DNA Probe Assay Research Articles

Immunochemical detection using the light-addressable potentiometric sensor

Rapid, sensitive and flexible assay systems are needed for immunoassays, receptor-ligand binding studies and DNA probe assays. Filtration capture and sensor detection offer several advantages to these areas. Although dependent on the affinity of the specific binders employed, the sensitivity of these techniques can be in the order of 10(-12) M, and total assay time can be less than 15 minutes.

Oral histoplasmosis in India: a case report and an overview of cases reported during 1968–92

Oral histoplasmosis in a 30-year-old male with no history of travel outside India is described. An ulcerating lesion was located on the hard palate. A chest X-ray was normal. Based on physical examination, regional lymph nodes, liver and spleen were not involved. The diagnosis was established by demonstrating yeast-like budding cells in a biopsy of the lesion and by isolating Histoplasma capsulatum in pure culture. The identity of the isolate was confirmed by a chemiluminescent DNA-probe assay and the exoantigen test. A review of the Indian literature from 1968 to 1992 revealed the occurrence of 25 authentic cases of histoplasmosis in India. In 19 cases, lesions were confined to the oral cavity confirming prior observation that histoplasmosis in Indian patients tends to occur primarily in extrapulmonary sites, particularly the oral cavity.

Rapid Diagnosis of Coccidioidomycosis With a DNA Probe to Ribosomal RNA

Although nucleic acid probe hybridization assays were previously exclusively used as a tool in the research setting, such assays have recently become commercially available for the detection of a variety of infectious microorganisms. We used a commercially available DNA hybridization probe test that targets organism-specific ribosomal RNA sequences to rapidly diagnose a patient with disseminated coccidioidomycosis. The natural amplification inherent to such DNA:RNA probe systems obviates the need for electrophoretic separation and amplification steps, which are often required in more traditional DNA:DNA probe assays. With this probe, culture confirmation was obtained within 48 hours after the clinical specimens were received. Rapid DNA hybridization probe techniques have wide application in infectious diseases, especially those characterized by slow culture growth of pathogens such as deep fungi and atypical mycobacteria.

Detection of JC virus by polymerase chain reaction and colorimetric DNA hybridization assay

A procedure for rapid detection of JC virus (JCV) using the polymerase chain reaction is described. The procedure was tested in eight HIV-1-seropositive patients with progressive multifocal leukoencephalopathy. One-step DNA extraction using a chelating resin was carried out on clinical samples of cerebrospinal fluid (CSF), urine and brain tissue. After amplification, PCR products were detected by a DNA hybridization method. Microplates were coated with a specific probe and hybridized PCR products were revealed by a commercial colorimetric immunoassay. Using this procedure JC virus DNA was detected in all CSF specimens from patients with progressive multifocal leukoencephalopathy. This sensitive and rapid (24 h) procedure could greatly facilitate use of the DNA probe assay for detection of JC virus in clinical laboratories.

Direct DNA probe assay for Neisseria gonorrhoeae in pharyngeal and rectal specimens.

The direct detection of gonococcal DNA in rectal and pharyngeal specimens was evaluated by using a DNA probe-based assay (Gen-Probe, Inc., San Diego, Calif.). Rectal (234) and pharyngeal (608) swab specimens were obtained from 249 men and 372 women attending sexually transmitted disease clinics in Las Vegas and Reno, Nevada. The prevalence of gonococcal infection by culture at the pharyngeal and rectal sites was 2.9% (16 of 548 specimens) in women and 2.7% (8 of 294 specimens) in men. No false-positive reactions were observed among the 234 rectal specimens tested. Two probe-positive, culture-negative specimens were detected among the 361 pharyngeal specimens obtained from women. Both of these samples were confirmed as Neisseria gonorrhoeae by a probe competition assay. The overall correlation of the DNA probe test with pharyngeal and rectal cultures was 99.4% (837 of 842 cultures), with a sensitivity of 87.5% (21 of 24 cultures) and specificity of 99.7% (816 of 818 cultures). The positive and negative predictive values of the DNA assay were 91.3 and 99.8%, respectively. The direct DNA probe assay provides an alternative to culture screening for rectal and/or pharyngeal gonococcal infections.

