An array-based melt curve analysis method for the identification and classification of closely related pathogen strains.

Hassibi, Arjang; Ebert, Jessica; Bolouki, Sara; Anemogiannis, Alexander; Mazarei, Gelareh; Li, Yuan; Johnson, Kirsten A; Van, Tran; Mantina, Pallavi; Gharooni, Taraneh; Jirage, Kshama; Pei, Lei; Sinha, Ruma; Manickam, Arun; Zia, Amin; Naraghi-Arani, Pejman; Schoolnik, Gary; Kuimelis, Robert G

PCR-based techniques are widely used to identify disease causing bacterial and viral pathogens, especially in point-of-care or near-patient clinical settings that require rapid results and sample-to-answer workflows. However, such techniques often fail to differentiate between closely related species that have highly variable genomes. Here, a homogenous (closed-tube) pathogen identification and classificationmethod is described that combines PCR amplification, array-based amplicon sequence verification, and real-time detection using an inverse fluorescence fluorescence-resonance energy transfer technique. The amplification is designed to satisfy the inclusivity criteria and create ssDNA amplicons, bearing a nonradiating quenchermoiety at the 50-terminus, for all the related species. The array includes fluorescent-labeled probes which preferentially capture the variants of the amplicons and classify themthrough solid-phase thermal denaturing (melt curve) analysis. Systematic primer and probe design algorithms and empirical validationmethods are presented and successfully applied to the challenging example of identification of, and differentiation between, closely related human rhinovirus and human enterovirus strains.

POLYMERASE chain reaction; PATHOGENIC fungi; PATHOGENIC bacteria; SINGLE-stranded DNA; RHINOVIRUSES

Biology Methods & Protocols, 2018, p1

2396-8923

Academic Journal

10.1093/biomethods/bpy005