid emission of light, while the amplicons specific to HIV-1 and HCV are detected by using probes with slower kinetics of light emission.…”

Stains, labels and detection strategies for nucleic acids assays

“…Chlamydia trachomatis and Neisseria gonorrhoeae) in the same assay tube. 118 The photoprotein apoaequorin undergoes a calcium ion triggered reaction in the presence of coelenterazine that produces a £ash of blue light (maximum 469 nm). Recombinant apoaequorin (detection limit 51amol) labels are mainly used coupled to streptavidin or to anti-hapten antibodies (e.g.…”