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PubMed1983

Isolation of a genomic clone for bovine pancreatic trypsin inhibitor by using a unique-sequence synthetic DNA probe.

Unique-sequence synthetic DNA probes, based on the known amino acid sequence of bovine pancreatic trypsin inhibitor, were constructed from oligodeoxynucleotides. In genomic Southern blot experiments, these probes were shown to hybridize specifically to discrete restriction fragments. A synthetic probe also was used to isolate a cloned BPTI gene from a bovine genomic library. DNA sequence analysis of this clone indicated that the BPTI coding region was neither preceded by a start codon nor immediately followed by a termination codon. This suggests that the mature form of BPTI may be produced through proteolytic processing from a larger polypeptide precursor.

Amino Acid Sequence↗

PubMed1988

A specific DNA probe for the identification of Campylobacter jejuni.

A 6.1 kb DNA probe for the human enteric pathogen Campylobacter jejuni has been isolated from a genomic library constructed in the plasmid vector pBR322 in Escherichia coli. The DNA sequence used as a probe was identified from recombinant plasmids following immunological screening of transformants using polyclonal antisera to whole cells and to membrane antigens of C. jejuni. Restriction endonuclease fragment mapping of C. jejuni DNA inserts from three of the recombinant plasmids showed an overlapping DNA fragment. One of these recombinant plasmids, when used as a DNA probe in Southern hybridization, specifically hybridized with chromosomal DNA from all of the C. jejuni strains tested. Hybridization was not detected at high stringency between the DNA probe and chromosomal DNA from any other Campylobacter species tested except weakly with the chromosomal DNA of strains of Campylobacter coli. Hybridization was also not detected with chromosomal DNA from a range of other enteric bacteria likely to be encountered in faecal material. The intensity of hybridization with C. coli could be increased by reducing the stringency of hybridization.

Campylobacter fetus↗

PubMedDate not supplied

Ribosomal DNA-probes differentiate five cryptic species in the Anopheles gambiae complex.

This study describes the use of ribosomal DNA probes to identify the species of individual mosquitoes in the Anopheles gambiae complex, a group of six morphologically identical mosquito species among which are two of the principal vectors of malaria in Africa. The DNA probes are sequences of DNA derived from the ribosomal genes of An. gambiae. Each probe reveals a different sized restriction enzyme fragment specific to each of the five species in the complex that were examined in this study: An. gambiae, An. arabiensis, An. quadriannulatus, An. melas and An. merus. The probes detect highly repeated sequences of DNA, thus the method is sufficiently sensitive to be applied to a small portion of a mosquito. Furthermore, because the DNA can be extracted from desiccated or alcohol preserved specimens, the test is compatible with other mosquito assays performed on dried specimens such as blood meal and malaria sporozoite antigen ELISAs. Determination of the nucleotide sequences that underlie the species-specific restriction enzyme site differences detected by these probes will lead to the development of synthetic DNA probes that can be used to identify an individual mosquito to species on the basis of a simple dot-blot or squash-blot.

Animals↗

PubMed1987

Non-radioactive hepatitis B virus DNA probe for detection of HBV-DNA in serum.

A diagnostic test has been developed to detect hepatitis B virus (HBV) DNA in human sera. This test involves a dot-blot technique in which non-radioactive nucleic acid labelled with 2-acetylaminofluorene (AAF) is used as probe. Two series of human sera from 228 blood donors and 113 HBsAg chronic carriers were tested by hybridization with the same DNA probe labelled either with AAF or with 32P. A correlation between the techniques was observed for 328 sera (96%), and using the non-radioactive test it was possible to detect 56 (86%) of the 65 HBV-DNA-positive patients. A comparative study of the HBeAg/anti-HBe status and the presence of HBV-DNA was carried out on the sera from 113 HBsAg chronic carriers, 65 of which were positive for HBeAg and 29 of which were positive for anti-HBe antibodies. With the AAF test, 44 of the HBeAg-positive sera were positive, while 5 of the anti-HBe-positive sera were positive. This study shows that, although this non-radioactive test is slightly less sensitive than the radioactive hybridization assay (RHA), it can be used for a survey of HBV carriers. Dissociation of the viral multiplication and the HBe/anti-HBe status was identified with the AAF test as well as with the RHA. It would therefore appear that the AAF test described here may be used for the extensive survey of HBV multiplication.

