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PubMed2000

[Studies on detection methods of two DNA probes in Mycobacterium tuberculosis].

Two different biotinylated DNA probes which are highly specific to M. tuberculosis(MT) were made and studied. One probe is a 20 bp oligonucleotide labeled with biotin at 5' end, the other is a long DNA probe produced by PCR amplification procedure allowed for the incorporation of biotin labeled UTP. The two probes were hybridized with MT genome DNA and a 317 bp PCR product amplified from IS6110 sequence of MT, and then detected by alkaline phosphatase conjugates through colorimetric reaction. The detection sensitivity and specificity of the two probes were comparatively studied. The hybridization condition including concentration of probe, temperature of hybridization and washing filter thereafter were also investigated preliminary. The detection limit of the oligonucleotide probe and the 188 bp PCR probe were 100 ng and 6 ng of DNA respectively in detection of M. T genome, and 400 pg and 50 pg of DNA respectively in detection of PCR products of MT. The two probes can be only hybridized to MT and BCG, but not with other 24 mycobacterium or non-mycobacterium tested. The optimal hybridization temperature and washing filter temperature of oligonucleotide were 42 degrees C and 60 degrees C respectively; and that of 188 bp probe, 68 degrees C and 60 degrees C-68 degrees C. Generally the specificity of two probes were all high, but the sensitivity of 188 bp DNA probe was 7-16 times that of the oligonucleotide probe. The higher sensitivity, lower hybridization background and faster revelation of the 188 bp DNA probe made it a better choice in detection of MT.

Biotin↗

PubMed1993

Characterization of two DNA probes specific for Serpulina hyodysenteriae.

Two DNA probes, one 1.1- and one 0.75-kb probe, specific for Serpulina hyodysenteriae were isolated from a genomic library generated from virulent S. hyodysenteriae 5380. These probes are highly specific and react with all S. hyodysenteriae strains tested. Under stringent conditions, the DNA probes did not react with the nonpathogenic species Serpulina innocens or with other species of enteric bacteria, including Escherichia coli. Both probes are able to detect S. hyodysenteriae in colony blot hybridizations, and when applied to fecal specimens, they can detect 10(4) S. hyodysenteriae cells in 0.1 g of seeded fecal matter. Both probes can detect S. hyodysenteriae in fecal specimens from swine with clinical signs of swine dysentery after experimental challenge and from swine from a herd with an acute outbreak of swine dysentery. These probes have application as a diagnostic tool in veterinary microbiology.

Animals↗

PubMed1993

A survey of clinical isolates of Enterobacteriaceae using a series of DNA probes for aminoglycoside resistance genes.

DNA probes specific for the aminoglycoside adenylyltransferase ANT(2"), the aminoglycoside phosphotransferases APH(3')-I and APH(3')-II, and the aminoglycoside acetyltransferases AAC(3)-I, AAC(3)-V and AAC(6')-I were used to screen 151 clinical isolates for the presence of these resistance determinants. The two most common resistance genes in the study isolates were those encoding ANT(2") and APH(3')-I. The phenotypic pattern of aminoglycoside resistance, as determined by a modified disk diffusion test correlated well with the genotypes of the organisms as established using DNA probes, although strains carrying the ANT(2") gene could express one of several phenotypes.

Acetyltransferases↗

PubMed2001

Identification of Streptococcus salivarius by PCR and DNA probe.

AIMS: To establish species-specific PCR and DNA probe methods for Streptococcus salivarius and to clarify the distribution of dextranase in oral isolates of Strep. salivarius. METHODS AND RESULTS: A pair of PCR primers and a DNA probe were designed based on the nucleotide sequence of the dextranase gene of Strep. salivarius JCM5707. Both the PCR primer and the DNA probe specifically detected Strep. salivarius but none of the other oral streptococci (23 strains of 13 species). The primer and the probe were capable of detecting 1 pg and 1 ng of the genomic DNA, respectively, purified from Strep. salivarius JCM5707. All oral isolates (130 strains from 12 subjects) of Strep. salivarius from human saliva were positive by both methods. CONCLUSION: The present PCR and DNA probe methods are highly specific to Strep. salivarius and are useful for the its detection and identification of this bacterium. The dextranase widely distributes among oral isolates of Strep. salivarius. SIGNIFICANCE AND IMPACT OF THE STUDY: The DNA sequence of a dextranase gene present in the genome of Strep. salivarius is useful as the target DNA of the species-specific PCR and DNA probe.

