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A highly sensitive immuno-PCR assay for detecting Group A Streptococcus
Huining Liang1, Susan E Cordova, Thomas L Kieft
1Biology Department, New Mexico Institute of Mining and Technology, Socorro, NM 87801, USA.
Journal of Immunological Methods
|September 13, 2003
Summary
A novel hybrid assay combining immuno-polymerase chain reaction (immuno-PCR) and ELISA techniques offers highly sensitive detection of Group A Streptococcus (Strep A). This robust method achieves detection limits down to one-thousandth of a cell, even with interfering bacteria present.
Area of Science:
- Microbiology
- Immunology
- Biotechnology
Background:
- Group A Streptococcus (Strep A) causes significant human infections.
- Accurate and sensitive diagnostic methods are crucial for timely treatment.
- Existing diagnostic techniques may lack the required sensitivity or specificity.
Purpose of the Study:
- To develop a highly sensitive and specific diagnostic assay for pathogenic Group A Streptococcus.
- To combine the strengths of immuno-polymerase chain reaction (immuno-PCR) and enzyme-linked immunosorbent assay (ELISA) for bacterial detection.
- To evaluate the assay's performance in the presence of potential interfering substances.
Main Methods:
- A hybrid immuno-PCR/ELISA assay was developed using sonicated bacterial cells coated onto microtiter plates.
- Biotinylated anti-Group A monoclonal antibody (mAb) was used, linked via a streptavidin (STV) bridge to biotinylated reporter DNA.
- Reporter DNA was amplified using PCR, electrophoresed, and quantified for signal detection.
Main Results:
- The assay achieved a minimum detection limit of approximately 1/1000th of a Streptococcus pyogenes cell.
- High sensitivity was maintained even in the presence of 100,000 Escherichia coli cells.
- No cross-reactivity was observed with other Streptococcus species.
- A direct antibody-DNA linkage method yielded sensitivity of approximately 10 cells.
- The assay showed robustness with minimal inhibition from clinical and food samples, though soil extracts were slightly inhibitory.
Conclusions:
- The developed hybrid immuno-PCR assay is highly sensitive, specific, and robust for detecting Group A Streptococcus.
- This method demonstrates significant potential for clinical diagnostics and serves as a model for other immuno-PCR applications.
- The assay's adaptability suggests broad applicability for detecting various bacteria in diverse sample types.