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Rapid, Simple, and Highly Specific Detection of Streptococcus pneumoniae With Visualized Recombinase Polymerase
Fang Wang1, Yan Wang1, Xia Liu1
1Department of Central Laboratory, The Second People's Hospital of Lianyungang City (Cancer Hospital of Lianyungang), Affiliated to Jiangsu University, Lianyungang, China.
Abstract:
Streptococcus pneumoniae is a major pathogen that causes microbiological illness in humans. The introduction of polyvalent vaccines has resulted in a significant decrease in pneumococcal-related mortality. However, pneumococcal infections continue to be a leading cause of death in children under the age of 5 and adults over the age of 65 worldwide. A speedy and highly sensitive diagnostic tool is necessary for routine adoption to adequately manage patients and control the spread of infection. In this study, we investigated a new nucleic acid amplification technique, isothermal recombinase polymerase amplification (RPA), which amplifies DNA at 37°C under isothermal conditions with high specificity, efficiency, and rapidity. Using the autolysin gene lytA as the molecular diagnostic target, an RPA primer-probe combination was designed and optimized for the detection of S. pneumoniae. This RPA reaction produced amplification products labeled with specific chemical markers, to be detected with gold-nanoparticle-based lateral flow strips (LFS), reducing the reliance on equipment and trained personnel. The high specificity of the RPA-LFS technique was demonstrated with the specific detection of 22 strains of S. pneumoniae but not 25 closely related pathogenic bacteria. The assay showed good sensitivity, and detected S. pneumoniae down to 3.32 colony-forming units/μL. When used on clinical samples, the assay provided accurate and consistent results compared with PCR. The compliance with the culture-biochemistry method was 98.18% and the kappa index was 0.977. These results reveal that the RPA-LFS test significantly improved S. pneumoniae identification, particularly in resource-limited areas.
Insights
A new recombinase polymerase amplification (RPA) and lateral flow strip (LFS) test rapidly and accurately detects Streptococcus pneumoniae. This nucleic acid amplification technique offers a sensitive and specific diagnostic tool, especially for resource-limited settings.
Area of Science:
- Microbiology
- Molecular Diagnostics
- Biotechnology
Background:
- Streptococcus pneumoniae remains a leading cause of death globally, particularly in young children and the elderly.
- Existing diagnostic methods require specialized equipment and trained personnel, hindering widespread adoption.
- There is a critical need for rapid, sensitive, and accessible diagnostic tools for pneumococcal infections.
Purpose of the Study:
- To develop and validate a novel nucleic acid amplification technique for the rapid detection of Streptococcus pneumoniae.
- To assess the specificity, sensitivity, and clinical accuracy of the developed assay.
Main Methods:
- Investigated isothermal recombinase polymerase amplification (RPA) for DNA amplification at 37°C.
- Designed and optimized an RPA primer-probe combination targeting the lytA gene of S. pneumoniae.
- Utilized gold-nanoparticle-based lateral flow strips (LFS) for visual detection of RPA products.
Main Results:
- The RPA-LFS assay specifically detected 22 strains of S. pneumoniae without cross-reactivity with 25 other pathogenic bacteria.
- The assay demonstrated high sensitivity, detecting down to 3.32 colony-forming units/μL of S. pneumoniae.
- Clinical sample analysis showed high concordance with PCR (98.18% compliance, kappa index 0.977).
Conclusions:
- The developed RPA-LFS test is a highly specific, sensitive, and rapid method for identifying Streptococcus pneumoniae.
- This assay offers a valuable diagnostic solution, particularly for resource-limited settings, improving patient management and infection control.
