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PubMed1977

High performance liquid chromatographic separation and direct ultraviolet detection of phospholipids.

A fast and efficient method for the separation of (phospho)lipids by high performance liquid chromatography using n-hexane, 2-propanol, water mixtures as the solvent system is described. The lipid separation occurs on a LiChrosorb Si-60 (10 micron) column and the individual components are monitored directly by ultraviolet absorption at 206 nm. Of a total lipid extract from erythrocytes a complete separation is achieved of cholesterol, phosphatidic acid, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, lysophosphatidylcholine and lysophosphatidylethanolamine, whereas phosphatidylcholine and sphingomyelin are only partly separated under these circumstances. Furthermore, a mixture of synthetic phospholipids, i.e. 1,2-dilauroyl-sn-glycero-3-phosphatidic acid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-1'-sn-glycerol and 1,2-dioleoyl-sn-glycero-3-phosphocholine has been completely resolved. In addition to separation of phospholipids in different classes, separation of molecular species can also be achieved in some cases, as is shown for 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine and 1,2-didocos-13'-cis-enoyl-sn-glycero-3-phosphocholine.

Animals↗
PubMed2001

Rapid detection of 3500Q and 3531 mutations and MspI polymorphism in exon 26 at the apolipoprotein B gene.

Several environmental and genetic factors are associated with high levels of cholesterol. Hypercholesterolemia is the main phenotype of Familial Defective Apolipoprotein B and Familial Hypercholesterolemia that are caused by mutations at the apolipoprotein (apo) B and LDL receptor genes, respectively. Identification of the specific genetic alteration associated with hypercholesterolemia is an important issue in clinical diagnosis of high risk for CAD. Apo B gene mutations and polymorphisms are usually screened by SSCP, DGGE, and heteroduplex, which must be confirmed by DNA sequencing or by direct detection using PCR techniques. In this study, we have optimized a PCR-RFLP procedure for identification of 3500Q and 3531 mutations and MspI polymorphism at the apo B gene. The technique can be performed in a single reaction, using the restriction endonuclease MspI for simultaneous detection of 3500Q mutation and MspI polymorphism, and NsiI for detection of 3531 mutation. The procedure was validated by analysis of control DNA samples from individuals carrying these mutations. Screening of 186 Brazilian hypercholesterolemic individuals showed that the frequency of the M-allele (7.8%) of MspI polymorphism was similar to that found in other individuals with CAD. However, neither 3500Q nor 3531 mutations were detected in this group. In conclusion, this procedure is simple and rapid, being easily introduced in clinical laboratories for direct detection of the more frequent mutations at the apo B gene associated with hypercholesterolemia.

Alleles↗
PubMed2004

Direct electrical detection of hybridization at DNA-modified silicon surfaces.

Electrochemical impedance spectroscopy was used to investigate the changes in interfacial electrical properties that arise when DNA-modified Si(111) surfaces are exposed to solution-phase DNA oligonucleotides with complementary and non-complementary sequences. The n- and p-type silicon(111) samples were covalently linked to DNA molecules via direct Si?C linkages without any intervening oxide layer. Exposure to solutions containing DNA oligonucleotides with the complementary sequence produced significant changes in both real and imaginary components of the electrical impedance, while exposure to DNA with non-complementary sequences generated negligible responses. These changes in electrical properties were corroborated with fluorescence measurements and were reproduced in multiple hybridization-denaturation cycles. The ability to detect DNA hybridization is strongly frequency-dependent. Modeling of the response and comparison of results on different silicon bulk doping shows that the sensitivity to DNA hybridization arises from DNA-induced changes in the resistance of the silicon substrate and the resistance of the molecular layers.

Biosensing Techniques↗
PubMed2002

Measurement of urinary N-acetyl-S-(N-methylcarbamoyl)cysteine by high-performance liquid chromatography with direct ultraviolet detection.

