Cyclopentane peptide nucleic acid: Gold nanoparticle conjugates for the detection of nucleic acids in a microfluidic format.

Routine patient testing for viral infections is critical to identify infected individuals for treatment and to prevent spreading of infections to others. Developing robust and reliable diagnostic tools to detect nucleic acids of viruses at the point-of-care could greatly assist the clinical management of viral infections. The remarkable stability and high binding affinity of peptide nucleic acids (PNAs) to target nucleic acids could make PNA-based biosensors an excellent starting point to develop new nucleic acid detection technologies. We report the application of cyclopentane-modified PNAs to capture target nucleic acids in a microfluidic channel, and the use of bioorthogonal PNAs conjugated to gold nanoparticles as probes to semi-quantitatively signal the presence of a target nucleic acid derived from HIV-1. The basic results presented could be used to develop more advanced devices to detect nucleic acids from viruses such as HIV, SARS-CoV-2, and a wide range of other human diseases.

Selection and phylogenetics of salmonid MHC class I: wild brown trout (Salmo trutta) differ from a non-native introduced strain.

We tested how variation at a gene of adaptive importance, MHC class I (UBA), in a wild, endemic Salmo trutta population compared to that in both a previously studied non-native S. trutta population and a co-habiting Salmo salar population (a sister species). High allelic diversity is observed and allelic divergence is much higher than that noted previously for co-habiting S. salar. Recombination was found to be important to population-level divergence. The α1 and α2 domains of UBA demonstrate ancient lineages but novel lineages are also identified at both domains in this work. We also find examples of recombination between UBA and the non-classical locus, ULA. Evidence for strong diversifying selection was found at a discrete suite of S. trutta UBA amino acid sites. The pattern was found to contrast with that found in re-analysed UBA data from an artificially stocked S. trutta population.

Transforming microbial genotyping: a robotic pipeline for genotyping bacterial strains.

Microbial genotyping increasingly deals with large numbers of samples, and data are commonly evaluated by unstructured approaches, such as spread-sheets. The efficiency, reliability and throughput of genotyping would benefit from the automation of manual manipulations within the context of sophisticated data storage. We developed a medium- throughput genotyping pipeline for MultiLocus Sequence Typing (MLST) of bacterial pathogens. This pipeline was implemented through a combination of four automated liquid handling systems, a Laboratory Information Management System (LIMS) consisting of a variety of dedicated commercial operating systems and programs, including a Sample Management System, plus numerous Python scripts. All tubes and microwell racks were bar-coded and their locations and status were recorded in the LIMS. We also created a hierarchical set of items that could be used to represent bacterial species, their products and experiments. The LIMS allowed reliable, semi-automated, traceable bacterial genotyping from initial single colony isolation and sub-cultivation through DNA extraction and normalization to PCRs, sequencing and MLST sequence trace evaluation. We also describe robotic sequencing to facilitate cherrypicking of sequence dropouts. This pipeline is user-friendly, with a throughput of 96 strains within 10 working days at a total cost of < €25 per strain. Since developing this pipeline, >200,000 items were processed by two to three people. Our sophisticated automated pipeline can be implemented by a small microbiology group without extensive external support, and provides a general framework for semi-automated bacterial genotyping of large numbers of samples at low cost.