2-Acetylaminofluorene↗

PubMed2005

DNA probe identification of bacteria colonizing internal surfaces of the implant-abutment interface: a preliminary study.

BACKGROUND: Currently, there is limited knowledge concerning the specific genus and species of bacteria that may colonize internal surfaces of the implant-abutment interface (IAI) of two-stage dental implants. The purpose of this study was to use DNA probe analysis to identify those periodontopathic bacteria that may inhabit the internal surfaces and healing abutment screw-threads of the IAI of dental implants in situ. METHODS: Following osseointegration, bacterial samples for DNA probe analysis were obtained from 54 two-stage hydroxyapatite plasma spray-coated implants in 32 patients. Using sterile paper points, samples were obtained from the IAI of 43 implants and the screw-threads of healing abutments in the other 11 implants. DNA probes were available to detect the following microbes: Actinobacillus actinomycetemcomitans, Tannerella forsythensis, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, and Treponema denticola. RESULTS: All samples taken from healing abutment screw-threads were negative for all target microbes. The aggregate percentage of positive results for each target microbe for samples from internal surfaces of the IAI were: 41.9%, A. actinomycetemcomitans, 60.5%, T. forsythensis; 44.2%, C rectus; 60.5%, E. corrodens; 48.8%, F. nucleatum; 46.5%, P. gingivalis; 55.8%, P. intermedia; and 51.2%, T. denticola. In addition, no significant differences were noted between colonization of individual microbial species when comparing anterior to posterior and maxillary to mandibular implant sites. CONCLUSIONS: Moderate to high levels of eight different periodontopathic microbes inhabiting the internal surfaces of the IAI of 43 two-stage implants in partially edentulous patients were identified by DNA probe analysis. The microbes colonized these surfaces within 25 days following the second stage surgery and placement of the healing abutment. In contrast, all samples obtained from screw-threads of 11 healing abutments were DNA probe negative. These findings appear to support those of other investigations demonstrating the translocation of bacteria from residual dentition to implants.

Bacteria↗

PubMed2005

Multiplex PCR-DNA probe assay for the detection of pathogenic Escherichia coli.

A multiplex PCR-DNA probing assay was developed to detect four major Escherichia coli virotypes. Six highly specific polymerase chain reaction (PCR) primer sets and DIG-labeled chemiluminescent probes were designed to target the Shiga-like toxin I and II genes (stxI and stxII) of verotoxigenic E. coli (VTEC), heat-stable and heat-labile toxin genes of enterotoxigenic E. coli (ETEC), adherence factor (EAF) of enteropathogenic E. coli (EPEC) and a fragment of the invasiveness plasmid (IAL) of enteroinvasive E. coli (EIEC). The primer pairs generate products of 350, 262, 170, 322, 293 and 390 bp in length, respectively. The multiplex primers and probes were tested for specificity against 31 pathogenic E. coli strains, nine nonpathogenic E. coli and non-E.coli enteric and environmental bacterial strains. The results showed a high degree of specificity of the primers and probes for strains from corresponding virotypes and no reaction with the nontarget bacterial strains. The proposed multiplex PCR-DNA probing assay provides rapid and specific detection of four major virotypes of E. coli.

Adhesins, Bacterial↗

PubMed1990

Use of a centromere-specific DNA probe (p82H) in nonisotopic in situ hybridization for classification of micronuclei.

The DNA probe p82H was used to visualize centromeric DNA in micronuclei (MN) of human cells. Slides prepared from cultures treated by the aneugen (causing aneuploidy) colcemid showed significantly more MN with centromeric signals than those treated by the clastogen (causing chromosome breakage) bleomycin. These results indicate that in situ hybridization with the alphoid p82H DNA probe is a suitable method with which to distinguish between MN containing whole chromosomes and acentric fragments, and hence allows one to discriminate between the clastogenic and aneugenic effects in MN formation.

Aneuploidy↗

PubMed1992

Identification and mapping of six microdissected genomic DNA probes to the proximal region of mouse chromosome 1.

