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PubMed2002

Detection of genetically modified maize by the polymerase chain reaction and capillary gel electrophoresis with UV detection and laser-induced fluorescence.

In this paper, the possibilities of capillary gel electrophoresis (CGE) to detect transgenic maize in flours are shown. The method is based on the extraction and amplification by the polymerase chain reaction (PCR) of a specific DNA fragment from transgenic maize and its subsequent analysis by CGE with UV detection or laser-induced fluorescence (LIF). Some useful considerations regarding the optimization of DNA extraction and amplification conditions are given. Also, a comparison is established between the two CGE protocols for DNA detection based on ultraviolet absorption (CGE-UV) and LIF (CGE-LIF). The requirements, advantages, and limitations of both CGE methods are discussed. To our knowledge, this is the first paper on the use of CGE-LIF to detect transgenic food.

DNA Primers↗

PubMed2004

Detection and quantitation of genetically modified maize (Bt-176 transgenic maize) by applying ligation detection reaction and universal array technology.

We have applied the ligation detection reaction (LDR) combined with a universal array approach to the detection and quantitation of the polymerase chain reaction (PCR) amplified cry1A(b) gene from Bt-176 transgenic maize. We demonstrated excellent specificity and high sensitivity. Down to 0.5 fmol (nearly 60 pg) of PCR amplified transgenic material was unequivocally detected with excellent linearity within the 0.1-2.0% range with respect to wild-type maize. We suggest the feasibility of extending the LDR/universal array format to detect in parallel several transgenic sequences that are being developed for food applications.

Bacillus thuringiensis Toxins↗

PubMed2005

Development of a sensitive high-performance liquid chromatography-diode array detection assay for the detection and quantification of the beauveria metabolite oosporein from submerged culture broth and bio-control formulations.

A sensitive high-performance liquid chromatography-diode array detection (HPLC-DAD) assay is described for the detection and quantification of the Beauveria metabolite oosporein from fungal culture broth and two biocontrol agent formulations. In all cases, analyte recovery was achieved with a Britton-Robinson buffer system at pH 5.5 diluted with methanol 3:7 (v/v) (BR5.5-MeOH). The HPLC-DAD assay, using a binary solvent gradient with acidic modifiers and detecting the metabolite at 287 nm, showed linearity over 3 orders of magnitude and a limit of detection (LOD) of 6.0 +/- 2.3 microg of oosporein/L of BR5.5-MeOH. The oosporein content of the representative fungal culture broth samples and two Beauveria formulations (Melocont-Pilzgerste and Melocont-WP) was found to be 504.7 +/-13.6 mg of oosporein/L of culture filtrate, 7.4 +/- 0.6 mg of oosporein/kg of Melocont-Pilzgerste, and 38.2 +/- 1.3 mg of oosporein/kg of Melocont-WP with recovery rates of 93 +/- 2, 99 +/- 8, and 92 +/- 3%, respectively.

Benzoquinones↗

PubMed2006

High-sensitivity blood-based detection of breast cancer by multi photon detection diagnostic proteomics.

We have developed several new methods for blood-based cancer detection by diagnostic proteomics. Ultrasensitive methods of immunoassay using multiphoton-detection (IA/MPD) increase sensitivity by 200- to 1,000-fold (1 femtogram/mL). This has allowed the measurement of cancer biomarkers with very low concentrations in blood that could not be measured for full patient cohorts with conventional immunoassays. Sensitivity and specificity in cancer detection have been found to be potentiated by use of immunoassay panels which include tissue-specific cancer biomarkers as well as cytokines and angiogenic factors. The ultrasensitive immunoassays revealed that patient to patient variations in the concentrations of individual biomarkers in blood can extend over many orders of magnitude (up to six) and that the distributions of biomarker concentrations over patient cohorts are non-Gaussian. New methods of data analysis which correlate abundances of multiple, different biomarkers have been developed to deal with such data sets. Sensitivity and specificity of about 95% have been achieved for blood-based detection of breast cancer in pilot studies on 250 patients and 95 controls. Pilot studies indicate that this methodology may also allow differentiation of malignant breast cancer from benign lesions and can provide similar sensitivity and specificity for other epithelial cancers such as prostate cancer, ovarian cancer and melanoma. The methods developed for selection, application, and evaluation of very high sensitivity biomarker panels are expected to have general relevance for diagnostic proteomics.

