Detection of targets
A target and labeling technology, applied in the field of analysis and detection of microorganisms and molecules, can solve the problems of lack of clinical specificity, lack of high clinical sensitivity and specificity to distinguish colonized patients, and low sensitivity of rapid toxin immunoassays.
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example 1
[0154] Example 1: Detection of Clostridium difficile toxin B in a sample
[0155] The method uses digital imaging to detect molecules labeled with fluorescently dyed nanoparticles without the need for amplification. A fluorescent nanoparticle label is illuminated to emit photons, which are collected using a 1:1f / 4 relay lens. The light emitted by the particles strikes a small cluster of pixels on the digital camera's CMOS chip, creating white spots in the final image. At low analyte concentrations, digital counting of individually labeled targets yields better signal-to-noise ratios than simply integrating the signal from the entire detection region. Non-magnified imaging allows imaging of large fields of view, enabling detection of small numbers of target molecules in large sample volumes within milliseconds.
[0156] The sample is first mixed with a diluent and a target-specific immunoreagent consisting of fluorescent and magnetic particles coated with complementary antibo...
example 2
[0157] Example 2: Evaluating Analysis Performance.
[0158] To assess the analytical sensitivity of the C. difficile toxin B test in fecal matrices, a pooled stool sample containing 14 randomly selected clinical samples when tested by the real-time PCR C. difficile test was used A negative result was given. Pooled samples spiked with C. difficile toxin B were tested in a series of two-fold dilutions. The detection limit of Clostridium difficile toxin B obtained by this method is 45pg / ml ( Figure 9 ). Similar results were also observed when using different pools of PCR-negative stool samples. At a toxin B concentration of 45 pg / ml, the reaction contained an approximately 100-fold excess of magnetic particles compared to the number of toxin B molecules. At this analyte concentration, the magnetic and fluorescent particles must be tethered together by a single toxin B molecule on average, demonstrating that the method detects individual molecules by imaging without amplifica...
example 3
[0160] Example 3: Detection and Mitigation of Matrix Effects by Assay Controls
[0161] Positive and neutralizing assay controls were designed to facilitate detection and subsequent mitigation of sample matrix effects. Assay control and toxin B tests were performed in parallel on equal aliquots containing the mixture of clinical sample and assay reagents. Positive controls contained defined amounts of incorporated toxin (100 pg). A deviation of the positive control signal from expected results indicates negative assay interference (assay inhibition). Neutralizing controls contain toxin B neutralizing antibodies that sequester toxin B in clinical samples, making it undetectable in the assay. In this way, the neutralization control differentiates specific signals originating from toxin B in the sample from non-specific signals. Nonspecific signals may be caused by analyte-independent deposition of fluorescent particles or autofluorescent sample components on the detection sur...
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