DNA Primers↗

PubMed1990

DNA probes distinguish geographical isolates and identify a novel DNA molecule of Babesia bovis.

A genomic DNA library of Babesia bovis was screened to identify DNA probe candidates for direct detection of the parasite. Two sequences, Bo6 and Bo25, had the highest sensitivity and further analysis revealed unique characteristics of each of these. Neither sequence hybridized detectably to bovine DNA. Bo6 detected 100 pg of both a Mexican and an Australian isolate of B. bovis, but Bo6 also detected 1.0 ng of Babesia bigemina DNA under identical conditions. A unique characteristic of Bo6 is that it hybridizes to an apparent 7.4-kilobase DNA in undigested genomic DNA of both B. bovis and B. bigemina. The sequence is well conserved between the 2 geographic isolates of B. bovis, but it is apparently divergent in B. bigemina. Bo25 did not hybridize detectably to bovine or B. bigemina DNA. This sequence detected 100 pg of homologous B. bovis Mexican isolate DNA, but the sensitivity was reduced to 1 ng for the Australian isolate DNA. The restriction enzyme profile of the Bo25 sequence in genomic DNA differed markedly in the number, size, and intensity of bands between the 2 B. bovis geographic isolates tested. Thus, the Bo25 sequence can distinguish geographic isolates of B. bovis.

Animals↗

PubMed1999

Thermodynamic basis of the enhanced specificity of structured DNA probes.

Molecular beacons are DNA probes that form a stem-and-loop structure and possess an internally quenched fluorophore. When they bind to complementary nucleic acids, they undergo a conformational transition that switches on their fluorescence. These probes recognize their targets with higher specificity than probes that cannot form a hairpin stem, and they easily discriminate targets that differ from one another by only a single nucleotide. Our results show that molecular beacons can exist in three different states: bound to a target, free in the form of a hairpin structure, and free in the form of a random coil. Thermodynamic analysis of the transitions between these states reveals that enhanced specificity is a general feature of conformationally constrained probes.

Base Sequence↗

PubMedDate not supplied

Overview of principles and current uses of DNA probes in clinical and laboratory medicine.

Fundamental considerations of deoxyribonucleic acid (DNA) probe hybridization reaction including concept of stringency is reviewed. Restriction fragment length polymorphism (RFLP) and its detection using DNA probes are discussed together with the importance of RFLP in genetic linkage analysis. Methodological considerations, such as limitations in sample preparation and improvements effected in techniques, cloning DNA, and labelling of DNA probes, are addressed. Principles of hybridization technology and blotting techniques are reviewed. Amplification of DNA by the polymerase chain reaction (PCR), labelling with PCR, detection of PCR products and separation procedures prior to use of PCR are discussed. Other amplification methods and in-situ hybridization (ISH) procedures are reviewed. Selected current applications of DNA probes in the area of infectious diseases, genetic diseases, HLA typing, paternity and forensic testing are briefly discussed. Emphasis is placed on the use of DNA probes in the area of oncology. Background on activation of proto oncogene, loss of tumor suppressor genes, chromosomal translocation, and chromosome analysis are provided for understanding DNA probe assays for ph1 positive leukemias and applications of Interphase cytogenetic technique in oncology. Finally, analytical strategies for detection of tumor suppressor genes are addressed.

DNA Probes↗

PubMed1990

Construction and applications of DNA probes for detection of polychlorinated biphenyl-degrading genotypes in toxic organic-contaminated soil environments.

Several DNA probes for polychlorinated biphenyl (PCB)-degrading genotypes were constructed from PCB-degrading bacteria. These laboratory-engineered DNA probes were used for the detection, enumeration, and isolation of specific bacteria degrading PCBs. Dot blot analysis of purified DNA from toxic organic chemical-contaminated soil bacterial communities showed positive DNA-DNA hybridization with a 32P-labeled DNA probe (pAW6194, cbpABCD). Less than 1% of bacterial colonies isolated from garden topsoil and greater than 80% of bacteria isolated from PCB-contaminated soils showed DNA homologies with 32P-labeled DNA probes. Some of the PCB-degrading bacterial isolates detected by the DNA probe method did not show biphenyl clearance. The DNA probe method was found to detect additional organisms with greater genetic potential to degrade PCBs than the biphenyl clearance method did. Results from this study demonstrate the usefulness of DNA probes in detecting specific PCB-degrading bacteria, abundance of PCB-degrading genotypes, and genotypic diversity among PCB-degrading bacteria in toxic chemical-polluted soil environments. We suggest that the DNA probe should be used with caution for accurate assessment of PCB-degradative capacity within soils and further recommend that a combination of DNA probe and biodegradation assay be used to determine the abundance of PCB-degrading bacteria in the soil bacterial community.

