A highly sensitive immuno-PCR assay for detecting Group A Streptococcus

Abstract

A highly sensitive hybrid assay, based on immuno polymerase chain reaction (immuno-PCR) and enzyme-linked immunosorbent assay (ELISA) techniques, was developed for the detection of pathogenic Group A Streptococcus (Strep A). Cells were disrupted by sonication and then coated onto the walls of Maxisorp microtiter plates. Next, biotinylated anti-Group A monoclonal antibody (mAb) was bound to the antigen and then linked, via a streptavidin (STV) bridge, to biotinylated reporter DNA. After extensive washing, the denatured reporter DNA was transferred to PCR tubes, amplified, electrophoresed, and used as the signal for detection of bacteria. The minimum detection limit of this assay is the equivalent of approximately one one-thousandth of a Streptococcus pyogenes cell, even in the presence of 100,000 Escherichia coli cells. The combination of multiple antigens per cell and PCR amplification provides the extreme sensitivity in this immuno-PCR assay. No cross-reaction was found with other Streptococcus species. We also directly linked the anti-Group A monoclonal antibody to DNA using succinimidyl 4-[ N-maleimidomethyl]-cyclohexane-1-carboxylate (SMCC). The sensitivity using directly linked antibody-reporter DNA was approximately 10 cells. Because this assay could be adapted for detection of many different bacteria in a variety of sample types, we tested the potential for interference from substances that could be present in clinical, food, and environmental samples. Sonicated meat or human plasma did not inhibit detection; however, extracts of concentrated soil samples were somewhat inhibitory. This highly specific, sensitive, and robust assay could be applied to clinical detection of Group A Streptococcus and serves as a model for other immuno-PCR assays.