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PubMed2026

A homogeneous immunoassay based on AlphaLICA technology for detecting florfenicol residues in animal-derived foods.

Florfenicol (FF), a broad-spectrum amide antibiotic widely used in livestock, poultry, and aquaculture, poses potential threats to food safety and public health due to its residual accumulation. In this study, a novel homogeneous immunoassay based on Amplified Luminescent Proximity Homogeneous Assay (AlphaLICA) technology was developed for the first time for rapid screening of FF residues in milk and egg matrices. By covalently immobilizing the FF-BSA conjugate and goat anti-mouse IgG onto luminescent and photosensitive microspheres, respectively, the method achieved wash-free, homogeneous quantitative detection through a competitive immunoreaction. Under optimized conditions, the assay exhibited a linear range of 0.2-16.2 ng mL-1, with a limit of detection of 9.7 pg mL-1 and a limit of quantification of 183 pg mL-1. The intra- and inter-batch coefficients of variation ranged from 3.08% to 5.70% and 2.44% to 7.09%, respectively. Spike recovery rates in milk and egg matrices ranged from 93.18% to 107.17% (RSD &#x2264; 5.57%). Cross-reactivity with 11 other common antibiotics, including chloramphenicol and thiamphenicol, was below 0.1%, demonstrating excellent specificity. Comparative analysis with a commercial ELISA kit showed high consistency (r2 = 0.9332, p < 0.001). With high sensitivity, strong specificity, simple operation, and a detection time of only 10 min, this method provides a reliable technical platform for high-throughput, rapid monitoring of FF residues in milk and egg matrices.

Journal Article↗
PubMed1998

[Molecular genetic diagnostics. Technology for detecting mutations in DNA].

Analyses of the genetic material in both inherited and acquired diseases such as cancer are already part of the repertoire of many laboratories. Over the last ten to fifteen years research has revealed a very complex human DNA, and shown that the molecular basis for disease is both intricate and variable. Methods for detecting the various categories of genetic changes are needed. These methods should be simple, quick and easy to perform, and cost-effective, and at the same time be both specific and sensitive. No single technology can fulfil all these criteria, and a variety of methods has therefore been introduced. In this overview we describe the different methods and their strengths and limitations.

Cost-Benefit Analysis↗
PubMed2001

Miniaturization of a hepatitis C virus RNA polymerase assay using a -102 degrees C cooled CCD camera-based imaging system.

Innovations in detection technologies have allowed us to develop a novel assay in 1536-well plate format and assess the advantages of screen miniaturization compared with conventional high-throughput compound screening in 96- or 384-well plates. An HCV RNA polymerase assay has been miniaturized in 1536-well plates by using a new detection technology known as LEADseeker homogeneous imaging system. It uses a -102 degrees C cooled charge-coupled device (CCD) camera and newly designed scintillation proximity microparticles. The miniaturized assay used europium-doped streptavidin-coated yttrium oxide (YO(x)) or polystyrene (PS) microspheres to capture biotin-labeled [(3)H]RNA product transcripts. Beads in proximity to the radioisotope convert the emitted beta(-) particles into photons having wavelengths in the red region of the visible spectrum, optimal for detection by the CCD camera. Because the camera collects light from all wells of the plate simultaneously, 1536-well plates are imaged as rapidly as 384-well plates, on the order of 10 min per plate. The assay has a signal to background of approximately 20-fold, satisfactory for high-throughput robotics screening. The enzyme kinetics and potency of a known inhibitor were similar to those obtained from the conventional assay using scintillation proximity assay (SPA) beads and a scintillation plate counter. Furthermore, the newly developed microbeads (emitting at 610 to 620 nm) are less prone to quenching effects caused by yellow-colored compounds, than conventional SPA beads or scintillation fluid (emitting at 400 to 480 nm region). Thus, the LEADseeker imaging system is a useful new tool for miniaturization of assays for high-throughput screening.

Cold Temperature↗
PubMed2005

Advanced qRT-PCR technology allows detection of the cholecystokinin 1 receptor (CCK1R) expression in human pancreas.

