Characterization and Validation of a Lyophilized Loop-Mediated Isothermal Amplification Method for the Detection of Esox lucius.

In many ways, globalization is beneficial, but in one way, it promotes the spread of alien (invasive) species through international trade and transport. In different habitats, Esox lucius (northern pike) can be considered a regionally alien species, and this fish tends to establish a higher density population than desired in fresh water. Early identification of such invasive species using sensitive and quick methods is important to be able to take immediate measures and avoid environmental problems. Loop-mediated isothermal amplification (LAMP) has emerged as the best DNA/RNA detection technique, without any expensive equipment and could be used to detect environmental DNA (eDNA). However, the reagents for amplification are not stable at ambient temperature for field applications. Therefore, this work aims to lyophilize the entire reaction mixture as a single microbead, with enzyme, and LAMP primers towards the detection of mitochondrial cytochrome B (Cyt B), a housekeeping gene in Esox lucius. Analytical and molecular techniques were performed to characterize and validate the lyophilized beads, respectively. The lyophilized beads were stored at two different temperatures, at 20 °C and 4 °C, and tested for biological activity after different time intervals. The result shows that lyophilized beads are bioactive for almost 30 days when stored at 20 °C, while beads at 4 °C did not lose their bioactivity after storage for up to one year. This study will be particularly useful for conducting on-site LAMP analyses in the field, where resources for freezing and storage are limited.

Comparative evaluation of loop-mediated isothermal amplification and PCR for detection of Esox lucius housekeeping genes for use in on-site environmental monitoring.

Esox lucius (northern pike) is an invasive species in fresh water and causes extreme impacts in the local habitat. Northern pike easily replaces the local native species and disrupts the regional ecosystem. Traditionally, in connection with environmental monitoring, invasive species are identified using PCR through species-specific DNA. PCR involves many cycles of heating to amplify the target DNA and requires complex equipment; on the contrary, loop-mediated isothermal amplification (LAMP) entails isothermal amplification, which means the target needs to be heated to only one temperature between 60 and 65°C. In this study, the authors conducted a LAMP assay and a conventional PCR assay to determine which technique is less time consuming, more sensitive and reliable for use in real-time and on-site environmental monitoring. Mitochondrial gene cytochrome b, an essential factor in electron transport; histone (H2B), a nuclear DNA responsible for the chromatin structure; and glyceraldehyde 3-phosphate dehydrogenase involved in energy metabolism are taken as the reference genes for this article. The results show that LAMP is more sensitive and less time consuming than the conventional PCR, and thus it can be used for the detection of northern pike in aquatic ecosystems related to environmental monitoring.

Effect of Silver-Containing Titania Layers for Bioactivity, Antibacterial Activity, and Osteogenic Differentiation of Human Mesenchymal Stem Cells on Ti Metal.

Biomaterials with better osteogenic capacity, rapid osteo-integration, and higher mechanical strength are undoubtedly preferred for successful bone implant development. A porous sodium hydrogen titanate layer was formed on Ti metal by NaOH treatment, and the Na+ ions were replaced by Ag+ ions by subsequent AgNO3 treatment that formed silver-containing hydrogen titanate. Heat treatment at 600 °C transformed sodium hydrogen titanate into sodium titanate with sheet-like morphology, whereas silver-containing hydrogen titanate was converted to anatase TiO2 with an elongated rod-like structure. Further increment in temperature lead to the formation of rutile TiO2 with distracted network morphology. Between these two, the anatase TiO2 was ascertained to be bioactive by being capable of forming bonelike apatite in simulated body fluid within a period of 12 h. The concentration of silver on Ti metal was further optimized for better antibacterial activity against S. aureus and biocompatibility toward bone cells. A detailed investigation of thus optimized silver-containing Ti metal on the proliferation and differentiation of multipotent human mesenchymal stem cells further proved their biocompatibility nature and facilitation of osteogenic differentiation, thereby conferring those as ideally suited materials for bioimplant development in bone tissue engineering.