Nonisotopic DNA Probe Research Articles

A non-isotopic probe-hybridization assay for residual DNA in biopharmaceuticals

Although most biopharmaceuticals are highly purified, there is a theoretical concern that such recombinant products could be contaminated with oncogenic or bacterial DNA. A crucial part of the control of such biologicals is to ensure they do not contain more residual DNA than a safety limit suggested by the regulatory agency. Currently, the FDA has suggested a 100 pg per dose limit for residual DNA. DNA probes labeled with a radioisotope such as 32P have been commonly used in hybridization tests. Because of the radiation safety concern, we chose to develop a procedure for assessing DNA levels by either a dot or slot blot hybridization technique using a nonisotopic DNA probe and immuno-enzymatic detection. A minimum detectable limit (MDL) of <10 pg DNA mg −1 protein can be achieved. Method validation data demonstrated that the precision, reproducibility, and robustness of this approach are appropriate for quality control.

In Situ Hybridization as a Method to Study the Regulation of Gene Expression in Paramecium

A method for whole cell in situ hybridization in Paramecium was developed, which allowed us to visualize both the cytoplasmic mRNA and the transcripts in the macronucleus, and to estimate their respective variation throughout the cell cycle. Nonisotopic DNA probes, labeled with digoxigenin (DIG), were prepared by PCR from a central part of two genes of P. tetraurelia, respectively coding for a β‐tubulin and the 51A surface antigen (51A‐sAg). The probes were detected with an FITC‐conjugated anti‐DIG antibody. While no fluorescence is observed in the micronuclei, mRNA are detected in the macronucleus, as discrete spots which disappear after RNase H treatment, in the cytoplasm where they are uniformly distributed, and in the post‐autogamous fragments of the old macronucleus. The intensity of the cytoplasmic labeling observed with the two probes shows a clear and different cell cycle‐dependent modulation. For the β‐tubulin mRNA, a peak is observed at the beginning of mitosis, while for the 51A‐sAg, the peak occurs later, during cytokinesis. The macronuclear labeling, which likely reflects the transcription rate, seems roughly constant for the 51A‐sAg. In contrast, for the β‐tubulin genes, the intensity of the macronuclear signal shows a peak that just precedes the cytoplasmic peak.

The intracellular polymerase chain reaction for small CMV genomic sequences within heavily infected cellular sections

The indirect intracellular polymerase chain reaction (in situ PCR) combines the potential sensitivity of the polymerase chain reaction (PCR) with the high specificity and morphological preservation of in situ hybridization (ISH). This study describes a method for the amplification of small, specific cytomegalovirus (CMV) genomic sequences [100 base pairs (bp)] within large formalin-fixed, paraffin-embedded tissue sections. A heat-resistant glue surrounds the section, creating a well which contains a relatively large volume of isotonic reaction solution without a covering mineral oil layer; this optimizes morphological preservation, permits the evaluation of large sections, and allows both denaturation steps and up to 40 cycles of in situ PCR to be performed, whilst progressively concentrating the reaction solution by evaporation during thermal cycling. ISH was performed using a non-isotopic DNA probe with specificity for CMV, with or without prior in situ PCR amplification, both for samples on slides (fibroblasts and lung) and in suspension (fibroblasts). Samples on slides were evaluated by both blinded studies and image analysis, comparing the intensity of signal (P < 0.003) and the numbers of positive cells detected (P < 0.007), with or without intracellular amplification. Cells in suspension were analysed by blinded studies on cytospins and by gel electrophoresis of cell lysates. Successful intracellular amplification was achieved in this high copy model.

Analysis and Characterization of Mycoplasma gallisepticum Isolates from Pennsylvania

Because Mycoplasma gallisepticum F strain vaccine can be pathogenic in chickens and is pathogenic in turkeys, we monitored the spread of MG F strain into unvaccinated flocks by screening field and experimental isolates. Thirteen MG isolates obtained from various sources in Pennsylvania were screened using several techniques capable of differentiating between MG strains. DNA restriction enzyme analysis (REA), Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein profiles, non-isotopic DNA probes, and a monoclonal antibody specific for F strain were used to characterize each of the 13 isolates. Three of the 13 isolates were identical to F strain; two of these were obtained from challenge studies, and one was a field isolate from a multiple-age commercial egg farm where the F strain vaccine had been used in the past. The remaining 10 isolates were different from MG F strain but were quite similar if not identical to each other according to REA; SDS-PAGE protein profiles show similarities between the 10 isolates. The results suggested that F strain vaccine is not a major cause of field outbreaks of MG in Pennsylvania.