OBJECTIVES: To help clarify the controversy over the detection of expression of the cholecystokinin 1 receptor (CCK1R; CCKAR) in human pancreas. METHODS: Applied qRT-PCR to detect CCK1R expression using the SYBR green/Smart Cycler II and the QZyme oligonucleotide/ABI PRISM 7500 systems to detect CCK1R expressed message in highly purified cDNAs from human pancreas and other tissues. Samples of normal pancreas were obtained at operation (pancreaticoduodenectomy; Whipple's procedure) and used to ascertain the expression of CCK1R in human tissue and investigate donor individual variability in expression levels by semi-quantitative RT-PCR and scanning densitometry. RESULTS: We present molecular evidence obtained with advanced qRT-PCR technology that clearly establishes CCK1R expression in human pancreas. Amplification variation in individual human samples is documented here. By targeting different stretches of the sequence with several primer pairs, it was observed that SYBR green qRT-PCR failed to amplify efficiently over GGA-and GAA-rich nucleotide triplet regions, leading to false negative results. The QZyme system quantified the expression with the following distribution: stomach > small intestine approximately colon > brain approximately kidney > pancreas. CCK1R expression levels varied from undetectable, to high levels of expression, in individual samples collected from surgical specimens. CONCLUSION: CCK1R message can be conclusively detected and quantified in human pancreas cDNA by targeting the appropriate nucleotide sequence regions of this gene.

DNA, Complementary↗
PubMed1999

Technologies for detecting genetic polymorphisms in pharmacogenomics.

BACKGROUND: Pharmacogenomics is an emerging scientific discipline examining the genetic basis for individual variations in response to therapeutics. METHODS AND RESULTS: Genetic polymorphisms are a major cause of individual differences in drug response. Metabolic phenotyping can be accomplished by administering a probe drug or substrate and measuring the metabolites and clinical outcomes. However, this approach tends to be labor intensive and requires repeated sample collection from the individual being tested. Alternatively, genotyping allows determination of individual DNA sequence differences for a particular trait. Commonly used genotyping methods include gel electrophoresis-based techniques, such as polymerase chain reaction (PCR) coupled with restriction fragment length polymorphism analysis, multiplex PCR, and allele-specific amplification. Fluorescent dye-based high-throughput genotyping procedures are increasing in popularity, including oligonucleotide ligation assay, direct heterozygote sequencing, and TaqMan (Perkin Elmer, Foster City, CA) allelic discrimination. High-density chip array and mass spectrometry technologies are the newest advances in the genotyping field, but their wide application is yet to be developed. Novel mutations/polymorphisms also can be identified by conformation-based mutation screening and direct high-throughput heterozygote sequencing. CONCLUSIONS: Rapid and accurate detection of genetic polymorphisms has great potential for application to drug development, animal toxicity studies, improvement of human clinical trials, and postmarket monitoring surveillance for drug efficacy and toxicity.

Clinical Laboratory Techniques↗
PubMed2006

Sharpening the tools: a summary of a National Institutes of Health workshop on new technologies for detection of fetal cells in maternal blood for early prenatal diagnosis.

In 2003 the National Institute of Child Health and Human Development (NICHD) sponsored a workshop entitled "Sharpening the Tools", which was designed to explore the then current state of prenatal diagnosis and screening using fetal cells in maternal blood. The goals of the workshop were to: review the then current state of the field and assess present capabilities, identify future research needs and challenges in this area, identify promising new and innovative approaches for future exploration, and provide a written summary of the conference for public distribution. The workshop featured brief presentations by experts from a wide range of scientific fields and by innovative bioengineering and technology leaders from academic centers and private industry. The workshop was divided into presentations on target cells, target approaches for separation, genetic and protein analysis, and "out of the box" (bioengineering) approaches. The passage of time since the workshop has allowed an objective assessment of where the research has progressed. A 2006 update on the field is included at the end of the summary.

Blood↗
PubMed2001

New technologies to detect static and dynamic upper airway obstruction during sleep.

Increase in upper airway resistance is the main patho-physiological feature in the obstructive breathing disorders during sleep. Upper airway events may be divided into two main groups: static obstruction (apneas) and dynamic obstruction (hypopneas, flow limitation, and snoring). This classification is useful to provide better information about the patho-physiological mechanisms of obstruction and to better define the diagnostic tools necessary for detecting abnormal respiratory events during sleep. Detection of dynamic obstruction requires sensors with a good frequency response. As thermistors have a poor dynamic response, they are not efficient in detecting the dynamic obstruction but are good enough to detect static obstruction. Nasal prongs (NP) connected a to pressure transducer and the impedance signal measured by the forced oscillation technique (FOT) are relatively new tools to noninvasively investigate dynamic upper airflow obstruction during sleep. FOT provides a direct index of the magnitude of airway obstruction and, therefore, of the upper airway patency, even under conditions of no flow (apneas). NP are aimed at assessing flow. Thus, both techniques have a different scope. The main advantages of NP are that they are easy to use and do not require sophisticated technology, while FOT needs a more complex instrumentation. For clinical routine studies NP are probably the best and simplest method for assessing the different respiratory events during sleep. However, FOT would be particularly useful in selected applications such as assessing upper airway patency in some central apneas; interpreting the irregular pattern of breathing during REM sleep; in better characterizing the inspiratory flow-limited breaths classified as intermediate; and in studying upper airway mechanics.