A new high-performance liquid chromatographic (HPLC) method is described for the determination of urinary N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC), the final product of the conjugation reaction between a metabolic intermediate of N,N-dimethylformamide (DMF) and glutathione. Urine samples were purified by C(18) solid-phase extraction and then directly analysed by HPLC with an Aminex Ion Exclusion HPX-87H column maintained at 25 degrees C and a UV detector set at 196 nm. Under isocratic conditions (2.4 mM sulphuric acid, flow-rate=0.6 ml/min) AMCC eluted at 20.2 min. The reproducibility (C.V.%) was 1.3-2.7% (intra- and inter-assay, N = 5); the accuracy was 98.0+/-1.7% at 10 mg/l and 101.9+/-1.5% at 800 mg/l (mean+/-SD, N = 3). AMCC was measured in urine from 22 exposed subjects. A strong correlation was found between AMCC and environmental DMF [AMCC (mg/g creatinine)=3.40xDMF (mg/m(3)) + 3.07; r=0.95], while in the urine of 20 unexposed subjects the concentration of AMCC was constantly below the detection limit of the method (0.9 mg/l in urine). The method described appears to be useful for the biological monitoring of DMF exposure.

Acetylcysteine↗
PubMed2002

The expression of hepatitis-C virus antigen by immunohistochemical stain and polymerase chain reaction in corneas of seropositive corneal donors.

Background: Of the donor corneas rejected for transplantation, the largest group is that from donors testing seropositive for hepatitis C virus (HCV). In situations of severe shortage in supply of donor corneal tissue, we may consider the use of seropositive donors for transplantation if we can prove with high certainty the absence of HCV RNA in the donor corneal tissue. Polymerase chain reaction (PCR) is a highly sensitive and specific technique for direct detection of HCV RNA and can be used for this purpose. Nevertheless, it is not applicable for routine clinical use in most eye departments due to its unavailability and cost effectiveness. Purpose: To study the possible use of immunohistochemical method for detection of HCV antigen in corneal tissue of seropositive donors and correlate the results with those of PCR. Immunohistochemical methods have not yet been studied in donor corneal tissue. Materials and methods: Eight corneas of 4 seropositive and 8 corneas of 4 seronegative corneal donors were studied by immunohistochemical and PCR methods for the presence of HCV antigen in their corneal tissue and sera. Results: HCV RNA was not detected in the sera and corneal tissue of all seropositive and seronegative corneal donors by either PCR and immunohistochemical methods. Conclusion: Although the study is too small for conclusive results, the correlation between the immunohistochemical and PCR studies for direct detection of HCV antigen in corneal tissue of seropositive donors may raise the possibility of using the immunohistochemical method for screening of donor corneas for the detection of HCV antigen. A larger prospective study investigating the sensitivity, specificity and clinical applicability of the immunohistochemical method is warranted.

Journal Article↗
PubMed1997

Equine herpesvirus 4 DNA in trigeminal ganglia of naturally infected horses detected by direct in situ PCR.

Neuronal and lymphoid tissues of 15 randomly selected horses were analysed post mortem by liquid nested-PCR to study the tropism of equine herpesvirus 4 (EHV-4). In four animals the trigeminal ganglia and in one case the lung were positive. Using a direct in situ PCR the EHV-4 genome was localized in the nuclei of neurons and in the bronchiolar as well as alveolar epithelium of the lung. In none of these tissues could infectious virus or viral antigens be detected. Applying the more sensitive liquid RT-PCR, however, an acute infection was demonstrated in one of the trigeminal ganglia by amplification of viral transcripts coding for glycoprotein B. The failure to detect these transcripts in the other trigeminal ganglia and the lung indicates a latent infection. This report formally proves that, like other members of the Alpha-herpesvirinae, EHV-4 establishes latency in the trigeminal ganglia.

Animals↗
PubMed1998

Comparison of the ligase chain reaction with solid and liquid culture media for routine detection of Mycobacterium tuberculosis in nonrespiratory specimens.

