Background: Rapid and accurate diagnosis of the causative agents of infections is essential to support medical decisions in hospitals. The DiagCORE® Gastrointestinal Panel is a fast real-time PCR-based system for the qualitative in vitro detection and identification of the most common bacteria (14), viruses (6) and parasites (4) causing gastrointestinal infections using directly stool samples from patients. A key step during the development of in vitro diagnostics PCR-based systems is the assessing of the oligonucleotides assay reactivity (inclusivity) and their specificity.
Materials/methods: First an in silico analysis was performed following an in-house developed pipeline that was used to analyze all oligonucleotides included in the DiagCORE® Gastrointestinal Panel. This pipeline combined BLAST (Basic Local Alignment Search Tool) searches coupled with simulate_PCR software (Gardner et al., 2014). Hence, both desired and off-target PCR amplifications products were predicted considering different numbers of mismatches between oligonucleotides and pathogen target sequences and specific amplicon size. In a second approach all annotated target sequence available in the National Center for Biotechnology Information (NCBI) were used to construct a target sequence alignment for each pathogen to optimize oligonucleotides designs for the detection of all clinically relevant strains, genotypes and subtypes. Thirdly, in vitro testing of reference ATCC strains were performed to confirm in silico predicted cross-reactivity and specificity.
Results: The oligonucleotides included in the DiagCORE® Gastrointestinal panel showed in silico inclusivity rates ranging from 94.7-100% of total target sequences for all pathogens. Specificity in silico testing highlighted the risk of some potential cross-reactions for Campylobacter assay that was ruled out after in vitro PCR testing.
Conclusions: In conclusion, the results demonstrated that all relevant gastrointestinal pathogen strains intended to be detected with the DiagCORE® Gastrointestinal panel are represented in the inclusivity test, and no remarkable cross-reactivity was observed after in vitro validation. Finally, the in silico pipeline established is a useful method to test the overall potential reactivity of the oligonucleotide assays, which can be implemented to identify which organisms strains worth trying in vitro testing in inclusivity and exclusivity studies during the development of in vitro diagnostics systems.
Authors: Boher P, Ruiz M, Vara K, Pareja J, Van de Sand C*.