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PubMed1998

Use of polymerase chain reaction and restriction enzyme analysis to directly detect and identify Salmonella typhimurium in food.

A primer set of oligonucleotides (Salm 3 and Salm 4) from the invA gene of Salmonellae has been evaluated for the specific detection of Salmonella spp. by the polymerase chain reaction (PCR). This primer set amplified 33 Salmonella serovars but did not amplify 16 non-Salmonella bacteria. Moreover, after PCR amplification, it was possible to identify Salm. typhimurium by restriction enzyme analysis. The PCR-RE method developed could represent a helpful tool for detecting Salmonella spp., and for directly and rapidly identifying Salm. typhimurium in food.

Animals↗
PubMed1991

DNA recovery and direct detection of Tn5 sequences from soil.

Specific Tn5 sequences inserted in the genome of Enterobacter agglomerans were detected in EcoRI digested DNA directly recovered from soil 70 d after its inoculation with the bacteria, when these were no longer culturable on agar medium. A new method of DNA extraction from soil was used. No amplification of DNA sequences by PCR was needed.

DNA↗
PubMed2005

Direct detection of S-nitrosothiols using planar amperometric nitric oxide sensor modified with polymeric films containing catalytic copper species.

The direct amperometric detection of S-nitrosothiol species (RSNOs) is realized by modifying a previously reported amperometric nitric oxide gas sensor with thin hydrophilic polyurethane films containing catalytic Cu(II)/(I) sites. Catalytic Cu(II)/(I)-mediated decomposition of S-nitrosothiols generates NO(g) in the thin polymeric film at the distal tip of the NO sensor. Three different species are examined to create the catalytic layer: (1) a lipophilic Cu(II)-ligand complex; (2) Cu(II)-phosphate salt; and (3) small (3-microm) metallic Cu(0) particles. All three catalytic layers yield reversible amperometric response in proportion to the concentration of S-nitrosothiols (e.g., nitrosocysteine, nitrosoglutathione, S-nitroso-N-acetylcysteine, S-nitrosoalbumin) present in the aqueous test solution. Sensitivity toward the different RSNO species is dependent on the respective catalytic rates of decomposition of the RSNO species by reactive Cu(I), accessibility of the species into the polyurethane layer containing the catalyst, the level of reducing agents (ascorbate) used in solution to help generate reactive Cu(I) species, and the concentration of metal ion complexing agents present in the test solution (e.g., EDTA). Under optimized conditions, all RSNO species can be detected at < or =1 microM levels, with sensor lifetimes of at least 10 days for the sensors based on Cu(II)-phosphate and Cu0 particles. It is further shown that the new RSNO sensors can be used to assess the "NO-generating" ability of fresh blood samples by effectively detecting the total level of reactive RSNO species present in such samples.

Animals↗
PubMed1983

[Thin layer chromatographic test for the indirect and direct detection of the enzyme defect in histidinemia].

A thin layer chromatographic method for the direct and indirect detection of the enzyme defect in histidineemia is described. Histidin and urocanic acid are analyzed in 0,2-1 mg stratum corneum for specific screening. 5-10 mg skin biopsy material is needed for the direct measurement of the enzyme activity. This simple metabolic test is convenient even in normal hospital laboratories.

Amino Acid Metabolism, Inborn Errors↗
PubMed2000

Prenatal diagnosis of X-linked hyper-IGM syndrome by direct detection of mutation Q220X in the CD40L gene using PCR-mediated site directed mutagenesis.

We present the first report of prenatal diagnosis of X-linked hyper-IgM syndrome by PCR-mediated site directed mutagenesis (PSM) in a woman known to carry the Q220X mutation in the CD40L gene. Using the simple PSM assay, the Q220X mutation was identified by chorionic villous sampling (CVS) at 11 weeks' gestation and the pregnancy was terminated.

Adult↗
PubMed2002

Novel direct detection method for quantitative determination of intracellular nucleoside triphosphates using weak anion exchange liquid chromatography/tandem mass spectrometry.