Bacteria↗

PubMed1989

Evaluation of a prototype DNA probe test for the noncultural diagnosis of gonorrhea.

A prototype, nonisotopic, chemiluminescent DNA probe test called the Gen-Probe PACE (Probe Assay-Chemiluminescence Enhanced) system for Neisseria gonorrhoeae (Gen-Probe, San Diego, Calif.) was compared with conventional Martin-Lewis culture medium in JEMBEC plates for the laboratory diagnosis of gonorrhea. This 2-h noncultural assay is based upon the use of an acridinium ester-labeled DNA probe. The rRNA-directed DNA probe hybridizes with the target rRNA, and the hybridized probe is separated from the unhybridized probe through the use of magnetic microparticles. The esterified acridinium is hydrolyzed from the hybridized probe by the addition of an alkaline hydrogen peroxide solution, resulting in the production of visible light which is measured in a luminometer. The amount of light generated is directly proportional to the amount of gonococcal target rRNA present in the sample. A total of 407 clinical specimens (203 urethral and 204 endocervical) were collected from high-risk walk-in patients attending a sexually transmitted disease clinic. Separate patient specimens were collected for culture on Martin-Lewis medium in JEMBEC plates and for DNA probe assay. Statistical analysis of the overall comparative results showed that the DNA probe assay had a sensitivity, specificity, and positive and negative predictive values of 93, 99, 97, and 99%, respectively, in a patient population with a gonococcal disease prevalence of 21%. The results of this comparative study showed that the prototype chemiluminescent DNA probe assay is a rapid and reliable noncultural alternative for the laboratory diagnosis of gonorrhea.

DNA Probes↗

PubMed1993

Detection of recombinational mutations in cultured human cells by Southern blot analysis with minisatellite DNA probes.

Using the human acute monocytic leukemia cell line, THP-1, a hypermutability of minisatellite loci was demonstrated in cell culture by Southern blot analysis with minisatellite DNA probes. DNA was isolated from 98 subclones and hybridized to a panel of minisatellite probes consisting of three multilocus minisatellite probes (ML probes) and seven locus-specific minisatellite probes (LS probes). The Southern blot patterns of the hybridized subclones were compared with those of the parental THP-1. Four mutated bands with two ML probes and two mutated bands with two LS probes were detected. The mutation frequency was estimated roughly at 0.1% based on the total number of bands analyzed, and it was much higher than that expected for other DNA regions. Four of these mutations were thought to be alterations of repetitions caused by insertion or deletion of tandem repeats, and one mutant lost a complete minisatellite allele. The nature of the sixth mutant was unclear. Because of the hypermutability of minisatellite DNA, Southern blot analysis using minisatellite DNA probes can be used as a mutation assay system directly based on the DNA.

Base Sequence↗

PubMed1998

Rapid chair-side DNA probe assay of Bacteroides forsythus and Porphyromonas gingivalis.

This study compared a rapid, colorimetric DNA probe assay designed to be performed in a dental office within 40 min, with anaerobic culture and indirect immunofluorescence microscopy (IFM) for detection of Bacteroides forsythus and Porphyromonas gingivalis in subgingival plaque samples. The DNA probe assay used the Periodontal Microbial Identification Test (Saigene Corporation, Bothell, Washington, USA). B. forsythus was detected in 46 (52%), 49 (55%) and 39 (44%) of the samples by DNA probe, culture (at levels > or = 10(5)) and IFM, respectively. P. gingivalis was detected in 24 (27%), 18 (20%) and 29 (33%) of the samples by DNA probe, culture (at levels > or = 10(5)) and IFM, respectively. Results from the DNA probe assay were compared to culture. Culture negative, probe positive samples were re-evaluated by IFM, and IFM positive samples were considered positive in "resolved" data. Using resolved data. DNA probe detection sensitivity and specificity values for B. forsythus were 81% and 91% and for P. gingivalis were 80% and 95%, respectively. DNA probe test results were further compared with culture and IFM. For samples negative by both culture and IFM, probe specificity was 92% in 25 B. forsythus samples and 95% in 57 P. gingivalis samples. For samples positive by both reference methods, probe sensitivity was 82% in 27 B. forsythus samples and 73% in 15 P. gingivalis samples. B. forsythus was detected more frequently by culture compared with IFM; the reverse was observed for P. gingivalis. The rapid DNA probe assay for B. forsythus and P. gingivalis was comparable to cultivable and IF analyses.