The aim of this study was to compare the results of a commercial assay based on the ligase chain reaction [(LCR) LCx Probe System MTB; Abbott, USA] with those of culture in liquid medium (Septi-Chek AFB; Becton-Dickinson, USA) and culture on the egg-based Löwenstein-Jensen solid medium for the direct detection of Mycobacterium tuberculosis complex in nonrespiratory specimens. The results were analyzed according to the standard definition of a true-positive result. Two hundred thirty-five nonrespiratory samples routinely submitted to rule out tuberculosis were analyzed. All samples were smear-negative. Mycobacterial growth in either culture medium was detected in 18 (7.6%) specimens: Mycobacterium tuberculosis was recovered from seven (38.9%) specimens cultured on Löwenstein-Jensen medium and from 18 (100%) specimens cultured in Septi-Chek AFB. The LCR protocol was positive in 22 specimens. None of the LCR-negative controls showed positive results. All samples positive by culture on Löwenstein-Jensen medium were positive by culture in liquid medium and by the LCR assay. However, Mycobacterium tuberculosis was detected by culture in liquid medium in two specimens that were negative by the LCR assay, whereas six specimens negative by culture in liquid medium were positive by the LCR protocol; three of these were identified as true-positive results of the LCR assay. The sensitivity, specificity, and positive and negative predictive values were 33.3%, 100%, 100%, and 93.8%, respectively, for Löwenstein-Jensen medium; 85.7%, 100%, 100%, and 98.6% for the liquid medium; and 90.4%, 98.5%, 86.3%, and 99% for the LCR assay. These findings indicate that the LCR assay may be a valid method of high diagnostic yield for direct detection of Mycobacterium tuberculosis complex in nonrespiratory specimens.

Body Fluids↗
PubMed2002

Determination of membrane Peptide orientation by 1H-detected 2H NMR spectroscopy.

We demonstrate the application of the proton inverse detected deuteron (PRIDE) NMR technique to the measurement of the orientation of membrane-bound peptides with enhanced sensitivity. Gramicidin D, a transmembrane peptide, and ovispirin, a surface-bound peptide, were used as model systems. The peptides were 2H-labeled by 1H/2H exchange and oriented uniaxially on glass plates. The directly detected 2H spectra of both peptides showed only a strong D(2)O signal and no large quadrupolar splittings. In contrast, the PRIDE spectrum of gramicidin exhibited quadrupolar splittings as large as 281 kHz, consistent with its transmembrane orientation. Moreover, the large D(2)O signal in the directly detected 2H spectra was cleanly suppressed in the PRIDE spectrum. For ovispirin, the 1H indirectly detected 2H spectrum revealed a 104 kHz splitting and a zero-frequency peak. The former reflects the in-plane orientation of most of the helix axis, while the latter results from residues with a magic-angle orientation of the N-D bonds. These are consistent with previous 15N NMR results on ovispirin. The combination of PRIDE and exchange labeling provides an economical and sensitive method of studying membrane peptide orientations in lipid bilayers without the influence of D(2)O and with the ability to detect N-D bonds at the magic angle from the bilayer normal.

Antimicrobial Cationic Peptides↗
PubMed2006

Development and evaluation of a rapid, simple, and sensitive immunochromatographic assay to detect thermostable direct hemolysin produced by Vibrio parahaemolyticus in enrichment cultures of stool specimens.