A novel analytical method has been developed for direct quantification of intracellular nucleoside triphosphates (NTPs). Lysates of human peripheral blood mononuclear cells (PBMCs) were extracted by protein precipitation, and the filtered extracts were analyzed by weak anion exchange liquid chromatography (WAX-LC) coupled to detection by mass spectrometry (MS). Compared with ion pairing (IP)-LC/MS/MS, the only MS-compatible direct detection method for NTPs currently available, the new method completely avoids the usage of ion-pairing reagents and has a shorter analytical time of only 2 min. The method was validated and is being used to determine the amount of the triphosphate metabolite of D-D4FC (DPC817), an investigational HIV nucleoside reverse transcriptase inhibitor (NRTI), in human PBMC samples from clinical studies. By using a PE Sciex API 4000 triple quadrupole instrument operating in positive ion MRM mode, the method was able to achieve a lower limit of quantitation (LLOQ) of 5 fmol/10(6) cells in samples containing 3 x 10(6) lysed cells (6 fmol on-column). With minor adaptation, the method described here may be suitable for analyzing other NTPs. This paper also provides a discussion of the unique retention characteristics of WAX-LC, the principles of which may prove to be valuable for designing other forms of directly coupled ion-exchange (IX)-LC/MS methods suited for high sensitivity quantitative analysis.

Chromatography, Liquid↗
PubMed1992

Direct detection of Epstein-Barr virus in peripheral blood and comparison of Epstein-Barr virus genotypes present in direct specimens and lymphoblastoid cell lines established from nasopharyngeal carcinoma patients and healthy carriers in Hong Kong.

By means of a PCR assay, EBV was demonstrated directly in peripheral blood of previously infected individuals. The virus was detected in approximately 80% of specimens from EBV-seropositive individuals, but not in cord-blood lymphocytes by this method. When virus present in peripheral blood was compared to that observed directly in NPC biopsies or throat washings, it was distinct from that seen in biopsies in 4/15 cases (27%) and from that seen in throat washes in 1/22 cases (5%). The throat-wash virus differed from the biopsy virus in 3/20 cases (15%). The prototype F virus was found in 7/10 LCLs (70%) established from NPC patients' peripheral blood, but was only detected in 2/9 specimens (22%) directly analyzed by the PCR assay. This finding suggests selective isolation of prototype F EBV in spontaneous LCLs established from NPC patients.

Blotting, Southern↗
PubMed2003

Direct detection of leptospiral material in human postmortem samples.

Leptospiral culture, direct immunofluorescence, and the polymerase chain reaction (PCR) were used to detect leptospiral material in postmortem specimens collected from eight patients who died of leptospirosis. Diagnosis of leptospiral infection was based on clinical summary (premortem) and confirmed by serological analysis and/or culture of leptospires. Leptospiral culture was the least sensitive technique, yielding two isolates (3%) from 65 samples. Both isolates were from the aqueous humour and cerebrospinal fluid of the same patient. Direct immunofluorescence was of intermediate sensitivity for detection of leptospires, confirming the presence of leptospires in 11% (2 of 18) of tissue samples from three patients. PCR analysis was the most sensitive technique for detection of leptospiral material in tissue samples, being positive in 20% (11 of 56) of samples from eight patients. Both samples (cerebellum and liver) positive by immunofluorescence were also positive by PCR. The sensitivity of the PCR assay was 1-10 leptospires ml(-1) sample, and the assay was specific for Leptospira pathogenic species. Multi-system involvement was indicated based on successful amplification of leptospiral DNA from more than one tissue sample, which corroborated with the clinical and pathologic findings. The results suggest that in acute and/or fatal leptospirosis, the pathogenesis of the pathologic features are related to the presence of the organisms in the tissues. In conclusion, PCR combined with serology appears to be a useful tool for diagnosis of leptospirosis and may be invaluable in epidemiological studies.

Antigens, Bacterial↗
PubMed1998

Direct detection of 8-oxodeoxyguanosine and 8-oxoguanine by avidin and its analogues.

