An array-based melt curve analysis method for the identification and classification of closely related pathogen strains
| Autor: | T. Gharooni, Robert G. Kuimelis, Pejman Naraghi-Arani, Alexander Anemogiannis, Gary K. Schoolnik, Tran Van, Arjang Hassibi, Ruma Sinha, Kshama Jirage, Lei Pei, Kirsten A. Johnson, Jessica Ebert, Arun Manickam, Gelareh Mazarei, Sara Bolouki, Amin Zia, Pallavi Mantina, Yuan Li |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Computational biology Biology quencher Genome General Biochemistry Genetics and Molecular Biology Melting curve analysis law.invention 03 medical and health sciences law Pathogen Polymerase chain reaction Methods Manuscript enterovirus Amplicon melt curve rhinovirus 030104 developmental biology Förster resonance energy transfer point-of-care FRET pathogen classification Identification (biology) Primer (molecular biology) General Agricultural and Biological Sciences microarray inverse fluorescence |
| Zdroj: | Biology Methods & Protocols |
| ISSN: | 2396-8923 |
| Popis: | 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 classification method 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 quencher moiety at the 5ʹ-terminus, for all the related species. The array includes fluorescent-labeled probes which preferentially capture the variants of the amplicons and classify them through solid-phase thermal denaturing (melt curve) analysis. Systematic primer and probe design algorithms and empirical validation methods are presented and successfully applied to the challenging example of identification of, and differentiation between, closely related human rhinovirus and human enterovirus strains. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|