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PubMed2004

PCR-based detection of enterotoxigenic Staphylococcus aureus in the early stages of raw milk cheese making.

AIMS: To define PCR-based detectability of Staphylococcus aureus in raw milk and intermediate products of raw milk cheese making in the presence of a complex background microflora by targetting different specific genes harboured by a single strain. METHODS AND RESULTS: The strain Staph. aureus FRI 137 harbouring nuc, sec, seg, seh and sei genes was used in this study. Raw milk artificially contaminated by different concentrations of Staph. aureus FRI 137 was employed in dairy processing resembling traditional raw milk cheese making. Samples of milk and curds were PCR-analysed after DNA extraction by targetting all the above genes. The pathogen was detected when the initial contamination was 10(4) CFU ml(-1) by amplification of nuc and seh genes. 10(5) and 10(7) CFU ml(-1) were needed when seg or sei and sec genes were targetted, respectively. Enrichment cultures from raw milk and curd samples proved to increase the detection limit of 1 log on average. CONCLUSIONS: The direct detection of the pathogen in the raw material and dairy intermediates of production can provide rapid results and highlight the presence of loads of Staph. aureus potentially representing the risk of intoxication. However, every target gene to be used in the analysis has to be studied in advance in a system similar to the real case in order to determine the level of contamination potentially predictable. SIGNIFICANCE AND IMPACT OF THE STUDY: The detection in real dairy systems of significant loads of Staph. aureus by multiple targets PCR can be more accurate.

Animals↗
PubMed2000

Quantitative immunocapture PCR assay for detection of Campylobacter jejuni in foods.

The rapid detection of food-borne bacterial pathogens as part of a quality control program is necessary for the maintenance of a safe food supply. In this report, we present our findings for an immunocapture PCR method for the detection of Campylobacter jejuni in foods. The method permits direct detection of the pathogen without an enrichment step and can be performed in approximately 8 h. Assay results are quantitative, and one cell in a milliliter sample can be detected. Application of the method to spiked milk samples and chicken skin washes did not affect the sensitivity of the assay.

Animals↗
PubMed1994

[Vestibular function in bilateral idiopathic progressive sensorineural hearing loss].

To investigate vestibular function in patients with bilateral progressive sensorineural hearing loss (BPSHL), we examined 5 cases using electronystagmography. Cases 1, 2 and 3 were adult type, cases 4 and 5 were juvenile type. All patients had dizzy spells in the early stage of the disease, and showed spontaneous nystagmus. Bilateral reduction of caloric response and very low VOR (vestibulo-ocular reflex) gain on rotation testing were observed in cases 1, 2 and 3. Case 4 showed right canal paresis on the caloric test and left directional preponderance on the rotation test. Case 5 showed good responses to both tests. Optokinetic afternystagmus (OKAN) in cases 2, 3 and 5 was not brisk, and cases 1 and 4 showed directional preponderance of OKAN. Although OKAN is useful for detecting directional preponderance, it does not always reflect peripheral vestibular function. We suggest the existence of two types of BPSHL which correlate with vestibular function. One type is associated with high grade vestibular dysfunction while, in the other, vestibular function is fair.

Adolescent↗
PubMed2002

Advances towards integrated biodetection systems for environmental molecular microbiology.

To overcome many of the limitations associated with indirect detection methods, new techniques for the sensitive, specific, and direct detection of nucleic acids are required in order to accurately and quantitatively ascribe phenotype/function to uncultivated microorganisms. However, if advanced diagnostic and detection systems are going to be applied in environmental microbiology, future "biodetection" technologies and systems must be developed not from the point of view of the detector, but from the unique aspects of the environmental sample and the entire analytical process. This article highlights recent advances in nucleic acid-based technologies, and looks towards future advances that may address the broad needs and conditions imposed by environmental molecular microbiology.

Bacteria↗
PubMedDate not supplied

Evaluation of a newly developed office-based stool test for detecting Helicobacter pylori: an extensive pilot study.