8-Oxodeoxyguanosine is present in DNA from many tissues. The direct demonstration of 8-oxodeoxyguanosine as a potential biomarker of oxidative DNA damage has implications for the study of mutagenesis, carcinogenesis, and free radical toxicity. Avidin is shown here to bind with high specificity to this potentially mutagenic oxidized nucleoside, 8-oxodeoxyguanosine, and to the oxidatively modified base, 8-oxoguanine. The serendipitous finding that avidin bound to the nuclei of UVA-irradiated cells has led to the development of a technique which allows detection of the damage product in a manner analogous to that of immunological techniques. The technique has been shown to be applicable to isolated DNA and to DNA in fixed cellular material and postmortem tissue. Statistically different levels of damage can be demonstrated in both isolated DNA and cultured cells exposed to free radical generating systems using a 96-well plate-based methodology. The sensitivity of this method allows the detection of 10(-19) mol of 8-oxodeoxyguanosine. This novel usage of avidin conjugates applies also to its bacterial analogue, streptavidin, and to a lesser extent to the monoclonal antibody to biotin (the ligand bound by the parent compound). This finding has tremendous potential as a simple method analogous to immunotechniques for the direct detection of 8-oxodeoxyguanosine. From structural considerations we speculate that avidin would also bind to 8-oxodeoxyadenosine.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1990

Overlay technique for direct detection and identification of haemolytic Listeria on selective plating medium. Comparison of five media.

An overlay technique is proposed for the identification and counting of haemolytic Listeria colonies directly on selective plating media. The technique was applied to different Listeria-selective plating media. In pure culture studies with collection strains, the overlay technique was more efficient and reliable for detection haemolytic Listeria species compared with the incorporation of blood into the agar. The efficacy of the overlay technique for the direct detection of haemolytic colonies of Listeria from raw milk samples was related to agar selectivity. The best results were obtained with Listeria-selective agar medium modified (LSAMM). Catalase assay, together with reactions for aesculin and tellurite, were useful and reliable criteria for the identification of Listeria. All colonies on LSAMM which were positive for catalase, tellurite and aesculin while those displaying typical haemolysis corresponded in most cases to L. monocytogenes.

Animals↗
PubMed2006

Surface plasmon resonance biosensor for direct detection of antibody against Epstein-Barr virus.

This paper describes the direct label-free detection of antibodies against the Epstein-Barr virus (anti-EBNA) using a surface plasmon resonance (SPR) biosensor. The antibody detection was performed using the immunoreaction between anti-EBNA and a respective synthetic peptide (EBNA-1), which was conjugated with bovine serum albumin (BSA-EBNA) and immobilized on the sensor surface. Three immobilization chemistries for the attachment of BSA-EBNA were investigated to optimize ligand density and minimize loss of EBNA-1 immunoreactivity. The developed SPR biosensor functionalized with the optimal immobilization method was calibrated and characterized in terms of detection limit, reproducibility, regenerability and storability. It was demonstrated that the sensor is capable of detecting concentrations of anti-EBNA as low as 0.2 ng/ml (approximately 1 pM) both in buffer and 1% human serum and can be stored and regenerated for repeated use.

Antibodies↗
PubMed1997

Direct detection of Mycobacterium tuberculosis complex in clinical samples from patients in Norway by ligase chain reaction.