Six independent DNA probes, lambda Mm1C-150, lambda Mm1C-153, lambda Mm1C-156, lambda Mm1C-162, lambda Mm1C-163, and lambda Mm1C-165, have been isolated from a library of microdissected fragments from mouse chromosome 1, spanning cytogenetic bands C2 to C5. These DNA probes have been mapped by restriction fragment length polymorphism analysis with respect to 12 marker loci previously assigned to this portion of mouse chromosome 1, in a panel of 251 segregating Mus spretus x C57BL/6J interspecific backcross mice. The gene order and intergene distances were determined by segregation analysis to be centromere- lambda Mm1C-162-11.1 cM-Col3a1-8.8 cM-Len-2-2.6 cM-lambda Mm1C-163-1.6 cM-Fn-1-1.6 cM-Tp-1-0.8 cM-lambda Mm1C-165/Vil-0.4 cM-Inha-2.8 cM-lambda Mm1C-153-2.4 cM-lambda Mm1C-156-1.2 cM-Pax-3-5.6 cM-Akp-3-0.8 cM-Acrg-2.0 cM-Sag-0.5 cM-Col6a3-1.8 cM-lambda Mm1C-150-15.4 cM-Ren1,2. Four of these probes map within a chromosome 1 segment that is homologous to human chromosome 2q. Southern blotting analyses indicate that one of these anonymous probes, lambda Mm1C-165, detects DNA fragments highly conserved across species. These novel polymorphic probes should prove useful for linkage and physical mapping of this chromosomal region.

Animals↗

PubMed1994

Genetic DNA probes for Mycobacterium avium complex identification.

The acridinium ester labelled DNA probe specific for M. avium complex (MAC) was used for testing 30 strains of MAC previously identified by conventional procedures. The hybridization was positive in 26 strains, negative in 3 strains and one strain seemed to be contaminated when subcultured. The specific DNA probes allow to identify even the slow growing mycobacterial strains, in positive samples, within two hours while conventional methods usually take several weeks to show the result. A rapid confirmation of a mycobacterial species is invaluable for selecting an effective treatment as early as possible, which is extremely important e.g. in immunosuppressed patients, children with lymphadenitis, etc. Nevertheless, this method has the disadvantage of being too expensive for some laboratories, though questionable if repeated subcultures, technician's time and the delay in obtaining results are taken into account, and enabling to identify only a limited number of clinically significant mycobacterial species using commercially available probes.

Bacterial Typing Techniques↗

PubMed1990

The use of a specific DNA probe and polymerase chain reaction for the detection of Mycobacterium leprae.

A DNA probe encoding approximately 80% of the 18-kDa protein gene of Mycobacterium leprae was isolated and tested for specificity by assessing hybridization of the probe to genomic DNA from taxonomically related and unrelated DNA samples. The 360-base-pair (bp) probe was specific for M. leprae DNA and did not hybridize with genomic DNA from 18 species of bacteria nor with DNA from human, murine, and armadillo sources. Oligonucleotide primers were synthesized corresponding to the 5' and 3' ends of the 360-bp fragment to yield a fragment of similar size on amplification of M. leprae DNA by the polymerase chain reaction (PCR). A simple procedure for DNA extraction from M. leprae-infected tissues was developed that provided suitable template DNA for amplification. The PCR test was specific for M. leprae DNA from human and murine sources and detected M. leprae DNA in biopsies from leprosy patients and from control and uninfected human skin biopsy preparations seeded with as few as 100 M. leprae.

Animals↗

PubMed1992

Rapid generation of chromosome-specific alphoid DNA probes using the polymerase chain reaction.

Non-isotopic in situ hybridization of chromosome-specific alphoid DNA probes has become a potent tool in the study of numerical aberrations of specific human chromosomes at all stages of the cell cycle. In this paper, we describe approaches for the rapid generation of such probes using the polymerase chain reaction (PCR), and demonstrate their chromosome specificity by fluorescence in situ hybridization to normal human metaphase spreads and interphase nuclei. Oligonucleotide primers for conserved regions of the alpha satellite monomer were used to generate chromosome-specific DNA probes from somatic hybrid cells containing various human chromosomes, and from DNA libraries from sorted human chromosomes. Oligonucleotide primers for chromosome-specific regions of the alpha satellite monomer were used to generate specific DNA probes for the pericentromeric heterochromatin of human chromosomes 1, 6, 7, 17 and X directly from human genomic DNA.