Polysomnography↗
PubMed2004

Coronary artery spatial distribution of acute myocardial infarction occlusions.

BACKGROUND: Acute coronary occlusions leading to ST-segment elevation myocardial infarctions (STEMIs) are due primarily to rupture of atherosclerotic plaques. Present "vulnerable plaque" detection technology focuses on identifying individual plaques with no clear therapeutic plan beyond conventional risk factor reduction. We developed a spatial map of the distribution of acute coronary occlusions to test our hypothesis that plaque ruptures do not occur uniformly throughout the coronary tree. METHODS AND RESULTS: We analyzed 208 consecutive patients who presented to the Brigham and Women's Hospital with STEMI and mapped the location of the acute coronary occlusion. These occlusions were not uniformly distributed throughout each of the major epicardial coronary arteries but tended to cluster within the proximal third of each of the vessels (right coronary artery, P=0.001; left anterior descending artery, P=0.003; left circumflex artery, P=0.001). Furthermore, Poisson regression showed that for each 10-mm increase in distance from the ostium, the risk of an acute coronary occlusion was significantly decreased by 13% in the right coronary artery, 30% in the left anterior descending artery, and 26% in the left circumflex artery. CONCLUSIONS: Acute coronary occlusions leading to STEMI tend to cluster in predictable "hot spots" within the proximal third of the coronary arteries. Identification of these high-risk zones for acute coronary occlusions will lead to future advances in vulnerable plaque detection technology and potentially locally directed preventive strategies.

Coronary Angiography↗
PubMed2001

A whole new way of looking at things: the use of Dark Reader technology to detect fluorophors.

The Dark Reader optical system (Clare Chemical Research, Denver, CO, USA) uses relatively low intensity broad-band visible blue light in combination with broad-band optical filters to detect fluorescence with a level of sensitivity that often surpasses that of UV transilluminators and can rival that of laser-based scanners. Applications of DR (Clare Chemical Research) devices include the detection of DNA and SYBR-stained protein samples following, and also during, electrophoresis. Unlike laser-based imaging systems, the fluorescence is directly visible to the user as well as being fully compatible with charge-coupled device (CCD) and Polaroid camera-based detection and imaging. Additionally, the DR optical system functions well in multicolor fluorophor environments. Because the Dark Reader does not emit any UV light, the extent of DNA damage incurred when visualizing DNA samples is drastically reduced compared to the damage produced by a UV device and this can have a significant benefit on downstream cloning protocols. Furthermore, dye photobleaching is minimal, extending the length of time that a fluorescent sample is visible. The inherent flexibility of the DR optical system allows many different configurations of the Dark Reader to be constructed such as transilluminators, hand lamps and integrated transilluminator-electrophoresis units.

DNA↗
PubMed2003

Application of real-time polymerase chain reaction technology to detect prostatic bacteria in patients with chronic prostatitis/chronic pelvic pain syndrome.

To investigate the potential association between prostate infection and chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS), we used molecular approaches described in previous reports. These methods employed standard polymerase chain (PCR) reaction assays to provide a qualitative evaluation of prostatic bacterial species. Here, we report on the detection of prostatic bacteria using a real-time PCR. Template DNAs were examined from prostatic tissue samples from patients with CP/CPPS. Two PCR primer sets were used: one that amplifies a portion of all known bacterial ribosomal DNAs (16S rDNAs) and one that is specific for Escherichia coli as opposed to related, E. coli-like bacteria. The 16S rDNA real-time PCR assay detected bacterial DNAs in eight (26%) of 31 samples from patients with CP/CPPS, including three samples (10%) that were also positive by the E. coli real-time PCR assay. These E. coli positives were quantified at approximately 10(3) cfu/ml of tissue digested. Quantification, speed and specificity make real-time PCR a promising approach for the quantitative detection and identification of prostatic bacteria from CP/CPPS patients.

Biopsy↗
PubMed1987

DNA probe technology for detection of Haemophilus influenzae.

A 5 kb Haemophilus influenzae DNA fragment involved in penicillin-binding proteins expression was used as a probe for specific detection of H. influenzae strains. The 32p-labeled probe specificity was assessed by hybridization to bacterial dots and 75 strains were tested. All H. influenzae (18) and H. aegyptius (1) strains reacted very strongly with the probe. The H. influenzae serotypes tested (a, b, and non-typable strains) did not differ in their hybridization. Some hybridization was also found with the 12 other Haemophilus species tested as well as other Pasteurellaceae such as Actinobacillus lignieresii and Pasteurella multocida. Two other less related species (Klebsiella ozaenae and Providencia stuartii) also showed low hybridization. The probe detected as low as 10(5)-10(6) H. influenzae cells and 0.1 microgram of DNA in a dot sensitivity test. Hybridization to electroblotted, digested DNA from different species which reacted in the bacterial dot test revealed strong hybridization to H. influenzae and H. aegyptius only. This DNA probe should prove useful for H. influenzae and possibly H. aegyptius detection due to its high specificity and sensitivity under stringent hybridization conditions.