Thermostable direct hemolysin (TDH) is considered to be a major virulence factor in Vibrio parahaemolyticus, and most cases of V. parahaemolyticus diarrhea in humans are caused by tdh gene-positive strains. In the present study, we developed an immunochromatographic assay to detect TDH (TDH-ICA) and evaluated the utility of TDH-ICA for the diagnosis of V. parahaemolyticus diarrhea. TDH-ICA allowed the detection of 0.2 ng/ml of TDH within 10 min. Fecal homogenates were spiked with various numbers of tdh-positive V. parahaemolyticus organisms, and their enrichment cultures were tested with TDH-ICA. The results of detection of TDH in the enrichment cultures by TDH-ICA were in accord with the results of recovery of the spiked V. parahaemolyticus organisms from the enrichment cultures by plating onto thiosulfate-citrate-bile salts-sucrose agar. When enrichment cultures of 217 stool specimens from patients with diarrhea were tested with TDH-ICA, the TDH-ICA results showed 100% sensitivity and specificity compared to the results of isolation of V. parahaemolyticus from the stool specimens by a conventional bacterial culture test. Since TDH-ICA was able to detect TDH in a fecal enrichment culture within 10 min, TDH-ICA testing of a fecal enrichment culture could be completed rapidly and easily within approximately 16 h, including incubation time for the fecal enrichment culture. These results indicate that TDH-ICA is a rapid, simple, and sensitive TDH detection method and that TDH-ICA testing of a fecal enrichment culture is useful as an adjunct to facilitate the early diagnosis of V. parahaemolyticus diarrhea.

Animals↗
PubMed2006

Detection and enumeration of Salmonella enteritidis in homemade ice cream associated with an outbreak: comparison of conventional and real-time PCR methods.

Salmonellosis caused by Salmonella Enteritidis (SE) is a significant cause of foodborne illnesses in the United States. Consumption of undercooked eggs and egg-containing products has been the primary risk factor for the disease. The importance of the bacterial enumeration technique has been enormously stressed because of the quantitative risk analysis of SE in shell eggs. Traditional enumeration methods mainly depend on slow and tedious most-probable-number (MPN) methods. Therefore, specific, sensitive, and rapid methods for SE quantitation are needed to collect sufficient data for risk assessment and food safety policy development. We previously developed a real-time quantitative PCR assay for the direct detection and enumeration of SE and, in this study, applied it to naturally contaminated ice cream samples with and without enrichment. The detection limit of the real-time PCR assay was determined with artificially inoculated ice cream. When applied to the direct detection and quantification of SE in ice cream, the real-time PCR assay was as sensitive as the conventional plate count method in frequency of detection. However, populations of SE derived from real-time quantitative PCR were approximately 1 log higher than provided by MPN and CFU values obtained by conventional culture methods. The detection and enumeration of SE in naturally contaminated ice cream can be completed in 3 h by this real-time PCR method, whereas the cultural enrichment method requires 5 to 7 days. A commercial immunoassay for the specific detection of SE was also included in the study. The real-time PCR assay proved to be a valuable tool that may be useful to the food industry in monitoring its processes to improve product quality and safety.

Colony Count, Microbial↗
PubMed1994

The new diagnostic mycobacteriology laboratory.

Recent surveys in the USA show that many mycobacteriology laboratories continue to use less-than-optimum culture and susceptibility testing methods. This seems to be true for European countries as well. The past few years have brought significant changes to the clinical tuberculosis laboratory. High-performance liquid chromatography and direct detection of acid-fast bacilli in clinical specimens aim at the same goal: increased sensitivity and specificity of the diagnostic approach and reduction of turnaround time. This review outlines a brief comparison between contemporary traditional methods and the latest developments in the direct detection of acid-fast bacilli. If patient care and public health are always considered paramount, regardless of admission time, hospital type, etc., the current concept of services has several shortcomings. One way to manage this situation is to sort and allocate specimens according to a system of priorities. There is a growing realization that no single method by itself is the best. To streamline the best choice for laboratory diagnosis, an additional dynamic acid-fast network is presented: 'Point-of-Care,' 'Fast Track,' and 'Specialty' laboratories. The physician interacts with all three types of laboratories, so ongoing communication between the physician and the laboratory is essential. Laboratorians must work together in the formation of this dynamic acid-fast network to improve service rendered for our patients.