Adult↗

PubMed2000

The detection of Salmonella using a combined immunomagnetic separation and ELISA end-detection procedure.

The aim of this study was to develop a rapid immunoassay to detect Salmonella bacteria. Skimmed milk powder (SMP) in buffered peptone water was inoculated with six Salmonella strains (Salm. typhimurium, Salm. virchow, Salm. enteritidis, Salm. give, Salm. ealing and Salm. arizonae) at three inoculum levels (about 2-200 cfu 25 g(-1) SMP) and incubated (37 degrees C) overnight. Heat-treated salmonella cells were immobilized on paramagnetic particles and detected within 3 h using the Salmonella genus-specific monoclonal antibody M105 in a microtitre plate based assay. The rapid Salmonella detection method combining immunomagnetic separation and ELISA had a total isolation and detection time of less than 24 h, which is significantly shorter than the conventional techniques requiring 72-96 h. The technique had a sensitivity limit of 10(5)-10(6) cfu ml(-1).

Animals↗

PubMed1997

A comparison of two antigen-detection ELISA for detecting infection of Dirofilaria immitis in dogs.

A survey on 87 dogs necropsied in the Townsville region revealed 34 (39%) were infected with Dirofilaria immitis. Infected dogs had an average of 6.1 adult worms in the heart and associated blood vessels. The VetRED assay detected 23 of the 34 infected dogs (sensitivity 65%) and the Og4C3 ELISA detected 27 (sensitivity 80%). Sensitivity of the VetRED and Og4C3 ELISA increased to 88 and 94% respectively in dogs with three or more worms. Both tests detected correctly all uninfected dogs. Despite the higher accuracy of the Og4C3 ELISA, compared to the VetRED assay, it is unlikely to be used widely as a field test for heartworm unless it can be modified from its present plate ELISA format which takes 4 hours, into one which is more rapid and convenient. However, as a reference ELISA, it may well be worthwhile in situations which require considerable accuracy for detecting D. immitis infection.

Animals↗

PubMed2003

Clinical performance of computer-assisted detection (CAD system in detecting carcinoma in breasts of different densities.

OBJECTIVES: To determine the clinical performance of a computer-assisted detection (CAD) system in detecting carcinoma in breasts of different densities. MATERIALS AND METHODS: A total of 264 sets of bilateral screening mammograms taken in craniocaudal and medial-lateral oblique projections during the year 1997 were divided into four groups according to the BI-RADS density classification: fatty (pattern 1), scattered fibroglandular (pattern 2), heterogeneously dense (pattern 3) and extremely dense (pattern 4). Each group contained about 60% normal and 40% biopsy-proven cancer cases. Of the malignant cases, there were a mixture of mammographic findings including focal masses (<2.5 cm), asymmetrical density, architectural distortion or microcalcifications. Films with artefacts and obvious masses>2.5 cm were not included. The chosen cases were then digitized and analysed by the CAD system. Sensitivity was calculated as detection of cancer by at least one marker in at least one view. Specificity was calculated as the number of false-positive marks per image on normal cases. Statistical tests of significance were performed by using contingency tables and Chi square test. RESULTS: The CAD system detected 14 out of the total 15 cancer cases in totally fatty breasts with a sensitivity of 93.3% at a specificity of 1.3 false-positive marks per image. In breasts with scattered fibroglandular pattern, the sensitivity was 93.9% (31/33) and the specificity was 1.6 false-positive marks per image while in heterogeneously dense breasts, the sensitivity of the CAD system fell to 84.8% at a specificity of 1.6 false-positive marks per image. The sensitivity of the CAD system further dropped to 64.3% in markedly dense breasts while maintaining a specificity of 1.2 false-positive marks per image. The decrease in sensitivity in dense breast was found to be significant (p=0.046). CONCLUSION: The sensitivity of the CAD system deteriorated significantly as the density of the breast increased while the specificity of the system remained relatively constant.

