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

<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>_<it>0</it>) that does not require the assumption of equal reaction efficiency between unknowns and standard curve.</p> <p>Results</p> <p>The <it>Cy</it>_{<it>0 </it>}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>_{<it>0 </it>}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>_{<it>0 </it>}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>_{<it>0 </it>}method gave significantly more accurate and precise results.</p> <p>Conclusion</p> <p>Our results demonstrate that <it>Cy</it>_{<it>0 </it>}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

ELISA kit for determination of egg white proteins : collaborative study

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

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

Advances in the subseasonal prediction of extreme events: relevant case studies across the globe

Extreme weather events have devastating impacts on human health, economic activities, ecosysytems, and infrastructure. It is therefore crucial to anticipate extremes and their impacts to allow for preparedness and emergency measures. There is indeed potential for probabilistic subseasonal prediction on timescales of several weeks for many extreme events. Here we provide an overview of subseasonal predictability for case studies of some of the most prominent extreme events across the globe using the ECMWF S2S prediction system: heatwaves, cold spells, heavy precipitation events, and tropical and extratropical cyclones. The considered heatwaves exhibit predictability on timescales of 3-4 weeks, while this timescale is 2-3 weeks for cold spells. Precipitation extremes are the least predictable among the considered case studies. Tropical cyclones, on the other hand, can exhibit probabilistic predictability on timescales of up to 3 weeks, which in the presented cases was aided by remote precursors such as the Madden-Julian Oscillation. For extratropical cyclones, lead times are found to be shorter. These case studies clearly illustrate the potential for event - dependent advance warnings for a wide range of extreme events. The subseasonal predictability of extreme events demonstrated here allows for an extension of warning horizons, provides advance information to impact modelers, and informs communities and stakeholders affected by the impacts of extreme weather events