BACKGROUND/AIMS: Several strategies have been used to detect Helicobacter pylori (Hp) infection along two lines: 1) direct detection of the bacteria, and 2) detection of antigen-antibody assay against Hp. The non-invasive methods include ELISA test of serum, salivary and urine, urea breath test, and detection of Hp antigen in stool. The latter method (HpSA) has been proven reliable and better than the ELISA test, for it can detect current Hp infection and is suitable for post-treatment follow-up. Now, a new commercial kit, ImmunoCard STAT HpSA (Meridian Bioscience Europe) has been developed to detect stool Hp antigen. It is simpler and less time-consuming than HpSA. The aim was to examine whether ImmunoCard STAT HpSA is qualified for diagnosis of Hp infection. METHODOLOGY: 253 patients (163 men, 90 women, mean age: 53.3 +/- 13.9 y/o, range: 19-89 y/o) were enrolled in this study. All of them had undergone gastroendoscopy and urea breath test. 207 patients were diagnosed with peptic ulcer and 46 with gastritis. Stool samples were collected within 3 days of their visit for gastroendoscopy and were sent for the Immunocard test. RESULTS: 118 patients were diagnosed with Hp infection and of these, 113 were interpreted as positive by means of the Immunocard test. Among the other 135 patients without Hp infections, 123 were interpreted as negative by means of the Immunocard test. Sensitivity and specificity were 95.8% and 91.1%, and positive and negative predictive values were 90.4% and 96.1%. CONCLUSIONS: The ImmunoCard STAT HpSA had high sensitivity, and specificity and could be used for mass screening. We concluded that it is a rapid, simple, cheap, reliable, and non-invasive strategy to detect current Hp infection and can be used in post-Hp eradication follow-up in Taiwan.

Adult↗
PubMed2004

Detection of aquatic colloids in drinking water during its distribution via a water pipeline network.

Laser-induced Breakdown Detection (LIBD) is a highly sensitive method for the direct detection of nano-particles (colloids). During the detection process plasmas are generated on single particles by a focused laser beam, the resulting plasma light emissions are detected optically. The method is based on the difference in breakdown thresholds of liquid and solid matter, it is lower for solid material. The laser pulse energy is adjusted precisely so that in the pure liquid no breakdown events occur, and only in the presence of colloids is the breakdown threshold in the focal volume exceeded. The spatial distribution of several thousand recorded plasma flashes within the focal volume reveals the mean particle diameter. The evaluation of the number of breakdown events per number of laser pulses results in a breakdown probability, together with the particle size the concentration is calculated using specially-designed computer software. Compared to conventional laser light scattering methods the LIBD is approximately 6 orders of magnitude more sensitive for particles smaller than ca. 0.05 pm. Together with laser light obscuration the LIBD technique has been used successfully for the quantification of aquatic nano-particles during drinking water processing and its distribution via a pipeline network of nearly 1,700 km total length. In addition, the particulate content of several brands of mineral water has been investigated.

Colloids↗
PubMed1991

[The value of computerized tomography for neurosurgical treatment of traumatic frontobasal cerebrospinal fluid fistulas. 300 cases].

300 patients with traumatic frontobasal CSF-fistulae were treated neurosurgically from 1967 to 1989. CT was introduced in 1980, allowing a definitive detection of intracranial lesions including appraisal of cerebral edema, direct detection of fistulae (metrizamide-CT) and of brain hernias and far better imaging of osseous lesions, also of fine structures, such as a better apprasial of paranasal sinus pathology. Classical radiology is, however, still indispensable for imaging certain fracture types and localization of a pneumocranium. Computer Tomography allows an improvement of postoperative results, influencing indication, timing of operation, such as surgical approach.

Brain Injuries↗
PubMed1996

[Evaluation of a new detection method of Mycobacterium tuberculosis in clinical specimens by amplification of ribosomal RNA and its clinical application].