Bacteriological Techniques↗
PubMed1994

Cytomegalovirus and cervical cancer: failure to detect a direct association or an interaction with human papillomaviruses.

To investigate the possibility of human cytomegalovirus (HCMV) involvement in the aetiology of cervical carcinoma or in the development of a more clinically aggressive cancer cell phenotype, biopsies of cervical cancer from 103 women undergoing primary therapy for invasive (Stage Ia to IV) cervical cancer were investigated using a polymerase chain reaction (PCR) designed to detect sequences from the IE mtrII region of HCMV. PCR assays were also used on the same specimens to identify the presence of common human papillomavirus (HPV) types associated with cervical cancer (HPV 16, 18, 31, 33, 52, and 58). Of the 103 cancers examined, only 4 contained detectable HCMV DNAs, a proportion lower than that found by another Australian group investigating the cervical carriage of HCMV in women with normal cervices. In contrast, 89 of these cancers were positive for HPV DNA sequences, with HPV 16 (65/103) and HPV 18 (17/103) being most commonly detected. Three of the 4 HCMV-positive tumors were also positive for HPV 16 DNA. An examination of the relevant histopathological and clinical data revealed no evidence to support a contention that cancers positive for HCMV are associated with any unusual histologic cell types or with aggressive clinical behavior.

Adenocarcinoma↗
PubMed1985

Three theories of stroboscopic motion detection.

The three theories derive from three different paradigms. Suprathreshold judgements of perceived quality of motion in multi-flash displays are modelled by space-time Fourier analysis of the motion stimulus. Stroboscopic motion is perceived as being different from real motion to the extent that the additional Fourier components in stroboscopic motion are detectable. Stroboscopic motion of dots along conflicting paths leads to perceptual competition. The theory to describe perceptual I solution derives and proves the uniqueness of strength functions computed only from the time and from the distance between successive points on each path. Time-strength and motion-strength add to determine path-strength; only the strongest path is perceived. Motion-direction detection in continuously drifting two-flash combinations of sinusoidal gratings is described by elaborated Reichardt detectors (ERDs) that compute the covariance of temporal events in two adjacent locations. Other apparently different, detectors that account for direction-detection data are shown to be equivalent to ERDs.

Light↗
PubMed1999

Cloning of Mycoplasma synoviae genes encoding specific antigens and their use as species-specific DNA probes.

A genomic library of Mycoplasma synoviae (MS) was generated by using bacteriophage lambda gt11 as a cloning and expression vector. Identification of recombinant clones highly specific to MS was achieved by screening the library for expression of MS proteins with polyclonal antiserum that had been preadsorbed with 6 heterologous avian mycoplasma species antigens. Expression of the recombinant clones in Escherichia coli followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the total cell lysates and immunoblot yielded a predominant reactive fusion protein of 165 kD. Two clones (MS2/28 and MS2/12) that yielded inserts of different size were selected. The 2 MS DNA inserts were subcloned in a plasmid vector, labeled with digoxigenin, and used as probes for the specific recognition of several MS strains. A high degree of conservation was demonstrated for the MS2/12 and MS2/28 genes in tested MS strains. In addition, neither DNA fragment recognized any other avian mycoplasma species (M. gallisepticum, M. meleagridis, M. gallinarum, M. iners, M. anatis, and M. iowae), thus indicating their high specificity to MS. The sensitivity of the slot blot hybridization method using digoxigenin-labeled MS2/12 and MS2/28 probes for direct detection of MS from broth cultures of field isolates was 10(5) colony-forming units/ml. These results demonstrate the effectiveness of adsorbed antisera for the isolation of species-specific mycoplasma DNA and the potential for its use as probes for the specific and direct detection of MS from broth cultures of field isolates.

Animals↗
PubMed1998

Detection and direct typing of herpes simplex virus in perianal ulcers of patients with AIDS by PCR.

