Optimizing single-tube nested PCR for enhanced genotyping of single cells and in vitro produced bovine embryos.

Single-tube nested PCR (STnPCR) is a technique that improves nested PCR, reducing potential contamination and false-positive results, enhancing the amplification sensitivity. Despite being commonly used for the detection of microorganisms, STnPCR can be a valuable tool for bovine genotyping, encompassing essential targets as ROSA26 and TSPY, pivotal in the fields of animal reproduction, genetic improvement, and transgenic research. The objective of this study was to improve and innovate STnPCR for gene detection in cattle. We aimed to detect the ROSA26 and TSPY genes using low-concentration DNA samples, including single cells, small cell groups (one to five cells), in vitro-produced embryos, and bovine tissue samples. Moreover, we refined STnPCR for gene detection in up to single cells by conducting sensitivity testing with different concentration ratios of internal and external primers. Successful amplification of the ROSA26 and TSPY genes was achieved across all tested primer concentrations, even in single cells, with more consistent results observed at lower primer concentrations. Additionally, simultaneous gene amplification was achieved through STnPCR multiplexing, representing the first study of multiplex STnPCR in cattle. These outcomes not only confirm its effectiveness in detecting genetic markers for animal genetic improvement and transgenic elements but also pave the way for its widespread adoption in reproductive studies in bovines.

Quantifying microplastics in fishes: The first case study contrasting the perspective of untrained and experienced researchers.

Microplastic studies hold a low explored potential for citizen science and environmental education, but methodological issues challenge data produced by non-specialists. We compared microplastic abundance and diversity in the red tilapia Oreochromis niloticus recovered by untrained students with those recovered by researchers that have experience of three years studying the incorporation of this pollutant by aquatic organisms. Seven students dissected 80 specimens and performed digestion of digestive tract in hydrogen peroxide. The solution was filtered and inspected under a stereomicroscope by the students and by two expert researchers. A control treatment consisted of 80 samples handled only by experts. The students overestimated the abundance of fibers and fragments. Striking differences in abundance and richness of microplastics were verified between the fish dissected by students and by expert researchers. Therefore, citizen science projects involving the uptake of microplastics by fish should provide training until a satisfactory level of expertise is reached.