Adult↗

PubMed1989

Laboratory and clinical evaluation of a commercial DNA probe for the detection of Legionella spp.

We prospectively compared a commercially available Legionella DNA probe with culture and direct immunofluorescence. The analytical sensitivities of the DNA probe and direct immunofluorescence were equal. Both tests detected 4 X 10(3) CFU of Legionella pneumophila or Legionella micdadei per ml in the pulmonary secretions of experimentally infected guinea pigs. The diagnostic sensitivity of the reagent was evaluated by using 809 samples of respiratory secretions. Of 51 DNA probe-positive specimens, 31 came from patients with culture-confirmed legionellosis. Two culture-positive specimens had negative DNA probe tests. The sensitivity and specificity of the DNA probe were 93.9 and 97.4%, respectively. The sensitivity and specificity of direct immunofluorescence were 68.9 and 99.6%, respectively. The low specificity of the DNA probe resulted in an unacceptable positive predictive value (60.8%). False-positive DNA probe tests were not due to nonspecific binding of the probe or to technical problems but were associated with one lot of probe reagent. Most of the false-positive probe tests had values near the threshold value of greater than or equal to 4.0 suggested by the manufacturer. Raising the threshold value for a positive test to 7 lowered the sensitivity to 69.2% but raised the specificity to 99.2%. At this level, the performances of the DNA probe and direct fluorescent-antibody testing were equivalent. Respiratory secretions from patients receiving therapy for culture-confirmed Legionella infection remained DNA probe positive for up to 8 days, even though cultures and/or direct immunofluorescence tests often became negative. The DNA probe test is a satisfactory replacement for direct immunofluorescence but cannot replace culture for the laboratory diagnosis of Legionella infections.

Animals↗

PubMed1994

Nucleotide imbalance and polymerase chain reaction: effects on DNA amplification and synthesis of high specific activity radiolabeled DNA probes.

Synthesis of radiolabeled DNA probes via polymerase chain reaction (PCR) is a convenient alternative to the more conventional methods of random primer-labeling and nick translation. PCR requires less template and allows the synthesis of radiolabeled probes from specific sequences contained within cloning vectors and genomic DNA. Under nucleotide imbalance conditions where the concentration of the radiolabeled nucleotide was 0.825 microM and the other dNTPs were each > 25 microM, amplification by Taq DNA polymerase was inhibited. Reducing the concentrations of the unlabeled dNTPs resulted in greater yields of amplification product with maximal yield obtained when the concentration of three unlabeled nucleotides was two- to eightfold higher than that of the limiting labeled nucleotide. When we utilized this amplification method for synthesis of an 800-bp glyceraldehyde-3-phosphate (GAPDH) dehydrogenase probe, 87% of the added [32P]dCTP was incorporated into amplification product. Application of this method for synthesis of high specific activity probes ( > 4 x 10(9) cpm/micrograms) up to 2.6 kb in length is demonstrated and utility of the 800-bp GAPDH probe for hybridization to Northern blots for detection of GAPDH mRNA is presented.

Base Sequence↗

PubMed1987

Use of single (4B-2) and repetitive copy (pS4) deoxyribonucleic acid (DNA) probes to characterize translocated Y DNA in a pedigree with recurrent abortion.

Probes for unique and repetitive copy deoxyribonucleic acid (DNA) are available to detect and characterize Y DNA. The probe pS4 detects repetitive copy DNA mapped to Yq12. The upper limits of the pS4 sequences are defined by the upper limits of C-banding. The probe 4B-2 is a recombinant phage construct developed from a Y library and contains a unique copy 3.3 kb Eco RI fragment mapped to Yq11. A family was ascertained through a pregnant female who had a history of four consecutive abortions and two normal daughters. Cytogenetic analysis revealed the mother and one of her daughters to have a 46,XX,15p+ karyotype. Amniocytes were karyotyped as 46,XY,15p+. Genomic DNA from controls, mother, daughters, and amniocytes was digested with Mbo I and hybridized to 32P-labeled pS4 probe. DNAs from both 46,XX,15p+ females and 46,XY,15p+ amniocytes demonstrated a clear male-specific 2.3 kb band. Digestion of the same genomic DNAs with Eco RI and blot hybridization to 32P-labeled 4B-2 probe revealed the 3.3 kb male-specific band only in the 46,XY,15p+ amniocyte DNA. The additional sequences located on 15p segregating in the female members of this family correspond to Yq12. The effect of this additional DNA on gametogenesis is unknown.