Breast Neoplasms↗

PubMed1999

A comparison of invasive breast cancer detected in a screened population with invasive breast cancers detected at rapid access diagnostic facilities.

The objective of this study was to assess the impact of rapid access breast diagnostic facilities on the benefit of mammographic breast screening. Invasive cancers were compared for maximum diameter in two groups: group A--70 patients, with screen detected breast cancer aged 50-65, mean age 55.8; group B--46 patients, with symptomatic breast cancer aged 50-65, mean age 56.7. Group A patients had all been invited for screening and included interval cancers and cancers in lapsed attenders. Group B patients were detected at rapid access diagnostic facilities. In group A (screened) the invasive cancers had a mean diameter of 17.8 mm, with a median of 15 mm. Group B (unscreened) consisted of invasive cancers with a mean diameter of 25.2 mm, and a median of 25 mm. Thirty-two (46%) of the screened cancers were found to be less than 15 mm compared to 5 (11%) of the unscreened cancers, using the Wilcoxon Rank Sum Test, P<0.001. Although rapid access modern diagnostic facilities for breast cancers offer the ability to detect early cancers (11% in this survey), screen detected invasive cancers remain significantly smaller even when interval cancers and cancers in lapsed attenders are included.

Journal Article↗

PubMed1997

Diagnostic Detection of Herpes Simplex and Hepatitis C Viral Amplicons by Capillary Electrophoresis: Comparison With Southern Blot Detection.

Background: As a result of the large number of DNA-based clinical assays, there is intense interest in making polymerase chain reaction (PCR)-amplified DNA product analysis faster, more cost-effective, and more automated. Methods and Results: In this study, an evaluation of the use of capillary gel electrophoresis with laser-induced fluorescence detection is described as a means of analyzing postamplification PCR products from clinical specimens. Sixty-six herpes simplex virus and 152 hepatitis C virus amplicons were analyzed after PCR and reverse transcription PCR, respectively. It is shown that the use of a physical gel buffer system in a short capillary in conjunction with laser-induced fluorescence detection allows for sensitive detection of herpes simplex virus- and hepatitis C virus-specific DNA fragments in an expedient manner. Interinstrument and intercapillary reproducibility of the migration time was evaluated and found to be excellent. The advantages and disadvantages over agarose gel electorphoresis-Southern blot analysis are summarized. Conclusions: The advantages offered by capillary gel electrophoresis with laser-induced fluorescent detection including rapid and sensitive analysis, ease of setup, reduced cost, and possibility for automation, make this procedure a viable alternative to more labor-intensive agarose gel electrophoresis-Southern blot analysis as molecular diagnostic methodology.

Journal Article↗

PubMed1995

Comparison of antigen detection, polymerase chain reaction and culture for detection of Chlamydia trachomatis in genital infection.

An in-house polymerase chain reaction (PCR) test using ribosomal RNA gene primers was compared with chlamydia antigen detection (DIF) and culture for the detection of Chlamydia trachomatis. Five hundred and forty-eight fresh (unstored) genital swabs and 174 urines (collected at the same time) from patients attending a sexually-transmitted diseases clinic were examined. PCR, DIF and culture detected chlamydia in 43, 35 and 42 swabs respectively from the 43 resolved positive cases. The specificity on the resolved negative specimens was 100% for each of the tests. From the urines, PCR and DIF detected the organism in 16 and 15 cases respectively of the 23 resolved positive males tested but in only 2 and 3 cases respectively of the 9 resolved positive females tested. Specificities were 100% in all cases. Both of the non-culture tests manifested problems with urine due to inhibitory activity (in PCR test) or excessive debris (in DIF test) in about 5% of the specimens. Culture of the urines yielded sensitivities of 40% in the males and 22% in the females. Overall PCR was more sensitive than either culture or DIF on both urethral and cervical swabs and urines. The urines yielded less than three-quarters the number of positives that was obtained from the swabs and were considered to be an unsatisfactory specimen for chlamydial diagnosis. It is concluded that PCR is a satisfactory alternative to culture on genital swabs and may be preferable in situations where the viability of the organisms is in question. DIF remains useful because of its speed and simplicity but is insufficiently sensitive to be relied upon by itself.