Animals↗

PubMed1992

Adaptation of two commercially available DNA probes for the detection of E. coli and Staphylococcus aureus to selected fields of dairy hygiene--an exemplary study.

The application of two commercially available colorimetric DNA hybridization tests (GENE-TRAK E. coli and Staphylococcus aureus) to selected aspects of dairy hygiene was investigated. Bacterial isolates of different origin, naturally contaminated cheese varieties, nonfat dry milk, milk concentrates, artificially contaminated milk and raw milks from udder quarters were examined. Based on the observation that the sensitivity of the E. coli DNA probe was comparable to that of the beta-D-glucuronidase-based fluorescence reaction (with 4-methyl-umbelliferyl-beta-D-glucuronide) of E. coli strains in Fluorocult lauryl sulfate broth, a Most Probable Number technique for enumerating E. coli in cheese using the DNA probe was developed. Another specific DNA probe was applied for the detection of S. aureus as a mastitis agent. By using a modified sample preparation, specific diagnosis of this microorganism in milk from udder quarters was enabled within 6 hours. This procedure is recommended to be used in screening tests. Based on the examples presented the potential of these tests in several fields of hygiene was illustrated.

Animals↗

PubMedDate not supplied

Detection of enterotoxigenic Escherichia coli in faecal specimens by acetylaminofluorene-labelled DNA probes.

The present study describes acetylaminofluorene(AAF)-modified DNA probes for the identification of heat-labile (LT) and heat-stable (porcine STp and human STh) toxins from enterotoxigenic Escherichia coli (ETEC). AAF probes were compared with established biotinylated probes and bioassays. Ultracentrifugation was not necessary in the preparation of AAF-labelled probes, and the procedures, i.e. chemical modification of probes, hybridization and immunodetection steps, were optimized to detect ETEC by colony hybridization or direct dot blot techniques. The method combines chemical labelling (covalent attachment of AAF group to guanosine is achieved by treatment of DNA with N-acetoxy-2-acetylaminofluorene) and detection of the hybridized target DNA by means of anti-AAF monoclonal antibodies and alkaline phosphatase-labelled second antibodies.

2-Acetylaminofluorene↗

PubMed1991

Evaluation of DNA probe test for clinical diagnosis of chlamydial and gonococcal infections of the uterine cervix.

A DNA probe test (Gen-Probe) was compared with cell culture and an enzyme immunoassay method (Chlamydiazyme) for the clinical diagnosis of cervical chlamydial infection. A total of 127 specimens (95 before treatment and 32 after treatment) were obtained from 95 patients considered members of a high risk population. Forty-five (47.4%) of the 95 cases examined before treatment were positive for the DNA probe and the sensitivity, specificity, positive predictive value and negative predictive value of the test vs cell culture were 78.4, 93.4, 88.9, and 86.6%, respectively. Comparison of these data with those of Chlamydiazyme showed that the DNA probe method was a little less sensitive but much more specific. The kit could also detect gonococcal infection with sufficient accuracy in the same specimen as used for Chlamydia trachomatis, and was considered to be a useful tool as another noncultural alternative for clinical diagnosis of sexually transmitted chlamydial and gonococcal infections of the uterine cervix, although further improvement on the kit for Chlamydia trachomatis is expected.

Adolescent↗

PubMed1991

Isolation and characterization of Opisthorchis viverrini specific DNA probe.

An Opisthorchis viverrini specific repetitive DNA fragment was isolated from the O. viverrini genome. The cloned fragment, herein designated pOV-A6, contained tandemly repeated 334 bp O. viverrini DNA and has the potential to be a highly specific and sensitive DNA probe. Radioactively labelled pOV-A6 could detect as little as 25 pg of its own genomic DNA and five purified eggs by dot-blot hybridization. The presence of 50 O. viverrini eggs could be readily detected in samples containing faecal materials. The probe was highly specific for O. viverrini DNA and did not cross hybridize with DNAs prepared from related and unrelated parasites, enteric bacteria and a number of definitive and intermediate hosts. The probe has potential for screening a large number of samples and could be readily applied in epidemiological studies.

