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

RORγt + Treg to Th17 ratios correlate with susceptibility to Giardia infection

Funder: Fundacion Alfonso Martin EscuderoFunder: RCUK | Biotechnology and Biological Sciences Research Council (BBSRC); doi: https://doi.org/10.13039/501100000268Funder: Isaac Newton Trust; doi: https://doi.org/10.13039/501100004815Abstract: Infections with Giardia are among the most common causes of food and water-borne diarrheal disease worldwide. Here, we investigated Th17, Treg and IgA responses, and alterations in gut microbiota in two mouse lines with varying susceptibility to Giardia muris infection. Infected BALB/c mice shed significantly more cysts compared with C57BL/6 mice. Impaired control of infection in BALB/c mice was associated with lower Th17 activity and lower IgA levels compared with C57BL/6 mice. The limited metabolic activity, proliferation and cytokine production of Th17 cells in BALB/c mice was associated with higher proportions of intestinal Foxp3+RORγt+ regulatory T cells and BALB/c mice developed increased RORγt+ Treg:Th17 ratios in response to G. muris infection. Furthermore, G. muris colonization led to a significantly reduced evenness in the gut microbial communities of BALB/c mice. Our data indicate that differential susceptibility to Giardia infections may be related to RORγt+ Treg controlling Th17 activity and that changes in the microbiota composition upon Giardia infection partially depend on the host background

Inflammation-induced formation of fat-associated lymphoid clusters

Fat-associated lymphoid clusters (FALCs) are a type of lymphoid tissue associated with visceral fat. Here we found that the distribution of FALCs was heterogeneous, with the pericardium containing large numbers of these clusters. FALCs contributed to the retention of B-1 cells in the peritoneal cavity through high expression of the chemokine CXCL13, and they supported B cell proliferation and germinal center differentiation during peritoneal immunological challenges. FALC formation was induced by inflammation, which triggered the recruitment of myeloid cells that expressed tumor-necrosis factor (TNF) necessary for signaling via the TNF receptors in stromal cells. Natural killer T cells (NKT cells) restricted by the antigen-presenting molecule CD1d were likewise required for the inducible formation of FALCs. Thus, FALCs supported and coordinated the activation of innate B cells and T cells during serosal immune responses

Complete genome characterization of two wild-type measles viruses from Vietnamese infants during the 2014 outbreak

A large measles virus outbreak occurred across Vietnam in 2014. We identified and obtained complete measles virus genomes in stool samples collected from two diarrheal pediatric patients in Dong Thap Province. These are the first complete genome sequences of circulating measles viruses in Vietnam during the 2014 measles outbreak

Genome sequences of a novel Vietnamese bat bunyavirus

To document the viral zoonotic risks in Vietnam, fecal samples were systematically collected from a number of mammals in southern Vietnam and subjected to agnostic deep sequencing. We describe here novel Vietnamese bunyavirus sequences detected in bat feces. The complete L and S segments from 14 viruses were determined