Adolescent↗

PubMed1998

Strategies for rare-event detection: an approach for automated fetal cell detection in maternal blood.

This article explores the feasibility of the use of automated microscopy and image analysis to detect the presence of rare fetal nucleated red blood cells (NRBCs) circulating in maternal blood. The rationales for enrichment and for automated image analysis for "rare-event" detection are reviewed. We also describe the application of automated image analysis to 42 maternal blood samples, using a protocol consisting of one-step enrichment followed by immunocytochemical staining for fetal hemoglobin (HbF) and FISH for X- and Y-chromosomal sequences. Automated image analysis consisted of multimode microscopy and subsequent visual evaluation of image memories containing the selected objects. The FISH results were compared with the results of conventional karyotyping of the chorionic villi. By use of manual screening, 43% of the slides were found to be positive (>=1 NRBC), with a mean number of 11 NRBCs (range 1-40). By automated microscopy, 52% were positive, with on average 17 NRBCs (range 1-111). There was a good correlation between both manual and automated screening, but the NRBC yield from automated image analysis was found to be superior to that from manual screening (P=.0443), particularly when the NRBC count was >15. Seven (64%) of 11 XY fetuses were correctly diagnosed by FISH analysis of automatically detected cells, and all discrepancies were restricted to the lower cell-count range. We believe that automated microscopy and image analysis reduce the screening workload, are more sensitive than manual evaluation, and can be used to detect rare HbF-containing NRBCs in maternal blood.

Automation↗

PubMed2000

Detection of cervical antibodies to human papillomavirus type 16 (HPV-16) capsid antigens in relation to detection of HPV-16 DNA and cervical lesions.

A more sensitive luminescence immunoassay (LIA) for human papillomavirus type 16 (HPV-16) was developed and used to measure HPV-16 antibodies in cervical samples from 292 college-aged women who were examined at 4-month intervals. Of the 609 collected samples, IgG, IgA, and secretory piece-associated antibodies to HPV-16 were detected in 12%, 6%, and 8%, respectively, of samples tested. Cervical IgG antibodies were most strongly associated with HPV-16 DNA detected within the previous 12 months (odds ratio, 3.3; 95% confidence interval, 1.4-7.8). Secretory IgA (cervical IgA- and secretory piece-positive) was most strongly associated with detection of a squamous intraepithelial lesions 4-8 months earlier (odds ratio, 6.4; 95% confidence interval, 1.9-21.8). As with serum HPV-16 antibodies, there appears to be a several-month delay between cervical HPV infection and detection of cervical antibodies.

Adult↗

PubMed2002

Quantitative detection of increasing HIV type 1 antibodies after seroconversion: a simple assay for detecting recent HIV infection and estimating incidence.