Bacterial Proteins↗
PubMed2004

Application of a molecular beacon-real-time PCR technology to detect Salmonella species contaminating fruits and vegetables.

An oligonucleotide probe that becomes fluorescent upon hybridization to the target DNA (molecular beacon; MB) was evaluated in a real-time polymerase chain reaction (PCR) assay to detect the presence of Salmonella species. As few as 1-4 colony-forming units (CFU) per PCR reaction could be detected. The capability of the assay to detect Salmonella species from artificially inoculated fresh-cut produce such as cantaloupe, mixed-salad, cilantro, and alfalfa sprouts was demonstrated. A comparison of two commercially available kits utilizing MB-PCR (iQ-Check, Bio-Rad Laboratories) and conventional Association of Official Analytical Chemists (AOAC)-approved PCR (BAX, Dupont Qualicon) was performed on artificially inoculated produce. As few as 4 CFU/25 g of produce were detected after 16 h of enrichment in buffered peptone broth. These assays could be carried out entirely in sealed PCR tubes, enabling a rapid and high-throughput detection of Salmonella species in a large number of food and environmental samples. This is the first report of the application of MB probe being used for real-time detection of Salmonella species in whole and fresh-cut fruits and vegetables.

Colony Count, Microbial↗
PubMed1986

The use of solid-state image sensor technology to detect and characterize live mammalian cells growing in tissue culture.

Arguments are presented that approximately 30 000 cells have to be measured in a single experiment to measure radiobiological parameters in a low-dose survival assay. For this purpose, a fully automated device capable of detecting and recognizing individual live unstained mammalian cells at a rate of 1 cm2/min is required. Specifications of such a system are derived and evidence is presented which suggests that this can best be carried out using a solid-state image sensor in the form of a linear array of photodetectors. The outline of the design and other potential uses of such a device are discussed.

Animals↗
PubMed2005

Loop-mediated isothermal amplification: an emerging technology for detection of fish and shellfish pathogens.

Fish and shellfish diseases are a constant threat to the sustainability and economic viability of aquaculture. Early diagnosis plays a vital role in management of fish and shellfish diseases. Traditionally, various biochemical and serological tests have been used for fish disease diagnosis. However, the time and expertise required for such diagnoses makes it difficult for aquaculturists to easily adopt them under production conditions. Polymerase chain reaction and probe-based nucleic acid detection have become increasingly popular in fish and shellfish diagnostics. Recently, a novel technique called loop-mediated isothermal amplification (LAMP) has been developed, which is highly sensitive and rapid. LAMP has been used for the detection of bacterial, viral, fungal and parasitic diseases in both animal and plants. In aquaculture, LAMP-based detection of pathogens like Edwardsiella tarda, E. ictaluri, Nocardia seriolae, Tetracapsuloides bryosalmonae, white spot syndrome virus and infectious haematopoietic necrosis virus have been reported. In this review, the application of LAMP for the detection of aquaculture-associated pathogens is discussed.

Animals↗
PubMed2000

The inflammation meter: novel technology to detect the presence of infection/inflammation in patients without leukocytosis but with increased leukocyte adhesiveness/aggregation.

OBJECTIVES: To reveal the presence of infection/inflammation in patients with relatively normal white blood cell count (WBCC) by using the leukocyte adhesiveness/aggregation test (LAAT). METHODS: The LAAT was performed by using a simple slide test and image analysis (Inflamet), the WBCC, by an electronic cell analyzer, C-reactive protein, by Laser nephelometry and CD11b/CD18 by whole blood flow cytometry. RESULTS: Forty out of a cohort of 121 patients with nonviral acute febrile illness had a WBCC within normal limits. The intensity of the inflammatory response in these individuals as judged by either C-reactive protein, or fibrinogen concentrations, erythrocyte sedimentation or polymorphonuclear leukocyte CD11b/CD18 expression was similar to that observed in patients with a leukocytic response. Our present finding that 63% out of the group with documented infection/inflammation and no leukocytosis had a significantly increased LAAT suggest that the lack of leukocytosis is in part a pseudoleukopenia, or is associated with some degree of uncompensated tissue leukostasis. CONCLUSIONS: The lack of a leukocytic response in a patient with nonviral infection/inflammation is by no means a sign of a less inflammatory response. The increased state of leukocyte adhesiveness/aggregation might help to disclose the presence of inflammation in these individuals.

Adult↗