A new rapid M. tuberculosis detection method, the Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test (MTD), which allows direct detection of M.tuberculosis in clinical specimens by amplification of M.tuberculosis ribosomal RNA (rRNA), was evaluated. In the present study, MTD and conventional smear and culture examinations were performed on 225 sputum, 36 pleural fluid (PF), nine cerebrospinal fluid (CSF) and 41 bronchoalveolar lavage fluid (BALF) specimens. Among 225 sputum specimens from 132 patients, 20 culture-negative specimens from 14 patients and one specimen which was culture-positive for M.fortuitum on Ogawa's egg medium were MTD-positive. On the other hand, there were 6 M.tuberculosis culture-positive but MTD-negative specimens from 6 patients. The amount of bacilli in all six cases, however, was very few. The sensitivity of M.tuberculosis detection by MTD, as compared with Ogawa's egg medium and MB-Check, was 93 and 92%, respectively, in sputum specimens, and the specificity of MTD was 80 and 85%, respectively. Taking into account not only culture-positive specimens but also MTD-positive specimens from patients clinically diagnosed as active tuberculosis, the sensitivity and specificity of MTD were 93% and 99%, respectively. Among 36 PF specimens from 31 patients, of which 20 specimens from 16 patients were clinically diagnosed as tuberculous, only 4 from 4 patients were culture-positive for M.tuberculosis while 7 from 5 patients were MTD-positive. The results of MTD in 9 CSF specimens coincided well with those of MB-Check, but one MTD-positive specimen yielded a false-negative result with Ogawa's egg medium. Among 41 BALF specimens from 39 patients, only one was culture-positive while one culture-positive and 7 culture-negative specimens were positive on MTD. All these MTD-positive patients were later verified as having pulmonary tuberculosis either by transbronchial lung biopsy and other examinations, or by the clinical course of the disease, particularly the response to anti-tuberculosis drugs. Inconsistent results were seen in 21 sputum specimens from 15 patients, 4 PF specimens from 3 patients and 7 BALF specimens from 7 patients, all of which were culture-negative for M.tuberculosis but MTD-positive. None of these patients had any clinical findings inconsistent with diagnosis as tuberculosis. It was noted that MTD was very useful for rapid detection of M.tuberculosis in sputum as well as other clinical specimens. We observed the clinical course of 20 pulmonary tuberculosis patients by monthly examinations of sputum, using smears, Ogawa's egg medium, MB-Check and MTD. MTD-positivity rates fell down in parallel with decreased pulmonary tuberculous activity. During the clinical course, out of 61 MTD-negative specimens only 4 (6.6%) were culture-positive for M.tuberculosis, while out of 63 smear-and-culture-negative specimens 6 (9.5%) were MTD-positive. These data suggest that MTD is useful to assess the clinical activity and course of tuberculosis.

Adult↗
PubMed2006

Antibody-based surface plasmon resonance detection of intact viral pathogen.

Surface plasmon resonance (SPR) technique was used to directly detect an intact form of insect pathogen: the baculovirus, Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). An SPR sensor chip with three bio-functional layers was used to detect the intact AcMNPV: amine-reactive crosslinker with a disulfide bond that chemisorbs to gold film, Protein A, and a mouse IgG monoclonal antibody raised against a surface protein of the target viral pathogen. A two-channel (reference & test) micro-fluidic SPR system is used for reliable measurement. Bio-specific response to the AcMNPV is compared with the response for tobacco mosaic virus (TMV) as control. Successive exposure of the sensor chip to both viruses verifies a specific response to AcMNPV. This serves as a prerequisite to the development of a new type of viral pathogen detection sensors.

Antibodies, Viral↗
PubMed1985

Enzyme-linked immunosorbent assay for detection of enteric adenovirus 41.