A transcriptionally amplified DNA probe assay with ligatable probes and immunochemical detection

Transcriptionally amplified DNA probes are valuable tools in the development of sensitive nucleic acid-based diagnostic assays. Here we describe a model assay using a novel oligonucleotide hairpin probe that encodes a T7 RNA polymerase promoter. The hairpin probe and an adjacently hybridizing biotinylated capture probe were hybridized to target DNA and the duplex was captured onto streptavidin-coated magnetic particles. After ligation of the immobilized probes, which served to maintain specificity, the hairpin probe was transcribed by T7 RNA polymerase. The amplified RNA product was hybridized to the capture probe and bound to the streptavidin-coated magnetic particles. The immobilized heteroduplex was detected with an antibody-alkaline phosphatase conjugate specific for DNA:RNA hybrids, and the chemiluminescent substrate adamantyl-1,2-dioxetane phenyl phosphate. Ten attomoles of target DNA could be detected in a background of 5 micrograms of unrelated DNA. The chemiluminescent immunoassay was as sensitive as radioactive detection of specific product after gel electrophoresis.

Prevalence of Salmonella in raw meat used in diets of racing greyhounds.

One hundred twelve samples of commercial raw meat used in greyhound diets were collected and cultured for Salmonella using standard procedures. Fifty (44.64%) of these samples were positive for Salmonella. Salmonella typhimurium was the most frequently isolated serovar (48%), followed by S. newport (12.76%), S. agona (8.51%), and S. muenster (6.38%). The remaining 10 serovars recovered in this study represented 27.59% of the total Salmonella isolates. In addition, the meat samples were screened for Salmonella using a commercial DNA probe. Of the 106 samples tested, 70 (66.03%) were positive for Salmonella, which indicated that the DNA probe assay was more sensitive than the culture method for screening of Salmonella in raw meat. Antimicrobial susceptibility testing revealed that most of the Salmonella isolates were sensitive to a variety of antimicrobials, particularly amikacin and apramycin, and resistant to some others, such as clindamycin, erythromycin, penicillin, and sulfadimethoxine. The cumulative percentages of susceptibility (MIC50 and MIC90) of the Salmonella isolates were also determined. Most isolates were susceptible (MIC90) to low concentrations of gentamicin (2.0 micrograms/ml), imipenem (< or = 0.25 microgram/ml), and ciprofloxacin (< or = 0.5 microgram/ml). Marked resistance was found with the other antimicrobial agents. However, the high MIC values found for these isolates would not be achievable in vivo with the normal recommended doses of antimicrobial agents, so their use would not be beneficial. Numerous plasmid patterns were found in 17 randomly selected Salmonella isolates. Eight of the 17 isolates had 2-7 plasmids ranging from 2.4 to 15 kilobases in size. Eight isolates also exhibited large plasmids in the range of 50-60 and 95-105 kilobases.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA HYBRIDIZATION TECHNIQUES AND THEIR APPLICATION TO THE DIAGNOSIS OF INFECTIOUS DISEASES

Several commercial DNA probe assays are widely used in clinical microbiology laboratories. These include culture-confirmation assays for Mycobacterium species, which are recommended by CDC because they are both rapid and accurate, and culture-confirmation assays for N. gonorrhoeae. Probe assays for direct detection of N. gonorrhoeae and C. trachomatis are also widely used in public health and large reference laboratories. In many cases, the probe assays have decreased the time to identification of positive cultures and improved detection of these pathogens because they do not depend on the presence of viable organisms to achieve a positive result. Nucleic acid amplification assays hold promise for the rapid detection and identification of many infectious agents. PCR using universal primers enables researchers to identify new agents of disease that cannot be cultured in vitro; more importantly, PCR provides a method for detecting the presence of any bacterial species, including common organisms, in normally sterile body fluids, such as blood and cerebrospinal fluids. The use of such primers may well give PCR the broad-based approach needed to identify organisms in the clinical microbiology laboratory of the future.

Diagnostic efficacy of a DNA probe in pneumonia caused by Legionella species.

A commercial DNA probe kit (Gen-probe) for the detection of rRNA from legionellae was evaluated for its accuracy in diagnosing Legionnaires' disease in 167 patients with pneumonia. The test was performed on freshly obtained clinical respiratory tract samples. Cultures and direct immunofluorescence antibody (DFA) staining of the samples and serological tests were performed simultaneously for all patients. The probe assay result was positive in six patients; five of them had other laboratory evidence of disease (positive cultures or positive serological results or both). Depending on the diagnostic criteria, the probe test had a sensitivity of 31-67%, a specificity of 99% and positive predictive values of 67-83%. The diagnostic performance of the DNA probe assay in this study was superior to that of the DFA test. The results indicate that the examination of respiratory tract secretions by the Gen-probe kit is a suitable screening test for the diagnosis of Legionnaires' disease.

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.

