Nonisotopic Hybridization Assay Research Articles

Colorimetric Detection of the Tuberculosis Complex Using Cycling Probe Technology and Hybridization in Microplates

Cycling probe technology (CPT) is a simple signal amplification method for the detection of specific target DNA sequences. CPT uses a chimeric DNA-RNA-DNA probe that is cut by RNase H when bound to its complementary target sequence. In this study, a hybridization assay was developed to detect biotinylated CPT products that result from the amplification of a Mycobacterium tuberculosis complex sequence. The chimeric probe was specifically designed to avoid the formation of secondary structures. The chosen capture probe was perfectly complementary to and was the same size as OL2, one of the two CPT products. The assay was based on the observation that a long sequence, such as the initial probe, was destabilized when bound to a small capture probe as a result of steric hindrance. The capture probe preferentially bound OL2 rather than the long initial probe. We added a prehybridization step with a helper DNA to enhance this discrimination between the two sequences. Colorimetric detection was performed using a peroxidase-streptavidin conjugate. After optimization, the non-isotopic hybridization assay allowed the detection of around 10 amol of target DNA. Besides being faster and easier to perform, this detection method was compared to electrophoresis separation and gave similar results.

Detection of Chlamydia trachomatis in urethral and urine samples from symptomatic and asymptomatic male patients by the polymerase chain reaction.

To evaluate the commercially available polymerase chain reaction (PCR) assay Amplicor (Roche Molecular Systems, USA) for diagnosis of Chlamydia trachomatis infection, urethral and urine swabs from a total of 344 male patients were tested and the results compared with those obtained by the nonisotopic hybridization assay Pace 2 (Gen Probe, USA) for urethral samples and by the enzyme immunoassay EIA MicroTrak (Syva, USA) for urine. Discrepant results were analyzed by a repeated test run using a major outer membrane protein-derived primer PCR, by the probe competition assay, and by the direct immunofluorescence test (DIF). Thirty-nine men (11.3%) were chlamydia positive, based on the results of all tests from both sampling sites. The rate of detection of chlamydia in urethral specimens by Amplicor and the Pace 2 was 79.5% and 61.5%, respectively, while the rate of detection in urine sediment was 75% for both Amplicor and EIA. In the first run of the PCR, a high number of false-negative results for unfrozen samples was observed, decreasing the sensitivity of Amplicor in urine to 47.3%. The results of the study indicate that Amplicor detects more infected individuals compared with other tests and is suitable as an alternative diagnostic test for chlamydia infections, using not only urethral specimens but also urine specimens. However, the finding of false-negative results when using Amplicor on unfrozen samples must be further investigated.

Nonisotopic hybridization assay for determination of relative amounts of genotypic human immunodeficiency virus type 1 zidovudine resistance.

A nonisotopic hybridization assay for human immunodeficiency virus genotypic zidovudine resistance determination is described. Biotinylated PCR product was hybridized with enzyme-labeled probes for wild-type or resistant mutant sequences and detected colorimetrically or chemiluminescently in a microplate format. Changes in mutant-to-wild-type ratios allow for the monitoring of longitudinal patient samples.

Novel, ultrasensitive, Q-beta replicase-amplified hybridization assay for detection of Chlamydia trachomatis

A sensitive, nonisotopic hybridization assay termed "dual capture" is described. The assay rapidly and specifically detects very low levels of target nucleic acids and organisms. The assay is based on the principles of sandwich hybridization, reversible target capture, and Q-Beta replicase amplification. The assay can be completed in less than 4 h, and in the described model format, it detects Chlamydia trachomatis rRNA or rDNA. Up to 96 samples can be analyzed simultaneously. The assay employs two types of probes: a test-specific capture probe, which mediates the cycling of the target probe complex on and off derivatized magnetic beads, and a replicatable RNA detector molecule containing a sequence complementary to and adjacent to the capture probe site on the target. Following reversible target capture, detection of the signal is accomplished by replication of the detector molecule by Q-Beta replicase in the presence of propidium iodide. A specific assay signal can be detected from as few as 1,000 molecules above the background. In a limited study of 94 urogenital samples the assay detected five of the six culture-positive samples and did not detect the C. trachomatis target in 85 of the 88 culture-negative samples.

Diagnosis of viral hepatitis with a nonisotopic hybridization assay

The DNA enzyme immunoassay is an efficient method for the screening of PCR products derived from different hepatitis virus genomes, and allows to bypass both agarose gel electrophoresis and Southern blot hybridization with radioactively labeled probes. A wider application of this method will disclose new perspectives for the introduction of PCR in clinical laboratories.

