Rapid detection of bacterial pathogens using bacteriophage amplification coupled with surface-enhanced Raman spectroscopy lateral flow immunochromatography

Abstract

The current gold standards for bacterial identification involve culture-based methods that can take weeks to months to provide definitive results. Because of this lengthy time period, a need for rapid diagnostics aimed at pathogenic bacteria exists. To this end, a lateral flow immunochromatographic (LFI) assay was developed that exploited bacteriophages (phages) as secondary markers. Phages are useful in diagnostic assay because they specifically target a bacterial host and produce many progeny phages in a matter of hours. In a process called phage amplification (PA), phages were input below the detection limit of the LFI and if the targeted bacteria was present, the phages rapidly multiplied. This increase in phage concentration above LFI limit of detection indicated the presence of the target bacteria. The LFI devices presented here detected progeny phages using surface-enhanced Raman spectroscopy (SERS) nanoparticle reporters covalently linked with anti-phage antibodies. When interrogated with laser in the near IR range, a characteristic spectrum was produced by an organic reporter. This spectrum provided quantitative information of the LFI test line and eliminated dependence on visual determination. The PA SERS LFI system was first developed for the foodborne pathogen Listeria monocytogenes using phage A511. Several assay parameters were optimized, such as the conjugation of anti-A511 antibodies onto the SERS reporters, LFI construction and proper sample flow the SERS LFI device. L. monocytogenes was detected in as little as 2 hours and at concentrations as low as 1 × 105 colony forming units (cfu)/mL in 5 hours. PA SERS LFI for the detection of L. monocytogenes was further advanced to detect the pathogen in inoculated food matrices. Cantaloupe, ice cream, and two types of soft cheeses were inoculated with varying concentrations of L. monocytogenes and selectively enriched according to FDA protocol. After a 24 hour enrichment, 1 cfu/g was detected by PA SERS LFI in all foods tested. Finally, the PA SERS LFI strategy was adapted to detect the potential bio-warfare agent Yersinia pestis, using phage ϕA1122. Adjustments for the development a ϕA1122-specific SERS LFI included utilizing polyclonal rabbit anti-ϕA1122 antibodies for conjugation onto SERS reporters and for use as capture agents on the test line. PA SERS LFI detected Y. pestis in as short a time as 1 hour and at a concentration as low as 5.0 × 104 cfu/mL in 3 hours. The work presented in this dissertation represents a combination of several scientific principles to develop a rapid, specific, easy-to-use diagnostic tool for the detection of bacterial pathogens L. monocytogenes and Y. pestis. Detection time was reduced from 4-6 days by conventional culture methods to hours by exploiting phages for their specificity and rapid reproduction. LFI provides an inexpensive, simple analytical test to detect progeny phages produced from PA. Furthermore, quantitative analysis of the test line of LFI devices was achieved using spectroscopic SERS reporters, which eliminated the need for visual determination and lowered detection levels below visual levels.