The presence of herpes simplex virus type 1 (HSV-1) and HSV-2 in perianal ulcerations of 41 AIDS patients was assessed by virus culture and a type-specific PCR-based assay. HSV was isolated from the lesion site in 24 of 41 (58.5%) patients, and HSV DNA was detected by PCR in all 24 (100%) of these specimens. Additionally, PCR was used to detect HSV DNA in 12 of 17 (70.5%) HSV culture-negative samples. Thus, HSV genomic sequences could be demonstrated in 36 of 41 (87.8%) perianal ulcers in this series. Full agreement in HSV typing by either immunodot assay or PCR was seen in 24 samples that were positive by both virus culture and PCR. HSV-2 was demonstrated in 35 of 36 (97.2%) HSV-positive samples.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Gradients as visual primitives.

Following J. J. Gibson (1950), it is implicitly assumed in the literature that texture gradients are directly available as perceptual primitives. Yet, the depth response to compression gradients is poor compared with gradients of linear perspective. This may indicate that mechanisms for directly detecting the differential structure that constitutes a compression gradient do not exist. We tested this hypothesis outside the context of depth perception by measuring the speed with which participants could detect a gradient anomaly as a function of the number of elements in the gradient. Only in the case of linear perspective did anomalies "pop out." This was attributable to the emergent feature of alignment of the ends of the elements forming the gradient rather than the direct detection of its differential structure. It is argued that gradients are not perceptual primitives and that the poor depth response to compression in a variety of context (motion parallax, stereo, and perspective) therefore is not surprising.

Adult↗
PubMed1991

Detection of direct mutagenicity of cigarette smoke condensate in mammalian cells.

Mutagenicity of cigarette smoke condensate (CSC) and the acidic, basic and neutral fractions of CSC was examined in the AL hybrid cell, a Chinese hamster ovary cell containing one human chromosome 11. Since the human chromosome 11 is not necessary for survival of the AL cells, mutations involving large deletions and chromosomal loss by non-dysjunction are non-lethal events that are detectable by loss of human cell surface antigens (a1, a2 and a3) encoded by genes on chromosome 11p (a1 and a3) and 11q (a2) through an antibody-complement lysis assay. Exposure of AL cells to CSC without exogenous metabolic activation caused a dose-dependent cytotoxicity and mutagenicity. Mutagenicity also increased with time of incubation up to 3 h with a maximum of 300 a1- mutants/10(5) survivors (250% above background; P less than 0.0005) after incubation with 100 micrograms/ml CSC. Cytotoxicity and mutagenicity of CSC were inversely proportional to cell density. Fifty percent lethal doses for the acidic, basic and neutral fractions of CSC after 3 h of incubation were 30, 100 and 240 micrograms/ml respectively, and the acidic fraction at a concentration of 25 micrograms/ml induced 350 a1- mutants/10(5) survivors (230% above background; P less than 0.0005); the basic and neutral fractions were less mutagenic. These results indicate that CSC and fractions of CSC can directly produce a spectrum of mutations, through both deletional and non-dysjunctional mechanisms of a kind known to lead to inactivation of tumor suppressor genes.

Animals↗
PubMed1996

Heterozygote and mutation detection by direct automated fluorescent DNA sequencing using a mutant Taq DNA polymerase.

We describe a method for direct cycle sequencing of PCR fragments amplified from genomic DNA or cDNA. DNA sequencing template is amplified using PCR and oligonucleotide primers flanking the region of interest. The amplified fragment is directly cycle sequenced using fluorescent sequencing primers, Sanger dideoxy sequencing chemistry and an enzyme mixture of a mutant Taq DNA polymerase and thermostable pyrophosphatase. The sequence ladders produced are analyzed on a real-time, automated four-color sequencing system. The method produces sequence ladders from unpurified PCR fragments of sufficiently high quality such that heterozygotes can be reproducibly detected and identified by software that recognizes signal-strength patterns indicative of mixed-base positions.

Base Sequence↗