We have devised a simple enzyme immunoassay (EIA) that detects increasing levels of anti-HIV IgG after seroconversion and can be used for detecting recent HIV-1 infection. Use of a branched peptide that included gp41 immunodominant sequences from HIV-1 subtypes B, E, and D allowed similar detection of HIV-specific antibodies among various subtypes. Because of the competitive nature of the capture EIA, a gradual increase in the proportion of HIV-1-specific IgG in total IgG was observed for 2 years after seroconversion. This was in contrast to results obtained with the conventional EIA using the same antigen in solid phase, which plateaus soon after seroconversion. The assay was used to test 622 longitudinal specimens from 139 incident infections in the United States (subtype B) and in Thailand (subtypes B and E). The assay was also performed with an additional 8 M urea incubation step to assess the contribution of high-avidity antibodies. Normalized optical density (OD-n) was calculated (ODspecimen/ODcalibrator), using a calibrator specimen. An incremental analysis indicated that a cutoff of 1.0 OD-n and a seroconversion period of 160 days offered the best combination of sensitivity and specificity for classifying incident or long-term infections. The urea step increased the seroconversion period to 180 days with similar sensitivity and specificity. Separate analysis of B and E subtype specimens yielded the same optimal OD-n threshold and similar seroconversion periods. The assay was further validated in African specimens (subtypes A, C, and D) where the observed incidence was within 10% of the expected incidence. This assay should be useful for detecting recent HIV-1 infection and for estimating incidence among diverse HIV-1 subtypes worldwide.

AIDS Serodiagnosis↗

PubMed1997

The intraoperative detection of ovarian adenocarcinoma using radiolabeled CC49 monoclonal antibody and a hand-held gamma-detecting probe.

A phase II study to evaluate the safety and efficacy of the 125I-radiolabeled anti-TAG-72 monoclonal antibody, CC49, as a component of a system for the intraoperative detection of occult ovarian cancer deposits was carried out at the University of Nebraska Medical Center. Patients entered into the study were to have surgery for evaluation of their disease status. The primary objective of this study was to determine the ability of a gamma-detecting probe (GDP), the Neoprobe 1000, to intraopertively localize sites of disease not identified by traditional surgical or radiographic evaluation. It was postulated that improved detection of cancer foci might allow for therapeutic excision or might result in an alteration of subsequent treatment. Ten patients were enrolled in the study between May 1993 and March 1994. Nine of the patients were undergoing second-look surgery after completing primary chemotherapy. The remaining patient was having surgery to assess possible cancer recurrence. All patients received an intravenous injection of 2 mCi/1 mg 125I-radiolabeled CC49 without complication. After a mean of 24.5 days, the patients' background radiation counts were deemed low enough for accurate intraoperative cancer localization, and surgery was performed. Any visibly or palpably abnormal areas were biopsied after being evaluated with the GDP. Any areas suspicious for malignancy by GDP evaluation were also biopsied. Two patients without evident disease by radiographic or surgical examination had histologically confirmed metastases localized by the GDP. Four patients had obvious disease at surgery which was variably confirmed by the GDP; two of these patients had baseline elevations in circulating TAG-72 antigen levels that may have affected binding of antibody to the tumor. This system of radioimmunoguided surgery was well tolerated and practical in its application, and it permitted disease detection that resulted in potentially beneficial changes in patient management.

Adenocarcinoma↗

PubMed2003

HPLC separation of tetracycline analogues: comparison study of laser-based polarimetric detection with UV detection.

A sensitive and specific high-performance liquid chromatography (HPLC) method based upon laser-based polarimetric detection is developed for the determination of six tetracycline analogues. By interfacing the laser-based polarimeter online with an HPLC system, the specific rotation of each analogue is obtained as compounds elute from the separation system. The six structurally similar tetracycline analogues exhibit significant differences in specific rotations. The experiments suggest that specific rotation can be useful in identifying closely related tetracycline analogues. Linear relationships are found to be in the range of 0.342-0.0043 mg for the tetracycline analogues. Five of the six analogues exhibit excellent linearity (R(2) value >/= 0.99). The polarimetric results are compared with UV detection. The HPLC-laser-based polarimetric detection instrument is able to quantitate the studied tetracycline analogues with high precision, accuracy, and sensitivity, which make it useful for the development of a standard method for the determination of tetracyclines in biological specimens. The performance of the HPLC-polarimetric system for the analysis of tetracyclines in a biological matrix is evaluated. The selectivity of polarimetric detection provides a distinct advantage in the analysis of tetracycline analogues in milk. The HPLC-polarimetric system provides a rapid and sensitive technique that involves minimal sample cleanup and pretreatment for the analysis of tetracyclines in milk.