An enzyme-linked immunosorbent assay (ELISA) for direct detection of enteric adenovirus 41 (Ad41) in stool specimens was developed and compared with an Ad40-specific ELISA described previously [Johansson et al, 1980]. Rabbit antiserum to Ad41 was obtained by immunization with purified virions. To eliminate genus-specific reactivity the serum was passed through an immunosorbent column containing soluble adenovirus components of members of subgenera A to E. The anti-Ad41 serum still displayed high reactivity against Ad40 and had to be immunoabsorbed with soluble virus components of Ad40 to be rendered type-specific. The absorbed antiserum was used in an indirect ELISA and proved to be specific for Ad41. No heterotypic reactivity against Ad40 or Ad1 through Ad35 was found. The Ad41-specific ELISA proved to be of equal sensitivity to electron microscopy. The type-specific ELISAs for Ad40 and Ad41 were evaluated by testing 76 stool specimens containing enteric adenoviruses originating from England and Scandinavia. All specimens could be typed--41 (54%) as Ad40 and 35 (46%) as Ad41. These results were confirmed by DNA restriction site analysis. The type-specific ELISA proved to be a specific, sensitive, and a rapid technique for detection of Ad41 and allowed clear-cut discrimination from Ad40 in clinical specimens.

Adenoviruses, Human↗
PubMed2004

Evaluation of a new NASBA assay for the qualitative detection of hepatitis C virus based on the NucliSens Basic Kit reagents.

BACKGROUND: Direct detection of HCV RNA by nucleic acid amplification methods is an essential tool in the diagnosis of HCV infections. In-house developed methods based on reverse transcribed polymerase chain reaction (RT-PCR) are widely used but they are laborious and usually lack the standardization required by clinical laboratories. OBJECTIVES: To evaluate the sensitivity and the clinical performance of an HCV specific nucleic acid sequence based amplification (NASBA) assay based on the commercially available, NucliSens Basic Kit (bioMérieux) reagents. STUDY DESIGN: The analytical sensitivity of the Basic Kit-based HCV assay (BK-HCV) was determined using dilutions of the First World Health Organization International Standard for HCV RNA. The performance of the BK-HCV was evaluated at two study sites in comparison with in-house RT-nested PCR (RT-nPCR) by testing a total of 77 plasma specimens. Additional HCV laboratory tests such as Amplicor HCV v2.0 (Roche Diagnostics) and genotype were also included in the comparative analysis. RESULTS: The sensitivity of the BK-HCV was 100-150 IU/ml HCV RNA (85-100% hit rate). When evaluating the clinical performance, we found 96-100% correlation between BK-HCV and RT-nPCR, and 85-91% correlation between BK-HCV and Amplicor. The level of efficiency of the BK-HCV for detecting prevalent HCV genotypes was equal to in house RT-nPCR and Amplicor. CONCLUSIONS: The BK-HCV offers adequate sensitivity for diagnostic purposes and equivalent clinical performance to in-house RT-nPCR assays. The BK-HCV could become a suitable alternative to the in-house amplification methods, providing standardized reagents and procedures, plus rapid results to clinical laboratories.

Clinical Laboratory Techniques↗
PubMed1993

Non-radioactively labelled DNA probes for the detection of periodontopathogenic Prevotella and Porphyromonas species.

Digoxigenin-labelled synthetic DNA probes directed against the 16S rRNA were used for the direct detection of the periodontopathogenic bacteria Prevotella intermedia and Porphyromonas gingivalis in subgingival plaque by applying a DNA-RNA dot-blot hybridization procedure. The test was evaluated with 134 plaque samples from 26 patients with adult periodontitis or rapidly progressive periodontitis. The lower limit of detection was 10(4)-10(5) bacteria/specimen. A semiquantitative assessment of the two species in each sample and in the corresponding periodontal site was achieved by this technique. It is possible to examine 80-90 samples within two days with low material costs.

Adult↗
PubMed1998

[Evaluation of immunochromatography-based rapid detection kit for fecal Escherichia coli O157].