Solution hybridization and enzyme immunoassay for biotinylated DNA-RNA hybrids to detect enteroviral RNA in cell culture

A non-isotopic hybridization assay is described for detection of enteroviral RNA in cell culture. Two biotin-labelled cDNA probes, corresponding to 1 kb from the 5′ end and 3·5 kb from the 3′ end of the coxsackievirus B3 genome, were hybridized in solution with protease and detergent-treated cell culture suspensions. Labelled DNA-RNA hybrids were captured on microtiter plates coated with anti-biotin antibody and bound hybrids were measured with a β-galactosidase-labelled monoclonal antibody specific for DNA-RNA hybrids. Coxsackie B3 was detected at a concentration of 500 pfu ml −1. The limit of detection for other enteroviruses ranged from 10 3·3 to 10 5·8 pfu ml −1. The enteroviruses that could be detected included coxsackie B1 and 3, coxsackie A1-6 and 15, poliovirus types 1–3, and enteroviruses 7, 11, and 71. ECHO 22 was the only enterovirus, of those that were tested, that could not be detected. The solution hybridization reaction and enzyme immunoassay for DNA-RNA hybrids does not require the use of radiolabelled probes or extraction of RNA with phenol. The assay yields a quantitative endpoint, which avoids the subjectivity inherent in membrane-based methods. These features would make the assay more adaptable to clinical laboratories than other formats which have been devised for measurement of viral RNA.

Immunodetection of DNA with biotinylated RNA probes: A study of reactivity of a monoclonal antibody to DNA-RNA hybrids

A quantitative, nonisotopic hybridization assay which measures specific DNA-RNA hybrids is described. A biotinylated RNA probe is reacted in solution with a DNA target and the labeled hybrids are immobilized onto a solid phase surface with an antibiotin antibody. Bound hybrids are detected with a β-galactosidase-labeled monoclonal antibody against DNA-RNA hybrids and are quantitated with the addition of a fluorogenic substrate. In a model system using pSP65 or pGEM4 plasmids and transcripts, biotinylated RNA probes allowed detection of 5 pg of DNA in 10 6 pg of exogenous nucleic acids in 1000 min. Signals generated in the system depended on input target length. A nucleic acid target of 25 bases was still detectable in the assay. Human immunodeficiency virus type 1 (HIV-1) DNA was amplified in the polymerase chain reaction with Taq polymerase and a set of primers for the pol gene, one of which contained T7 RNA polymerase promoter sequences. A HIV-RNA probe of 326 bases was transcribed with T7 RNA polymerase using polymerase chain reaction (PCR) amplified DNA as a template. The RNA probe of 326 bases performed as well as a RNA probe of 2588 bases for detection of a DNA segment of 355 bp. For detection of dilutions of HIV-1 with PCR, a set of primers (outer set) was used for amplification of HIV-1 DNA. In a separate reaction a set of primers nested between the first set generated through PCR an amplified DNA fragment with the T7 promoter. This fragment was transcribed for the synthesis of a biotinylated RNA probe. This probe could then be reacted with material amplified with the outer set of primers. Ten copies of HIV-DNA could be detected with this procedure.

A sensitive nonisotopic hybridization assay for HIV-1 DNA

We have developed a microtiter-based sandwich hybridization assay for the detection of low copy number HIV-1 sequences. The assay employs a capture DNA sequence covalently coupled to microtiter wells through linker arms. The detection probe is a biotin-labeled DNA fragment derived from sequences adjacent to the capture sequence. After hybridization in the presence of sample nucleic acid, the detection probe remains bound only if the sample contained complementary sequences spanning the junction between capture and detection probes. The amount of detection probe bound is quantified by incubation with a peroxidase-streptavidin conjugate and a colorimetric peroxidase substrate. This assay has been combined with enzymatic target amplification to achieve sensitive detection of HIV-1 in patient samples. Following amplification of HIV-1 DNA using the polymerase chain reaction technique, a 190-bp product is produced. This product is easily and specifically quantified using the sandwich hybridization assay. The resulting test can detect one HIV-1-infected cell in 10 5 cells or about 30 molecules of HIV-1 DNA.

Time-resolved fluorometry for the identification of viral DNA in clinical specimens.

Time-resolved fluorometry was used for the detection of DNA probes labeled directly with europium (Eu3+) chelate. The sensitivity of this nonisotopic hybridization assay was 100 pg of homologous DNA. Equivalent results with reference tests were obtained in the detection of adenoviruses in clinical specimens.