Detection of BCR/ABL transcripts in chronic myeloid leukaemia by polymerase chain reaction and DNA enzyme immunoassay: a DNA probe assay without DNA labelling

The detection of the t(9;22) translocation, typical of chronic myeloid leukaemia (CML), can be accomplished by cytogenetical detection of Philadelphia (Ph1) chromosome or by molecular analysis of the bcr/abl fusion gene with nucleic acid probes after amplification by polymerase chain reaction (PCR). PCR-based approaches are now widely used for follow up of CML patients during therapy or after bone marrow transplantation (BMT). We describe here a microtitre, colorimetric assay (DNA Enzyme Immunoassay, DEIA) for analysis of t(9;22) translocation after enzymatical amplification of RNA from CML patients. This assay is based on the use of a monoclonal antibody specifically reacting with double stranded DNA, i.e. with hybridized DNA. The assay represents a nonisotopic alternative to other current hybridization assays and requires no modifications of primers, probe or target DNA.

Evaluation of the PACE 2 Neisseria gonorrhoeae assay by three public health laboratories

The Gen-Probe PACE 2 DNA probe assay for Neisseria gonorrhoeae was compared with conventional culture techniques in three Florida public health laboratories with 436 patients (271 females and 165 males). The prevalence rates based on culture were 19.9, 55.8, and 33.5% for females, for males, and overall, respectively. Twenty-seven probe-positive specimens gave negative culture results. Twenty of these specimens were resolved as true positives after retesting with a probe competition assay. The resolved sensitivity, specificity, positive predictive value, and negative predictive value were 99.4, 99.6, 99.4, and 99.6%, respectively.

Value of a DNA probe assay (Gen-Probe) compared with that of culture for diagnosis of gonococcal infection

The Gen-Probe PACE 2 system for Neisseria gonorrhoeae (GP), which uses a chemiluminescently labeled DNA probe, was compared with conventional culture as the method of reference. A total of 1,750 specimens were collected from 496 females and 623 males visiting the outpatient clinic of the Sexually Transmitted Diseases Department of the Westeinde Hospital, The Hague, The Netherlands, during the year 1991. The prevalences of gonorrhea culture-positive men and women were 14.9 and 7.7%, respectively. The overall positive rate was 8.7%. Sensitivity, specificity, and positive and negative predictive values of GP were 97.1, 99.1, 90.6, and 99.8%, respectively. A total of 12 of 13 patients with positive GP results and negative cultures may have had a gonococcal infection, a conclusion based on clinical symptoms, positive methylene blue smears, and high relative light unit ratios. The DNA probe test can be useful as a suitable screening and diagnostic test for gonorrheal infection in men and women. An advantage of using this DNA probe technique is that simultaneous testing for Chlamydia trachomatis of the same specimen is possible. We also examined whether (all) rRNA had disappeared after adequate treatment for gonococcal and/or chlamydial infection in 30 patients. None of those positive patients showed a positive result in the DNA probe assay after treatment.

Field testing an enzyme-linked synthetic oligonucleotide probe for identification of Anopheles gambiae s.s. and An. arabiensis.

Tests were carried out in Kenya to determine whether the enzyme-linked synthetic oligonucleotide probe (pAna 1) developed for identifying species of the Anopheles gambiae complex could be used under field conditions. The An. arabiensis male-specific pAna 1 probe was able to identify all male larval instars and adult males. However, the non-radioactive assay was not sufficiently sensitive to identify male sperm DNA in all the mated female An. arabiensis. Although the ratio of An. arabiensis to An. gambiae s.s. identified with pAna 1 in males during the dry season was in agreement with the ratio in half-gravid females identified cytogenetically, the ratios were different during the wet season. This study demonstrates that the enzyme-linked DNA probe assay is applicable under field conditions.

Preliminary evaluation of the ligase chain reaction for specific detection of Neisseria gonorrhoeae.

Rapid identification of Neisseria gonorrhoeae in clinical specimens is essential for effective control. Traditional culture requires a minimum of 24 h, and for some specimens harboring gonococci, the gonococci fail to grow or are misidentified. The recently described ligase chain reaction (LCR) is a highly specific and sensitive DNA amplification technique which was evaluated as an alternative to routine culture. Three LCR probe sets were used. Two of the probe sets were directed against the multi-copy Opa genes (Omp-II), while the third set was targeted against the multicopy Pilin genes. Each LCR probe set was evaluated with 260 microorganisms including 136 global isolates of N. gonorrhoeae, 41 isolates of N. meningitidis, and 10 isolates of N. lactamica; 26 nonpathogenic Neisseria strains; and 47 isolates of non-Neisseria species that may reside in clinical specimens. Amplification products were detected by using the IMx LCR format (Abbott Laboratories, Abbott Park, Ill.). Strains of N. gonorrhoeae were assayed at 270 cells per LCR (approximately 6.7 x 10(4) CFU/ml) with the Opa and Pilin probes, producing signals at least 21 and 15 times above background, respectively. In contrast, only background values were observed when testing the probe sets with 124 nongonococcal strains at 1.3 x 10(6) cells per LCR (approximately 3.2 x 10(8) CFU/ml). One hundred urogenital specimens were assayed by LCR, and compared with culture, the three probes were 100% sensitive (8 of 8) and 97.8% specific (90 of 92), resulting in an agreement of 98% (98 of 100). On the basis of the results of these preliminary studies, LCR has the potential to be an accurate and rapid DNA probe assay for the detection of N. gonorrhoeae in clinical specimens.

