No abstract

The act of selecting aptamers against blood serum leads to deep libraries of oligonucleotide sequences that bind to a range of epitopes in blood. In this study we developed an enriched aptamer library by performing positive selection against a pool of blood serum samples from transgenic mice (P301S) carrying the human tau gene and counter selecting against pooled blood serum from negative segregant (wild type) mice. We demonstrated that a large proportion of the aptamer sequences observed with next generation sequence (NGS) analysis were the same from selection round 5 and selection round 6. As a second step, we applied aliquots of the selection round 5 enriched library to blood serum from 16 individual mice for a single round of selection. Each of these individual libraries were characterized through NGS analysis and the changes in relative frequency in aptamers from transgenic mice versus wild type were used to construct a diagnostic fingerprint of the effect of the action of the transgene on the composition of blood serum. This study serves as a model for similar applications with human subjects.

In this study, we have used a direct immunoassay where the simple binding between antigen and an antibody is detected. Immunoassays were performed in a drop system, monitoring the frequency decrease of the quartz-crystal microbalance device because of mass increasing during immunoreaction. The QCM sensor was coated on both sides by gold electrodes, only one side of the crystal (liquid side) was in contact with the solution; the other side (contact side) was always dry. We tested a piezoelectric immunosensor for aflatoxin B1 (AFLA-B1) mycotoxin detection through the immobilization of DSP-anti-AFLAB1 antibody (AFLA-B1-Ab anti AFLAB1) on gold-coated quartz crystals (AT-cut/5 MHz). The DSP (3,3'-Dithiodipropionic-acid-diN -hydroxysuccinimide ester) was used for the covalent attachment of the proteins. The piezoelectric crystal electrodes were pretreated by DSP for 15 min, rinsed with water and dried in a gentle flow of nitrogen gas. Then the DSP-coated crystals were installed in a sample holder and exposed to the anti-AFLAB1 antibody and to the AFLA-BI. Frequency and resistance shifts (Δf and ΔR) were measured simultaneously. Δf versus AFLA-BI concentrations in the range of 0.5-10 ppb exhibited a perfect linear correlation with a coefficient of above 0.998.

No abstract

Agrobacterium tumefaciens-mediated gene transfer—actually the most used method to engineer plants—may lead to integration of multiple copies of T-DNA in the plant genome, as well as to chimeric tissues composed of modified cells and wild type cells. A molecular characterization of the transformed lines is thus a good practice to select the best ones for further investigation. Nowadays, several quantitative and semi-quantitative techniques are available to estimate the copy number (CN) of the T-DNA in genetically modified plants. In this study, we compared three methods based on (1) real-time polymerase chain reaction (qPCR), (2) droplet digital PCR (ddPCR), and (3) next generation sequencing (NGS), to carry out a molecular characterization of grapevine edited lines. These lines contain a knock-out mutation, obtained via CRISPR/Cas9 technology, in genes involved in plant susceptibility to two important mildew diseases of grapevine. According to our results, qPCR and ddPCR outputs are largely in agreement in terms of accuracy, especially for low CN values, while ddPCR resulted more precise than qPCR. With regard to the NGS analysis, the CNs detected with this method were often not consistent with those calculated by qPCR and ddPCR, and NGS was not able to discriminate the integration points in three out of ten lines. Nevertheless, the NGS method can positively identify T-DNA truncations or the presence of tandem/inverted repeats, providing distinct and relevant information about the transgene integration asset. Moreover, the expression analysis of Cas9 and single guide RNA (sgRNA), and the sequencing of the target site added new information to be related to CN data. This work, by reporting a practical case-study on grapevine edited lines, explores pros and cons of the most advanced diagnostic techniques available for the precocious selection of the proper transgenic material. The results may be of interest both to scientists developing new transgenic lines, and to laboratories in charge of GMO control.