Our aim was to investigate the use of DNA amplification with the ligase chain reaction (LCR) for detection of the Mycobacterium tuberculosis complex directly in human clinical specimens. The LCR assay employed was the Abbott LCx MTB Assay, which uses the gene encoding protein antigen b as the target template. Four hundred eighty-two samples from 457 patients in one clinical microbiology laboratory in Norway were processed by routine culture analysis (BACTEC culture), direct microscopy (Ziehl-Neelsen staining) and LCR. Of the 118 specimens containing cultivable M. tuberculosis, 106 (90.6%) were detected by LCR. Among the 364 culture-negative specimens, 356 samples were negative also by LCR and 8 (1.6%) were positive by LCR. In five of the eight LCR-positive and culture-negative samples, another sample from the same patient was M. tuberculosis culture positive and/or the patient had symptoms of tuberculosis. In comparison with culture, the sensitivity of LCR was 96.7% for smear-positive samples and 72.0% for smear-negative samples, respectively. For all samples combined, the sensitivity, specificity, and positive and negative predictive values were 90.2, 99.2, 97.4, and 96.7%, respectively. Challenging the M. tuberculosis LCR test with DNAs and cultures from strains of Mycobacterium ulcerans and Mycobacterium marinum, which are the mycobacterial species most closely related to the M. tuberculosis complex, resulted in all-negative test results. The sensitivity, specificity, and positive and negative predictive values of BACTEC culture in comparison with the LCR test and clinical criteria were 95.9, 100, 100, and 98.6%, respectively. A certain prioritization of samples subjected to the LCR assay should be based on clinical indications and risks with regard to infection transmission and patient isolation policy. More automation and lower expenses are generally desired for nucleic acid amplification kits. However, this M. tuberculosis LCR assay represents a valuable tool in routine mycobacterial diagnostics.

Bacteriological Techniques↗
PubMed1991

Carrier detection and prenatal diagnosis in 98 families of haemophilia A by linkage analysis and direct detection of mutations.

489 individuals from 98 families with a haemophilia A member were studied with restriction fragment length polymorphisms (RFLPs) for carrier detection and prenatal diagnosis. Five intragenic polymorphisms revealed with the restriction enzymes BclI, XbaI, BglI, HindIII and AlwNI and one extragenic multiallelic polymorphism (St14) at the DXS52 locus were used. The combination of the five intragenic polymorphisms did not add significantly more information than just the BclI and XbaI polymorphisms because of strong linkage disequilibrium. The sequences surrounding the intronic restriction sites of the BclI and XbaI RFLPs are known so they can be rapidly analysed using the polymerase chain reaction (PCR). 68.6% of the women were heterozygous for either the BclI or XbaI RFLP and this heterozygosity rate increased to 98.6% when the St14 extragenic polymorphism was included. Linkage analysis using these RFLPs led to the classification of over 90% of the women as carriers or normal and 98.6% of the carriers were heterozygous. Prenatal diagnosis was successful in the 16 foetuses tested and all could be classified as carrier, normal or haemophiliac. Five TaqI restriction sites in the coding region of the factor VIII gene can detect a C to T transition that results in an in-frame stop codon. These five sites were amplified by PCR in 119 haemophiliacs and tested for an abnormal TaqI restriction pattern. A stop codon was found in three haemophiliacs at exons 18, 22 and 24. The same analysis revealed three deletions, two involving the last exon 26 and one exons 23-26.

Alleles↗
PubMed2005

Direct detection of fetal cells in maternal blood: a reappraisal using a combination of two different Y chromosome-specific FISH probes and a single X chromosome-specific probe.

BACKGROUND: We have recently explored the detection of circulatory male fetal cells directly in maternal whole blood samples by fluorescence in-situ hybridization (FISH). In order to improve the efficacy of fetal cell detection, we have now examined whether this could be enhanced by the use of two different Y chromosome-specific FISH probes (alpha-satellite and classical satellite III regions) in combination with an X chromosome-specific FISH probe. METHODS: Nineteen maternal blood samples (median gestational age = 28 weeks, range = 12-37 weeks) were examined in a blinded manner. No enrichment procedure was performed. Following hypotonic treatment and Carnoy's fixation, total nucleated cells were examined by two color FISH with a single X and two Y chromosome-specific probes. Nine cases were examined in parallel by conventional XY-FISH. RESULTS: Fetal cell detection was superior when using two Y chromosome-specific probes (specificity = 75%; sensitivity = 91%) when compared to the conventional XY-FISH approach (specificity = 50%; sensitivity = 60%). CONCLUSIONS: Male fetal cells can be detected in most maternal blood samples examined. Specificity and sensitivity is improved when using a combination of single X and two Y chromosome-specific probes when compared to a conventional XY-FISH protocol.

Chromosomes, Human, X↗
PubMed1997

Rapid PCR with nested primers for direct detection of Campylobacter jejuni in chicken washes.