"Quix" is an immunochromatography-based direct detection kit for the E. coli O157 LPS antigen in the patient's stool. The present study was conducted to evaluate the efficacy of the kit for rapid diagnosis of enterohemorrhagic Escherichia coli (EHEC) O157 infection. Sensitivity of the kit was determined using a pure culture of a clinical isolate of E. coli O157. Analytical sensitivity was found to be 5 x 10(5) CFU/ml. When compared with the culture method using fecal samples of 64 patients and with bloody diarrhea, sensitivity and specificity were 95.0% (19/20) and 86.4% (38/44), respectively, and overall agreement to culture method was 89.1% (57/64). One patient was found positive by culture method while negative in the present method, where the sample contained a low number of the cells less than the detection limit. Four of the six patients with a negative result by culture method and positive in the present method, were confirmed E. coli O157 infection by positive IgM antibody response against the E. coli O157 LPS. The discrepancy between the two methods seemed to be attributable to antibiotic administration. In one patient, Salmonella urbana (O30(1)30(2)) was detected. The O30(1) antigen of this bacterium is well known to be identical to the E. coli O157 antigen. When the present method was compared with an ELISA-based E. coli O157 LPS antigen detection kit, sensitivity and specificity were 100% (11/11) and 82.1% (23/28), and overall agreement to ELISA method was 87.2% (34/39). From these findings, Quix is useful as a rapid diagnostic kit in the primary clinics, outpatient or bedside use. E. coli O157 LPS antigen in patient's fecal samples can be detected in about five minutes with this simple procedure. Early diagnosis using such a simple kit will largely contribute for the early treatment and prevention of severe complications of the E. coli O157 infection.

Antigens, Bacterial↗
PubMed1993

Detection of hepatitis B virus in plasma using flow cytometric analyses of polymerase chain reaction-amplified DNA incorporating digoxigenin-11-dUTP.

Blood donations are routinely screened by multiple serologic assays for antigens/antibodies associated with infection by blood-borne viruses, including hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency viruses (HIV-1 and HIV-2), and human T-cell lymphotropic virus (HTLV-I and HTLV-II). A direct detection of these viruses would be more effective for the prevention of transfusion-transmitted infections than the indirect measurement of the variable host immune response to these agents. Because the polymerase chain reaction (PCR) for viral gene amplification offers the most sensitive and direct means of detecting viruses in blood, we have developed a nonisotopic PCR procedure for the detection of HBV, chosen as a prototype. The problems, common to previously described PCR methods, of nucleic acid extraction and inhibition of the PCR by plasma proteins were overcome by isolation of HBV from plasma by means of 450-microns polystyrene beads covalently coated with monoclonal antibody to the Pre-S1 region of the viral envelope protein. Detergent lysis and proteinase K digestion of the immunocaptured virions isolated from plasma released the HBV DNA. A modified PCR-amplification protocol, incorporating digoxigenin-labeled dUTP in the amplified gene products followed by hybridization with a specific biotinylated oligonucleotide probe bound to streptavidin-coated 2.8-microns magnetic beads, allowed flow cytometric analyses of HBV-specific PCR products by means of antibodies to digoxigenin labeled with fluorescein isothiocyanate. The endpoint serial dilutions of pedigreed human plasma samples containing chimpanzee infectious dose (CID50) of 10(7) for adw and CID50 of 10(7.5) for the ayw subtypes were compared in repeated testing of PCR products by our immunoreactive bead (PCR-IRB) assay. HBV DNA was consistently detected in a 5 x 10(-10) dilution of each sample. In testing 20 coded specimens of blood donors, with or without serologic markers of HBV infection, the PCR-IRB was specific and more sensitive than the PCR analyses by slot blot hybridization with radioactive probe. The PCR-IRB assay can be adapted for simultaneous detection of multiple blood-borne viruses by an automated flow cytometric analysis system.

Bacterial Proteins↗
PubMed1992

Evaluation of the bead enzyme-linked immunosorbent assay for detection of cholera toxin directly from stool specimens.

A highly sensitive bead enzyme-linked immunosorbent assay (bead ELISA) for detection of cholera toxin (CT) was evaluated for direct detection of CT from stool specimens of patients with acute secretory diarrhea. Of the 75 stool samples examined, 59 yielded biochemically, and serologically confirmed strains of Vibrio cholerae O1. The bead ELISA was positive for CT in stool supernatants in 50 (84.7%) of the 59 samples from which V. cholerae O1 was isolated. In addition, the bead ELISA was positive for three stool specimens which were negative by culture. The free CT present in 48 of the 50 stool samples positive by culture for V. cholerae O1 and for CT by bead ELISA was completely absorbed by anti-CT immunoglobulin G. All of the 59 strains of V. cholerae O1 biotype eltor isolated in this study produced in vitro CT. The concentration of CT present in the bead ELISA-positive stool samples ranged between 26 pg/ml and greater than 100 ng/ml. This evaluation study demonstrates that the bead ELISA is a sensitive and simple method for direct detection of CT in nonsterile stool samples, and we recommend routine use of this assay for detection of CT in stool samples and culture supernatants in clinical and reference laboratories.

