24 research outputs found

    A new real-time PCR method to overcome significant quantitative inaccuracy due to slight amplification inhibition

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    <p>Abstract</p> <p>Background</p> <p>Real-time PCR analysis is a sensitive DNA quantification technique that has recently gained considerable attention in biotechnology, microbiology and molecular diagnostics. Although, the cycle-threshold (<it>Ct</it>) method is the present "gold standard", it is far from being a standard assay. Uniform reaction efficiency among samples is the most important assumption of this method. Nevertheless, some authors have reported that it may not be correct and a slight PCR efficiency decrease of about 4% could result in an error of up to 400% using the <it>Ct </it>method. This reaction efficiency decrease may be caused by inhibiting agents used during nucleic acid extraction or copurified from the biological sample.</p> <p>We propose a new method (<it>Cy</it><sub><it>0</it></sub>) that does not require the assumption of equal reaction efficiency between unknowns and standard curve.</p> <p>Results</p> <p>The <it>Cy</it><sub><it>0 </it></sub>method is based on the fit of Richards' equation to real-time PCR data by nonlinear regression in order to obtain the best fit estimators of reaction parameters. Subsequently, these parameters were used to calculate the <it>Cy</it><sub><it>0 </it></sub>value that minimizes the dependence of its value on PCR kinetic.</p> <p>The <it>Ct</it>, second derivative (<it>Cp</it>), sigmoidal curve fitting method (<it>SCF</it>) and <it>Cy</it><sub><it>0 </it></sub>methods were compared using two criteria: precision and accuracy. Our results demonstrated that, in optimal amplification conditions, these four methods are equally precise and accurate. However, when PCR efficiency was slightly decreased, diluting amplification mix quantity or adding a biological inhibitor such as IgG, the <it>SCF</it>, <it>Ct </it>and <it>Cp </it>methods were markedly impaired while the <it>Cy</it><sub><it>0 </it></sub>method gave significantly more accurate and precise results.</p> <p>Conclusion</p> <p>Our results demonstrate that <it>Cy</it><sub><it>0 </it></sub>represents a significant improvement over the standard methods for obtaining a reliable and precise nucleic acid quantification even in sub-optimal amplification conditions overcoming the underestimation caused by the presence of some PCR inhibitors.</p

    Diagnostic performance of PET/computed tomography versus PET/MRI and diffusion-weighted imaging in the N- and M-staging of breast cancer patients

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    Objective To provide a systematic review regarding the diagnostic performance of 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography/magnetic resonance imaging (PET/MRI) and diffusion-weighted imaging (DWI) compared to 18F-FDG PET/computed tomography (CT) focused on nodal and distant staging in breast cancer patients. Methods ThePubMedandEmbasedatabases were searched for relevant publications until April 2020. Two independent reviewers searched for eligible articles based on predefined in- and exclusion criteria, assessed quality and extracted data. Results Eleven eligible studies were selected from 561 publications identified by the search. In seven studies, PET/CT was compared with PET/MRI, and in five, PET/CT with DWI. Significantly higher sensitivity for PET/MRI compared to PET/CT in a lesion-based analysis was reported for all lesions together (77% versus 89%) in one study, osseous metastases (69-99% versus 92-98%) in two studies and hepatic metastases (70-75% versus 80-100%) in one study. Moreover, PET/MRI revealed a significantly higher amount of osseous metastases (90 versus 141) than PET/CT. PET/CT is associated with a statistically higher specificity than PET/MRI in the lesion detection of all lesions together (98% versus 96%) and of osseous metastases (100% versus 95%), both in one study. None of the reviewed studies reported significant differences between PET/CT and DWI for any of the evaluated sites. There is a trend toward higher specificity for PET/CT. Conclusion In general, there is a trend toward higher sensitivity and lower specificity of PET/MRI when compared to PET/CT. Results on the diagnostic performance of DWI are conflicting. Rather than evaluating it separate, it seems to have complementary value when combined with other MR sequences

    ELISA kit for determination of egg white proteins : collaborative study

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    An interlaboratory study was conducted in 11 laboratories to validate an ELISA method developed for the quantitative determination of egg white proteins (EWPs) in foods. The ELISA kit used for this study is based on sheep polyclonal antibody. It does not produce any false-positive results or cross-reactivity in a broad food matrix range with zero EWP content. All participants obtained the Egg ELISA Kit-native with standard operational procedure and the list of samples, as well as the samples and a protocol for recording test results. The study included 10 food samples. Four samples of food matrix with zero EWP content showed EWP content lower than the first standard (EWP content 0.5 mg/kg). One sample of food matrix with zero EWP content revealed EWP content higher than standard 3 (1.5 mg EWP/kg). Five food samples containing EWP as an ingredient tested positive and one negative. The statistical tests (Cochran, Dixon, and Mandel) and analysis of variance were used to evaluate the interlaboratory study results. Repeatability and reproducibility limits, as well as LOQ (1.4 mg EWP/kg) and LOD (0.43 mg EWP/kg), were calculated for the kit

    Hepatitis B virus exposure in human immunodeficiency virus seropositive Cuban patients

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    In order to estimate the prevalence of serological markers of exposure to Hepatitis B Virus (HBV), 295 subjects were selected at random from the National Registry of human immunodeficiency virus positive subjects. Evidence of exposure to HBV was defined as: testing Hepatitis B surface antigen (HBsAg) and anti-Hepatitis B core antigen (anti-HBc) positive or anti-HBc positive only. Overall, 133 (45.5%) were positive for anti-HBc and 15 (5.1%) resulted positive to HBsAg. Significant statistical association was found between male sex and exposure to HBV (p<0.01). Homosexual or bisexual behavior was found to be strongly associated to HBV exposure (p<0.001). In conclusion, the prevalence of HBV serological markers is higher in Cuban HIV positive subjects compared to the Cuban general population
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