Isothermal rolling circle amplification of virus genomes for rapid antigen detection and typing

Abstract

In this work, isothermal rolling circle amplification (RCA) of the multi-kilobase genome of engineered filamentous bacteriophage is used to report the presence and identification of specific protein analytes in solution. First, bacteriophages were chosen as sensing platforms because peptides or antibodies that bind medically relevant targets can be isolated through phage display or expressed as fusions to their p3 and p8 coat proteins. Second, the circular, single-stranded genome contained within the phage serves as a natural large DNA template for a RCA reaction to rapidly generate exponential amounts of double stranded DNA in a single isothermal step that can be easily detected using low-cost fluorescent nucleic acid stains. Amplifying the entire phage genome also provides high detection sensitivities. Furthermore, since the sequence of the viral DNA can be easily modified with multiple restriction enzyme sites, a simple DNA digest can be applied to detect and identify multiple antigens simultaneously. The methods developed here will lead to protein sensors that are highly scalable to produce, can be run without complex biological equipment and do not require the use of multiple antibodies or high-cost fluorescent DNA probes or nucleotides.