Methods for Selective Enrichment of Campylobacter spp. from Poultry for Use in Conjunction with DNA Hybridization

A DNA hybridization test was investigated for application to the detection of Campylobacter spp. in poultry samples. The test chemistry involves solution phase hybridization and detection by means of an enzymatically generated colorimetric endpoint. DNA probes used in the test system are targeted to unique sequences of ribosomal RNA and are specific for Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and Campylobacter fetus subsp. fetus. Initial experiments with pure cultures of C. jejuni established the sensitivity limit of the DNA hybridization assay at approximately 106–7 CFU per sample. Experiments were designed to define optimal conditions for recovery and selective enrichment of Campylobacter spp. from chicken carcasses for use in conjunction with the DNA hybridization assay. Following overnight enrichment, cultures were swabbed onto Campy-Cefex agar plates and allowed to incubate for 24 h. This overnight growth was then suspended and assayed with the DNA probe. The remainder of the overnight enrichment was centrifuged and the resulting pellet was analyzed. Thirty-eight chicken carcasses were assayed for Campylobacter spp. by DNA probe and culture methodology employing culture enrichment and selective plating. Culture procedures isolated Campylobacter spp. from 23 carcasses, while the DNA probe assay detected the organism from 21 carcasses. The DNA probe registered five “false” positives and seven “false” negatives relative to the cultural bacteriologic approach.

DNA probes for the detection of plant pathogenic bacteria

During the last few years there has been an increasing interest in exploiting the potential of DNA probes for identification and detection of phytopathogenic bacteria and there are now sufficient reports using representatives of the main relevant genera to show that DNA probes are of unequivocal value in identification. Detection of phytopathogenic bacteria is required under two main sets of circumstances: for diagnosis of diseased plant material and for plant hygiene purposes. The major difference is the demand for sensitivity and thus different DNA probe assays are relevance for the two conditions. Dot-blot assays are simple but not very sensitive. On the other hand, assays based on the polymerase chain reaction can be extremely sensitive. A common problem for many applications appears to be a negative influence on the assays from plant debris and/or background microflora. There are, however, several possibilities to counteract these effects. In this review, we discuss the recent progress made in this field.

Evaluation of a commercial DNA probe assay for the detection of Neisseria gonorrhoeae

The diagnostic value of a prototype ribosomal RNA-directed DNA probe system for Neisseria gonorrhoeae was evaluated in 119 men with urethritis. All 15 patients with positive results on Thayer-Martin culture were also positive with the DNA probe, while all 63 patients who had negative results on Thayer-Martin culture were also negative with the DNA probe. Thus the rate of agreement between the two tests was 100%. Positive DNA probe results were obtained in all 14 patients who were positive on an enzyme immunoassay, while negative DNA probe results were obtained in all 27 patients who were negative on the enzyme immunoassay. The agreement rate between these two tests was also 100%. The results of this study indicate that the DNA probe assay is a reasonable, noncultural alternative for directly detecting N gonorrhoeae in urogenital samples.

Chemiluminescent and Bioluminescent Assays for Polyadenylic Acid and Applications to DNA Probe Assay and Immunoassay

We developed a chemiluminescent (CL) assay for polyadenylic acid (poly A) based on the enzymatic cycling reaction following the conversion to ATP by pyruvate kinase/polynucleotide phosphoryrase enzyme reaction. Poly A was also measured with the bioluminescent (BL) assay using luciferase from firefly Photinuspyralis. The detection limits of poly A by CL and BL assay were 10 amol and 2 amol, respectively. We prepared the biotinylated poly A using biotin hydrazide and glutaraldehyde as a bifunctional coupling reagent to apply the poly A assay to a general avidin-biotin reaction system. When the CL assay and the BL assay of biotinylated poly A are applied to the DNA probe assay of λ-phage DNA and the immunoassay of thyroid-stimulating hormone (TSH) using avidin-biotin reaction as detection system, 500 pg assay−1 well of λ-phage DNA and 3 μU ml−1 of TSH were determined.

DNA probes and automation.

Current methods for DNA probe analysis in principle provide access to the total genetic information of any organism and permit applications in health care, for example. In practice, however, greatly improved efficiency of methods for decoding genetic information is required for both research and routine applications. This review describes recent progress towards automated DNA probe assays.