Rapid detection of Campylobacter jejuni by PCR directly from foods, without prior growth steps, would be beneficial for the poultry industry. We have previously reported a PCR assay that allows detection of this bacterium after 48 h growth on Campy cefex agar. We have now developed a more rapid nested PCR assay that specifically detects C. jejuni in chicken washes that have not undergone any lengthy growth steps prior to PCR. For the nested reaction, an external set of primers, C-1 and C-4, are used for 24 cycles. At this time, 1 microl of the PCR product is removed and added to a second reaction. The second PCR assay is run with C-1 and an internal primer, C-2, for 24 cycles. A single band on a 4% NuSieve agarose gel at 122 bp was apparent with C. jejuni cells at a sensitivity of 10(2) cfu ml-1. With this method chicken carcasses can be washed and C. jejuni identified all within 1 day. We detected C. jejuni in approximately 80% of four groups of chickens using this method. The identifications have been confirmed by standard microbiological techniques.

Agar↗
PubMed1992

Direct detection of hepatitis B virus from dried blood spots by polymerase chain reaction amplification.

The presence of hepatitis B virus DNA in the sera of individuals is the most definitive marker of an active viral infection. We have used polymerase chain reaction detection of hepatitis B virus DNA directly on whole blood dried as a spot on filter paper. The method is rapid, specific, and sensitive and has the ability to detect as little as 10 virus particles by ethidium bromide staining of the polymerase chain reaction-amplified products. The method is cost-effective, and the stability of the spots makes the collection and transportation of potentially infectious blood safe.

Base Sequence↗
PubMed1997

Direct detection of mutations in the breast and ovarian cancer susceptibility gene BRCA1 by PCR-mediated site-directed mutagenesis.

The tumor suppressor genes BRCA1 and BRCA2, which confer increased susceptibility to breast and (or) ovarian cancer, were recently identified. Mutation analysis of BRCA1 has demonstrated significant allelic heterogeneity; however, some distinct mutations have been detected in unrelated individuals. The most notable is the 185delAG mutation, which occurs at an estimated frequency of approximately 1% in individuals of Ashkenazi Jewish descent [1]. Although consensus has not been reached regarding clinical testing for mutations in BRCA1, a tiered strategy may be appropriate, in which direct testing for the more common mutations is one component. Specific alleles can be detected by using PCR-mediated site-directed mutagenesis (PSM), which alters the PCR products derived from either the wild-type or mutant allele to create or destroy a restriction endonuclease recognition site. Recognition sites are introduced by a base substitution in one of the primers. The alleles are then resolved by electrophoresis of the digested PCR products. We have applied this technique to the detection of four BRCA1 mutations: 185delAG, 5382insC, E1250X, and R1443X. Another mutation, 1294de140, can be resolved from the wild-type allele by high-resolution gel electrophoresis alone. The PSM technique is sensitive, does not require radioactivity, and is specific for individual mutations.

Alleles↗
PubMed1988

Laboratory diagnosis of Mycoplasma pneumoniae infection. 1. Direct detection of antigen in respiratory exudates by enzyme immunoassay.

Direct and indirect antigen capture enzyme immunoassays (Ag-EIA) have been developed for the detection of Mycoplasma pneumoniae in nasopharyngeal aspirates or sputum from respiratory infection. The sensitivity of the two Ag-EIA were similar, but the indirect method using polyclonal rabbit and guinea-pig antisera was more convenient. The Ag-EIA had a detection limit of 10(4-4.5) colony-forming units/ml of sample. It was specific for M. pneumoniae and gave a low level response with M. genitalium. There were no cross-reactions with 10 other species of mycoplasmas. Tests with a wide range of bacteria and chlamydia group antigen, representing agents sometimes found in the respiratory tract, were also negative. At the current level of development, the Ag-EIA detected about 90% of specimens that were also positive for culture; 43% of specimens from culture-negative--seropositive patients gave a positive result. The overall pattern of results indicated that while antigen detection is a quick and effective substitute for the slow culture method, serological examination for specific IgM antibody is also necessary to give a complete diagnostic coverage.

Antibody Specificity↗