Cholera Toxin↗
PubMedDate not supplied

Use of the Gen-Probe PACE system for the detection of Neisseria gonorrhoeae in urogenital samples.

The Gen-Probe PACE (Probe Assay-Chemiluminescent Enhanced) system for Neisseria gonorrhoeae was compared to Martin-Lewis medium in JEMBEC plates for the direct detection of N. gonorrhoeae in urogenital samples. This 2-hr, nonisotopic chemiluminescent test is based on the use of an acridinium ester-labeled DNA probe that binds with gonococcal target rRNA in a clinical sample. Following the separation of the hybridized probe from the unhybridized probe through the use of magnetic microparticles, the acridinium ester is hydrolyzed from the hybridized probe by the addition of an alkaline hydrogen peroxide solution, resulting in the production of 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 209 urethral and 203 endocervical specimens were collected from a high-risk, clinic population with a gonococcal disease prevalence of 24% during the study period. Statistical analyses of the overall results showed that, compared to culture, the Gen-Probe PACE System had a sensitivity, specificity, and positive and negative predictive values of 90%, 99%, 98%, and 97%, respectively. The comparative results of this study showed that the Gen-Probe PACE System for N. gonorrhoeae is a reasonable, noncultural alternative for detecting gonococci directly in urogenital specimens.

Cervix Uteri↗
PubMed2002

Solid-phase PCR in microwells: effects of linker length and composition on tethering, hybridization, and extension.

During the solid-phase PCR (SP-PCR), DNA oligonucleotides complementary to a soluble template and immobilized on a surface are extended in situ. Although primarily used for pathogen detection, SP-PCR has the potential for much broader application, including disease diagnostics, genotyping, and expression studies. Current protocols for SP-PCR in microwells are suitable for enzymatic detection of immobilized products, but yields are generally insufficient for direct detection of products using conventional fluorescent probes. Here, we quantitatively measure the outcome of tethering, hybridization, and solid-phase extension, and examine the effect of composition and length of the spacer at the 5' end of tethered oligonucleotides. Our results indicate that steric hindrance primarily affects polymerase activity rather than the efficiency of hybridization between the template and the tethered oligonucleotide. SP-PCR yields are significantly higher for a five-unit hexaethyleneglycol (HEG) spacer than for the more commonly used 10-residue deoxythymidine spacer. The optimal 5' HEG spacer resulted in a 60-fold increase in extension efficiency relative to a previously reported value for SP-PCR on a glass surface. Thus, optimized spacers should allow direct quantification of SP-PCR products, providing a simple, quantitative, and cost effective means of sample analysis for a variety of applications.

Arabidopsis↗
PubMed2006

Ultrasensitive native fluorescence detection of proteins with miniaturized polyacrylamide gel electrophoresis by laser side-entry excitation.

Direct detection of separated proteins inside polyacrylamide gels has many advantages compared to staining methods. Ultrasensitive native fluorescence detection of proteins with miniaturized 1-D and 2-D PAGE was achieved with laser side-entry excitation. The detection limit for R-phycoerythrin protein spots in 1-D SDS-PAGE with 532 nm excitation was as low as 15 fg, which corresponds to only 40,000 molecules. The average detection limit of six standard native proteins was 5 pg per band with 275 nm excitation. The dynamic range spanned more than three orders of magnitude. By using the same detection setup, approximately 150 protein spots from 30 ng of total Escherichia coli extraction were detected on a 0.8 cm x 1 cm gel in 2-D separation. The significant improvement in sensitivity for laser side-entry excitation comes from higher excitation power and lower background level compared with other excitation modes.

Electrophoresis, Gel, Two-Dimensional↗