Evaluation of a Nonisotopic DNA Assay Kit for Diagnosing Plasmodium falciparum Malaria in Madagascar

This study evaluated a nonisotopic DNA assay kit for diagnosing Plasmodium falciparum malaria in an area of Madagascar where all Plasmodium species of human malaria are present and where malaria is endemic. Blood samples from 440 healthy children and 20 healthy adults were processed and assayed in a single day in a blind protocol. The parasitemia levels of the four Plasmodium species were determined by microscopic examinations and by counts of numbers of malaria parasites per 1,000 white blood cells. Relative to P. falciparum infections, the DNA assay results agreed with those of microscopy for 207 positive and 239 negative samples; two samples were scored as positive by the DNA probe that were not detected by microscopy, and 12 samples were scored as positive by microscopy but were not detected by the assay. Relative to microscopy, the sensitivity of the assay was 95%, the specificity was 99%, and the effective sensitivity threshold of the DNA probe assay was approximately 30 parasites/mm3 of blood. The assay did not detect infections with either P. vivax, P. malariae, or P. ovale alone, but detected mixed infections of P. falciparum with either P. vivax or P. ovale. With this nonisotopic DNA probe assay, we were able to process large numbers of samples efficiently and to detect P. falciparum malaria infections with high sensitivity and specificity in a population that did not display overt disease symptoms.

Identification of Mycobacterium tuberculosis complex and Mycobacterium avium-intracellulare complex by using nonisotopic DNA probes

A total of 31 clinical isolates of mycobacteria consisting of 21 strains of M. avium-intracellulare complex (ten strains each of M. avium and M. intracellulare identified by specific 125I-labeled DNA probes and one strain of M. avium-intracellulare complex identified by conventional biochemical tests) and 10 strains of M. tuberculosis (identified by a specific 125I-labeled DNA probe) were subjected to identification by hybridization protection assay (HPA) with an acridinium-ester (AE) labeled DNA probe. The results thus obtained were in complete agreement with those obtained by 125I-labeled DNA probes (30 strains) or by conventional biochemical tests (one strain). HPA by the AE-labeled DNA probe is safe, simple and rapid, and can be done with ease in any clinical laboratory.

Performance of a nonisotopic DNA probe for detection of Chlamydia trachomatis in urogenital specimens

The Gen-Probe PACE 2 assay (GP), a nonisotopic DNA probe, was evaluated by using cell culture as the method of reference. Specimens were collected from 260 men and 482 women visiting the outpatient department for sexually transmitted diseases at the University Hospital in Rotterdam, The Netherlands. The prevalences of Chlamydia culture-positive men and women were 13.2 and 8.6%, respectively. Sensitivity values for the male and female patients were 70.6 and 92.7%, respectively. Specificity values for these groups were 98.2 and 97.7%, respectively. Sensitivity was significantly lower when the number of inclusions in cell culture was low. Samples which showed a discordance between cell culture and GP results were retested by the polymerase chain reaction. If the results of the polymerase chain reaction were considered as the points of reference, the sensitivity and specificity of both GP and cell culture could be calculated. The performance of GP for females was comparable to that of cell culture. In males, the sensitivity of GP was considerably lower than that of cell culture (77.2 versus 91.4%), while specificity was somewhat higher (99.6 versus 99.1%). Compared with cell culture, the GP is a relatively simple and rapid test that is suitable for diagnosing Chlamydia infections in urogenital specimens.