Animals↗

PubMed1993

Identification and enumeration of Listeria monocytogenes by nonradioactive DNA probe colony hybridization.

A plasmid containing the cloned listeriolysin gene of Listeria monocytogenes was used as a probe to identify Listeria strains by DNA colony hybridization. The probe DNA was labeled with horseradish peroxidase in the presence of glutaraldehyde. After the hybridization and wash procedures, the hybrid molecules were detected by luminescence, which resulted from the oxidation of luminol by a horseradish peroxidase-hydrogen peroxide-coupled reaction. Of the 150 Listeria strains and 16 non-Listeria strains examined, the probe hybridized only with L. monocytogenes. The technique was also used to enumerate L. monocytogenes in artificially contaminated foods.

Colony Count, Microbial↗

PubMed2001

Electrochemical detection of sequence-specific DNA using a DNA probe labeled with aminoferrocene and chitosan modified electrode immobilized with ssDNA.

The electrochemical detection of sequence-specific DNA using a DNA probe labeled with aminoferrocene (AFC) is reported. Sample ssDNA was immobilized on a chitosan modified glassy carbon electrode. A sequence-known DNA with 256 bp [obtained by polymerase chain reaction (PCR)] was successfully labeled with the electro-active reagent AFC by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide for the first time. This DNA probe labeled with AFC was applied to hybridize with a sequence-unknown DNA sample. Only the complementary sequence (cDNA) could form a double-stranded DNA (dsDNA) with the DNA probe labeled with AFC. The anodic peak currents (ipa) of the AFC bound to the dsDNA by differential pulse voltammetry were used for the determination of cDNA. The ipa of AFC was linearly related to the concentration of cDNA sequence between 1.0 x 10(-8) and 6.0 x 10(-6) mol L-1. The detection limit was 2.0 x 10(-9) mol L-1 using 3 sigma (where sigma is the standard deviation of blank solution, n = 11). The probe showed high sensitivity and selectivity.

Animals↗

PubMed1993

Detection of diarrheogenic Escherichia coli in children less than ten years old with and without diarrhea in New Caledonia using seven acetylaminofluorene-labeled DNA probes.

We report the use of seven acetylaminofluorene (AAF)-labeled DNA probes in evaluating the incidence of various Escherichia coli pathotypes in New Caledonia among 448 children with acute diarrhea (1,278 E. coli pathotypes studied) and 88 controls (264 E. coli pathotypes studied) in 1990. Diarrheogenic E. coli were detected using cloned gene probes for heat-labile and heat-stable enterotoxins, Shiga-like cytotoxins (SLTI and SLTII), the cell invasion phenotype (INV), and enteropathogenic-adherence factor (EAF). Isolates were also studied using bioassays and radioactive DNA probes as reference methods. Enterotoxigenic E. coli (ETEC) were isolated from only 5.36% of the patients; E. coli with localized adherence (LA) to HEp-2 cells was much more common in patients (14.4%) than in controls (3.4%; chi 2 = 7.54, P < 0.01), but most of the E. coli with an LA pattern were members of traditional enteropathogenic E. coli (EPEC) serogroups (chi 2 = 92.95, P < 0.001). Non-enteropathogenic E. coli with an LA pattern were weakly associated with diarrheal disease (8.9%). Escherichia coli with a diffuse or an aggregative pattern did not show a significant association with infantile diarrhea. Eight EPEC serogroups were identified and the frequency of positivity for the LA pattern was 70.5%; the EAF was significantly associated with the 0119:K9 serogroup. No enteroinvasive or SLT-producing E. coli were identified. An evaluation of the AAF probes in comparison with 32P-labeled probes and conventional bioassays was made during this epidemiologic survey. The positive and negative predictive values of the ETEC probes were 0.91 and 1, respectively (overall agreement = 99.8%).(ABSTRACT TRUNCATED AT 250 WORDS)

2-Acetylaminofluorene↗