Chromatography, High Pressure Liquid↗

PubMed1993

Diagnosis of congenital syphilis by combining Treponema pallidum-specific IgM detection with immunofluorescent antigen detection for T. pallidum.

Most infants at risk for congenital syphilis can be identified easily by a positive maternal serologic test for syphilis. However, a diagnosis of congenital syphilis can be difficult to make in an individual infant. Seven infants with delayed-onset congenital syphilis and 101 newborns at risk for congenital syphilis were evaluated for characteristic physical findings and laboratory-detectable abnormalities of congenital syphilis. By using a combination of Western blot for T. pallidum-specific IgM detection and immunofluorescent antigen detection for the identification of T. pallidum, a diagnosis of congenital syphilis was made in all 7 infants with delayed-onset congenital syphilis and 24 of the 101 newborns at risk for congenital syphilis. However, T. pallidum-specific IgM was negative in 6 of the 24 newborns with congenital syphilis. T. pallidum-specific IgM detection alone is inadequate for the diagnosis of congenital syphilis.

Antibodies, Bacterial↗

PubMed1989

Highly sensitive detection of DNA using enzyme-linked DNA-probe. 1. Colorimetric and fluorometric detection.

In order to develop non-radioactive oligonucleotide derivatives and to examine their utility as a diagnostic tool, namely as DNA-probe, an enzyme-linked oligonucleotide was synthesized. Oligonucleotide complementary to M13mp8 phage DNA was linked to alkaline phosphatase via a crosslinker and a spacer. M13mp8 phage DNA (single strand) immobilized on the nitrocellulose membrane was hybridized with the enzyme-linked oligonucleotide. The hybrid was detected with three detection methods; (1)colorimetric detection in solution, (2)colorimetric one on membranes, and (3)fluorometric one in solution. Methods(2) and (3) gave high sensitivities to detect as low as several to several tens attomoles of DNA and it was found that those methods with enzyme-linked oligonucleotides are potent for DNA-probe methodology from the viewpoint of automation.

Chromatography, Gel↗

PubMed2004

Prostate cancer detection in the prostate specific antigen range of 2.0 to 3.9 ng/ml: value of percent free prostate specific antigen on tumor detection and tumor aggressiveness.

PURPOSE: We evaluated the positive predictive value and cancer detection rate in the prostate specific antigen (PSA) range of 2.0 to 3.9 ng/ml and assessed the value of percent free (F) PSA (FPSA) on tumor detection and tumor aggressiveness in this low PSA range. MATERIALS AND METHODS: Of 3623 men who were attending the second round of screening within the European Randomized Study of Screening for Prostate Cancer, section Rotterdam 883 had PSA values of 2.0 to 3.9 ng/ml. These men were offered laterally directed sextant biopsy. FPSA was prospectively determined from pretreatment serum. Cancers were classified as prognostically favorable and unfavorable using biopsy results and other pretreatment diagnostic features. RESULTS: Using the PSA range of 2.0 to 3.9 ng/ml as a biopsy indication 126 cancers were detected, resulting in a positive predictive value of 17.1% and a cancer detection rate of 14.3%. By using percent FPSA and setting relative sensitivity at 95% 9% of biopsies could have been avoided. Unfavorable tumor characteristics were found in 46.9% of the men with T1C tumors. Mean percent FPSA was significantly lower in such men compared to men with favorable tumor characteristics. Of the men with percent FPSA lower than 10% 90% had unfavorable tumor characteristics. CONCLUSIONS: The PSA range 2.0 to 3.9 ng/ml is accessible for prostate cancer screening. Percent FPSA is of moderate value in avoiding unnecessary biopsies in the PSA range of 2.0 to 3.9 ng/ml. However, when assessing tumor aggressiveness in biopsy results, percent FPSA is predictive and can be used to select treatment options, such as watchful waiting.

Aged↗