Comparison of Gen-Probe DNA probe test and culture for the detection of Neisseria gonorrhoeae in endocervical specimens

A 2-h nonisotopic DNA probe assay for the direct detection of Neisseria gonorrhoeae in urogenital specimens has recently been modified (PACE 2; Gen-Probe, San Diego, Calif.). The new assay format was developed to increase the sensitivity of the assay and simplify procedural steps. In this study, the new DNA probe test was compared with a culture reference method for the detection of N. gonorrhoeae in endocervical specimens. The results of the DNA probe test were expressed as a ratio of relative light units (RLU) of the specimen/RLU of the cutoff recommended by the manufacturer. All patient samples with sample RLU/cutoff RLU ratios less than 0.7 were interpreted as negative, and ratios greater than 2.0 were interpreted as positive for gonorrhea. Samples with sample RLU/cutoff RLU ratios between 0.7 and 2.0 were repeated until two or more consistent negative or positive ratios were obtained. A total of 469 specimens were tested with an overall disease prevalence of 6.1%. Of the 469 patients tested, 5 specimens (1.0%) fell in this borderline region and were retested. If the manufacturer's recommended cutoff value had been used, the original DNA probe results would have resulted in two false-positives. Our data were analyzed for both symptomatic (prevalence, 11.7%) and asymptomatic (prevalence, 2%) women. The study indicated that with our modification of the manufacturer's endpoint interpretation, the DNA probe test was essentially equivalent to the culture method in terms of sensitivity, specificity, and positive and negative predictive values in both symptomatic and asymptomatic patient populations. The new DNA probe test can serve as a suitable screening and diagnostic test for the diagnosis of gonorrheal genital infections in women. Additionally, it offers the advantages of rapid turnaround time and ease of use and allows simultaneous testing for Chlamydia trachomatis on the same specimen.

Location of ribosomal genes in CHO cells; in situ hybridization with a non-isotopic DNA probe on G-banded chromosomes*

A novel in situ hybridization technique using sulfonated probes is described. This non-radioactive approach, which employs chemically modified DNA and immunocytochemical procedures, is compatible with pre-G-banding and allows a rapid localization of the hybridized sequences on chromosomal spreads with a high spatial resolution. Using this technique we have localised the Chinese hamster ribosomal genes in the telomeric region of ten chromosomes, and among them in the subtelomeric q region of the Z5 chromosome. These results are discussed, the genetic markers confirming and locating the origin of Z group chromosomes by rearrangements of Chinese hamster chromosomes.

Non-isotopic DNA probes for the identification of subgingival microorganisms.

The purpose of the present investigation was to examine the potential of non-isotopic DNA probes to identify pure cultures in predominant cultivable microbiota studies. Non-isotopic DNA probes to 7 subgingival species were prepared by 2 methods. In the first, biotin-labelled probes were prepared by nick translation. In the second, single-stranded DNA was covalently linked to horseradish peroxidase via polyethyleneimine. The relative sensitivities and specificities of these probes were tested against pure cultures of a range of subgingival species. Aliquots of broth cultures were standardized by optical densities, placed on nitrocellulose or Whatman 541 filters and then treated to lyse the cells, denature and fix DNA to the filter. Using a streptavidin-alkaline phosphatase detection system, 10(4)-10(5) cells were detected by homologous biotin-labelled probes. Horseradish peroxidase-labelled probes were approximately one order of magnitude less sensitive. Non-specific reactions with unrelated species, displayed by both biotin- and horseradish peroxidase-labelled probes, were eliminated by treatment of filters with proteinase K and organic solvents. Cross-reactions between closely related species could be discriminated by comparing reaction intensities of the test strains with probes to the each of the cross-reacting species involved. Thus, nonisotopic DNA probes could be used for the rapid identification of subgingival isolates. The technique could also be used for the recognition and grouping of strains of unknown species. A probe made to the strain of an unknown species may be used to rapidly screen hundred of unknown isolates for related strains.

A chemical method for introducing haptens onto DNA probes

We have developed a versatile chemical method of attaching hapten moieties onto DNA, for the construction of nonisotopic DNA probes. The DNA is reacted with N-bromosuccinimide at alkaline pH, resulting in bromination of a fraction of the thymine, guanine, and cytosine residues, with adenine modified to a lesser extent. The bromine is subsequently displaced by a primary amino group, attached to a linker arm. The other end of the linker arm has a detectable group preattached to it. We have labeled cloned hepatitis B viral (HBV) DNA with the hapten 2,4-dinitrophenyl (DNP) and used it in combination with a high affinity rabbit anti-DNP antibody, for the detection of hepatitis B DNA by slot blotting. This probe was sensitive enough to specifically detect 1 × 10 −17 mol (1 × 10 6 copies) of HBV DNA in total DNA from human serum.