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Rapid detection of Hemophilus influenzae and Streptococcus pneumoniae simultaneously using a duplex recombinase-aided
Lin Zhou1, Yuyan Xia1, Yanling Feng1
1Capital Center for Children's Health, Capital Medical University, Capital Institute of Pediatrics, Beijing, China.
Insights
A new duplex recombinase-aided amplification (RAA) assay rapidly detects two key bacteria causing childhood pneumonia. This simple, sensitive test aids early diagnosis of bacterial pneumonia in children.
Area of Science:
- Microbiology
- Molecular Diagnostics
- Pediatric Infectious Diseases
Background:
- Community-acquired pneumonia (CAP) is a major cause of death in children globally.
- Streptococcus pneumoniae and Haemophilus influenzae are primary bacterial culprits requiring hospitalization.
- Rapid, sensitive, and cost-effective diagnostic methods are crucial for effective CAP management.
Purpose of the Study:
- To develop a rapid, simultaneous detection method for S. pneumoniae and H. influenzae.
- To establish a highly sensitive assay for diagnosing bacterial CAP in children.
Main Methods:
- Developed a duplex recombinase-aided amplification (RAA) assay.
- Selected conserved lytA and omp6 genes as specific targets for S. pneumoniae and H. influenzae.
- Optimized reaction conditions, including temperature and probe concentrations.
Main Results:
- The duplex RAA assay provides simultaneous detection in under 10 minutes at 39°C.
- Achieved high sensitivity with limits of detection at 72 copies/reaction for S. pneumoniae and 35 copies/reaction for H. influenzae.
- Optimal probe concentrations determined as 0.6 μM for S. pneumoniae and 0.8 μM for H. influenzae.
Conclusions:
- Presents a rapid, accurate nucleic acid amplification assay for concurrent detection of major bacterial CAP pathogens.
- The assay's speed, simplicity, and sensitivity offer a promising tool for early etiological diagnosis.
- Facilitates timely and effective clinical management of childhood bacterial pneumonia.
Introduction:
Community-acquired pneumonia (CAP) remains a leading cause of mortality in children under five years of age worldwide. Streptococcus pneumoniae and Haemophilus influenzae are the most common bacterial pathogens causing CAP that requires hospitalization, highlighting the critical need for a simple, low-cost, and highly sensitive method for rapid diagnosis.
Methods:
We developed a duplex recombinase-aided amplification (RAA) assay for the simultaneous detection of S. pneumoniae and H. influenzae. Following comparative genomic analysis, the conserved lytA gene and omp6 gene were selected as the specific targets for S. pneumoniae and H. influenzae, respectively. The reaction conditions, including temperature and probe concentration, were optimized.
Results:
The established duplex RAA assay can be completed within 10 minutes at a constant temperature of 39°C, with an optimal probe concentration combination of 0.6 μM for S. pneumoniae and 0.8 μM for H. influenzae. The assay demonstrated high sensitivity, with a limit of detection of 72 copies per reaction for S. pneumoniae and 35 copies per reaction for H. influenzae.
Discussion:
This study presents a rapid and accurate nucleic acid amplification assay for the concurrent detection of two major bacterial pathogens in childhood CAP. The speed, simplicity, and sensitivity of the duplex RAA assay make it a promising tool for early and rapid etiological diagnosis in clinical settings.
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