BioTechniques最新文献

A disadvantage of colorimetric detection in nucleic acid amplification assays is the possibility that a colorblind individual may interpret colors differently than observers with full-color vision. Using an isothermal amplification assay, the ability of colorblind individuals to distinguish between positive and negative results for four dyes was tested. Five individuals with self-reported colorblindness and four with full-color vision reported their observations of the color of the solution. Although colorblind individuals may accurately interpret assay results, they were often not accurate in reporting the color. Hydroxynaphthol blue was the most problematic dye, and both phenol red and SYBR™ green were less troublesome. Consideration for colorblind individuals is warranted when developing an assay and training staff in its performance.

Skin and soft tissue infections caused by Pseudomonas aeruginosa are common acquired diseases in postpartum care. Many methods have been developed in recent years for detecting P. aeruginosa, but they are criticized for the drawbacks of labor-intensiveness, complicated operation and high cost. Here, a simple, sensitive and colorimetric assay for P. aeruginosa detection is described. The approach displays a green color for positive samples and colorless for target-free samples. The approach exhibits a wide detection range and a low limit of detection of 45 CFU/ml. Thus, the developed ligation-initiated multiple-signal amplification method may be used for on-site testing of pathogenic bacteria and assist in the early diagnosis of postpartum care skin infections.

[Formula: see text] Spatial genomics first emerged in the 1990s to map biological function data onto multidimensional images of embryo specimens and has continued to be instrumental in studying embryogenesis and understanding early human development in the decades since.

Working with recent isolates of human cytomegalovirus (HCMV) is complicated by their strictly cell-associated growth with lack of infectivity in the supernatant. Adaptation to cell-free growth is associated with disruption of the viral UL128 gene locus. The authors transduced fibroblasts with a lentiviral vector encoding UL128-specific-shRNA to allow the release of cell-free infectivity without genetic alteration. Transduced cells were cocultured with fibroblasts containing cell-associated isolates, and knockdown of the UL128 protein was validated by immunoblotting. Cell-free infectivity increased 1000-fold in isolate cocultures with UL128-shRNA compared with controls, and virions could be purified by density gradients. Transduced fibroblasts also allowed direct isolation of HCMV from a clinical specimen and cell-free transfer to other cell types. In conclusion, UL128-shRNA-transduced fibroblasts allow applications previously unsuitable for recent isolates.

The extraction of high-quality RNA from kenaf is essential for genetic and molecular biology research. However, the presence of high levels of polysaccharide and polyphenol compounds in kenaf poses challenges for RNA isolation. We proposed a simplified, time-saving and cost-effective method for isolating high quantities of RNA from various kenaf tissues. This method exhibited superior efficiency in RNA isolation compared with the conventional cetyltrimethylammonium bromide method and demonstrated greater adaptability to different samples than commercial kits. Furthermore, the high-quality RNA obtained from this method was successfully utilized for RT-PCR, real-time RT-PCR and northern blot analysis. Moreover, this proposed protocol also enables the acquisition of both high-quality and -quantity gDNA through RNase A treatment. In addition, the effectiveness of this approach in isolating high-quality RNA from other plant species has been experimentally confirmed.

Single cell cytometry has demonstrated plausible immuno-heterogeneity of mesenchymal stem cells (MSCs) owing to their multivariate stromal origin. To contribute successfully to next-generation stem cell therapeutics, a deeper understanding of their cellular morphology and immunophenotype is important. In this study, the authors describe MSCProfiler, an image analysis pipeline developed using CellProfiler software. This workflow can extract geometrical and texture features such as shape, size, eccentricity and entropy, along with intensity values of the surface markers from multiple single cell images obtained using imaging flow cytometry. This screening pipeline can be used to analyze geometrical and texture features of all types of MSCs across different passages hallmarked by enhanced feature extraction potential from brightfield and fluorescent images of the cells.

C-reactive protein (CRP) is a potential biomarker for evaluating inflammatory responses in patients receiving coronary artery bypass graft surgery. Here, the authors depict a sensitive and reliable colorimetric approach for CRP analysis. In this method, an aptamer specifically binds with CRP and an initiator sequence is released from an arch probe to activate signal amplification. The released initiator sequence hybridizes with the toehold section in the 'jellyfish' probe to form a blunt terminus to induce exonuclease III-assisted signal amplification. The method exhibited a low limit of detection of 1.32 ng/ml and high intraday and interday precision for CRP detection. In summary, this colorimetric approach may provide a potential alternative tool for the evaluation of inflammation in patients receiving coronary artery bypass graft and clinical diagnostics of disease.

Recombinant rabbit monoclonal antibodies (rabbit rAbs) have shown promise in various biomedical fields. However, it is challenging and costly to generate rabbit rAbs using traditional techniques. Here we describe a convenient and cost-effective method. Using this method, we generated rabbit rAbs against mouse soluble IL-6 receptor α with affinities in the range of 10^-9 to 10^-12 M. The presented method is suitable for industrial and academic scientists looking to customize rabbit rAbs for their research.

The Amerithrax investigation into anthrax letter attacks in the USA forever changed the game in microbial forensics. Here we review the techniques used, then and now, to neutralize bioterrorism threats. [Formula: see text].

With advancements in multicomponent molecular biological tools, the need for versatile, rapid and cost-effective cloning that enables successful combinatorial assembly of DNA plasmids of interest is becoming increasingly important. Unfortunately, current cloning platforms fall short regarding affordability, ease of combinatorial assembly and, above all, the ability to iteratively remove individual cassettes at will. Herein we construct, implement and make available a broad set of cloning vectors, called PlayBack vectors, that allow for the expression of several different constructs simultaneously under separate promoters. Overall, this system is substantially cheaper than other multicomponent cloning systems, has usability for a wide breadth of experimental paradigms and includes the novel feature of being able to selectively remove components of interest at will at any stage of the cloning platform.