Using a resequencing microarray as a multiple respiratory pathogen detection assay

Abstract

Simultaneous testing for detection of infectious pathogens that cause similar symptoms (e.g., acute respiratory infections) is invaluable for patient treatment, outbreak prevention, and efficient use of antibiotic and antiviral agents. In addition, such testing may provide information regarding possible coinfections or induced secondary infections, such as virally induced bacterial infections. Furthermore, in many cases, detection of a pathogen requires more than genus/species-level resolution, since harmful agents (e.g., avian influenza virus) are grouped with other, relatively benign common agents, and for every pathogen, finer resolution is useful to allow tracking of the location and nature of mutations leading to strain variations. In this study, a previously developed resequencing microarray that has been demonstrated to have these capabilities was further developed to provide individual detection sensitivity ranging from 10(1) to 10(3) genomic copies for more than 26 respiratory pathogens while still retaining the ability to detect and differentiate between close genetic neighbors. In addition, the study demonstrated that this system allows unambiguous and reproducible sequence-based strain identification of the mixed pathogens. Successful proof-of-concept experiments using clinical specimens show that this approach is potentially very useful for both diagnostics and epidemic surveillance.

FIG. 1.

Schematic of the processing protocol. Clinical samples (nasal swab or nasal wash specimens) were collected from patients presenting ARI symptoms. Nucleic acids were extracted from these samples, followed by reverse transcription. The products of reverse transcription were split into two multiplex PCR mixtures for amplification. PCR products from the two mixtures were combined after amplification, cleaned up, fragmented, and labeled. The labeled products were then hybridized to RPM v.1 chips. After the chips were washed and stained, the sequences generated from RPM v.1 were exported as FASTA-formatted files and further analyzed for pathogen identification using the automatic pathogen identification algorithm.