Abortion, Habitual↗

PubMedDate not supplied

Detection of enteric pathotypes of Escherichia coli by hybridization using six DNA probes.

A set of 6 DNA probes was tested to evaluate the incidence of various Escherichia coli pathotypes among 540 strains isolated in France from diarrhoeal stools of infants, children and adults. Enterotoxigenic E. coli were detected using 3 gene probes for enterotoxins LT, STaH and STaP. Enteroinvasive E. coli were detected using one DNA probe which specifically hybridizes with bacteria expressing the cell invasion phenotype "INV". They represented 1.5% and 1.1% of the total, respectively. An SLTI probe which contains the structural gene for the A subunit of Shiga-like toxin I was constructed to detect enterohaemorrhagic E. coli. Among the 5 strains detected, only 1 belonged to serotype O157:H7. An attempt was made to detect enteropathogenic E. coli (EPEC) using both an EPEC-adherence factor and the above mentioned SLTI probes. Under the experimental conditions, they did not appear to be efficient at detecting this pathotype.

Bacterial Adhesion↗

PubMedDate not supplied

[Detection of bovine infectious rhinotracheitis virus by hybridization using nonradioactive DNA-probes].

Biotin-labeled DNA probes for infectious bovine rhinotracheitis virus also known as bovine herpesvirus-1 (BHV-1) have been developed. The procedure is based on dot-blot hybridization using biotin-labeled bacteriophage M13 and plasmid probes containing cloned PstI and EcoRI-PstI restriction fragments of viral genome. The probes obtained were used to detect viral nucleic acids in specimens of bovine spermatic fluid or nasal swabs of calves. The method is simple and rapid, taking less than 24 h, and is highly specific and sensitive, this recommending it for practical veterinary.

Animals↗

PubMed1987

DNA probes for detection and identification of mycoplasmas (Mollicutes).

DNA probes are expected to prove a specific, sensitive, rapid and inexpensive means for diagnosis of mycoplasma infections, replacing procedures that depend on cultivation of the fastidious organisms. Probes made up of conserved genes, such as rRNA genes, do offer the advantage of identifying and distinguishing multiple species with a single labeled reagent. The mycoplasmal rRNA gene probe pMC5 was effective in detection and identification of mycoplasmas infecting cell cultures. However, use of pMC5 for detection of spiroplasmas and mycoplasma-like organisms (MLOs) in infected plants was hindered by hybridization of this probe with chloroplast rRNA genes. Moreover, for identifying species and strains by pMC5, a complex hybridization procedure--involving DNA purification, digestion, electrophoresis, and Southern blot hybridization--is required. More specific DNA probes, on the other hand, can identify specific Mollicutes by the much simpler, faster and more sensitive dot blot technique. Thus, a probe made of a cloned Spiroplasma citri plasmid could detect by this technique as little as 10 pg of S. citri DNA (equivalent to about 10(3) organisms) in infected plants and insects. DNA probes specific for Mycoplasma pneumoniae and M. genitalium were selected from genomic libraries and prepared in pUC13 by screening the libraries for inserts hybridizing only with DNA of the specific mycoplasma. The probes, labeled by nick translation with 32P-nucleotides, could detect as little as approximately 100 pg of the specific mycoplasmal DNA by dot blot hybridization. To eliminate radioactivity, the above DNA probes were labeled by biotinylation of sulfonation systems. Dot blot hybridization with these probes showed decreased sensitivity of detection by about one order of magnitude, and some nonspecific background reaction with large quantities of nonhomologous DNAs.

Animals↗

PubMed1987

DNA probes. Fruits of the new biotechnology.

The emergence of recombinant DNA technology in the 1970s has given rise to new diagnostic techniques based on DNA probes. The Food and Drug Administration's interest in these probes stems from its regulatory responsibilities regarding DNA probes as medical devices. The agency also sees DNA probes as valuable tools in meeting its mission of identifying, preventing, and resolving public health problems, especially in the area of food safety. The development of DNA probes for genetic diseases is raising medical and ethical questions that must be addressed by the medical community, whose ultimate responsibility must be to see that this new technology is used wisely, appropriately, and correctly.

Biotechnology↗