Oli2go: an automated multiplex oligonucleotide design tool.

The success of widely used oligonucleotide-based experiments, ranging from PCR to microarray, strongly depends on an accurate design. The design process involves a number of steps, which use specific parameters to produce high quality oligonucleotides. Oli2go is an efficient, user friendly, fully automated multiplex oligonucleotide design tool, which performs primer and different hybridization probe designs as well as specificity and cross dimer checks in a single run. The main improvement to existing oligonucleotide design web-tools is that oli2go combines multiple steps in an all-in-one solution, where other web applications only accomplish parts of the whole design workflow. Especially, the oli2go specificity check is not only performed against a single species (e.g. mouse), but against bacteria, viruses, fungi, invertebrates, plants, protozoa, archaea and sequences from whole genome shotgun sequence projects and environmental samples, at once. This allows the design of highly specific oligonucleotides in multiplex applications, which is further assured by performing dimer checks not only on the primers themselves, but in an all-against-all fashion. The software is freely accessible to all users at http://oli2go.ait.ac.at/.

Full pathogen characterisation: species identification including the detection of virulence factors and antibiotic resistance genes via multiplex DNA-assays.

Antibiotic resistances progressively cause treatment failures, and their spreading dynamics reached an alarming level. Some strains have already been classified as highly critical, e.g. the ones summarised by the acronym ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.). To restrain this trend and enable effective medication, as much information as possible must be obtained in the least possible time. Here, we present a DNA microarray-based assay that screens for the most important sepsis-relevant 44 pathogenic species, 360 virulence factors (mediate pathogenicity in otherwise non-pathogenic strains), and 409 antibiotic resistance genes in parallel. The assay was evaluated with 14 multidrug resistant strains, including all ESKAPE pathogens, mainly obtained from clinical isolates. We used a cost-efficient ligation-based detection platform designed to emulate the highly specific multiplex detection of padlock probes. Results could be obtained within one day, requiring approximately 4 h for amplification, application to the microarray, and detection.

Crosslinking of PCR primers reduces unspecific amplification products in multiplex PCR.

The polymerase chain reaction is not only essential for many DNA-based diagnostic methods but is also exploited in other molecular methods that require an upstream amplification step. Multiplex PCRs are especially attractive as they reduce the number of individual reactions. However, the multiplexing efficiency is impaired by primer interactions such as the formation of primer dimers. In this study, covalent crosslinking of primers via their 5'-ends was used to avoid those undesired effects. The specificity of the primers as well as the efficiency of the PCR could be increased upon primer crosslinking in reactions containing up to 34 primer pairs targeting the most important antibiotic resistance genes in a single multiplex reaction.

Highly Specific Ligation-dependent Microarray Detection of Single Nucleotide Polymorphisms.

The fast detection and characterization of pathogens are essential for an efficient treatment of infectious diseases. However, the development of improved and reliable diagnostic methods is still an ongoing process because not only pathogens but also their antibiotic resistances have to be identified. The gold standard today is, however, a cultivation-based characterization approach, which takes days until results can be evaluated. In patients with, for example, severe sepsis, the diagnostic test duration is a very critical parameter because a delay of treatment optimization increases the mortality rate significantly. In contrast, DNA-based molecular techniques can obtain results within a few hours. A further challenge in diagnostic laboratories is that patient samples have to be screened for hundreds of potential pathogens, antibiotic resistance genes, and virulence factors, which is achieved by using a number of specialized tests at the moment. Microarrays are outstandingly good for the simultaneous analysis of thousands of different genes and have become a popular tool in biological studies. Nevertheless, further optimizations of the microarray technology are required due to the obligatory DNA labeling and/or amplification steps and the effects of nonspecific DNA hybridization. Here, we describe a fast and highly specific solid-support-based DNA characterization method for pathogens and antibiotic resistance genes.