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A versatile loop-mediated isothermal amplification microchip platform for Streptococcus pneumoniae and Mycoplasma
Hua Wang1, Zhan Ma2, Juanxiu Qin1
1Department of Laboratory Medicine, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China.
Insights
A new microfluidic chip platform enables rapid, simultaneous detection of Streptococcus pneumoniae and Mycoplasma pneumoniae, crucial for diagnosing childhood pneumonia at the point of care. This low-cost, sensitive assay offers a vital tool for early intervention, especially in resource-limited settings.
Area of Science:
- Biotechnology
- Medical Diagnostics
- Microfluidics
Background:
- Community-acquired pneumonia (CAP) is a leading cause of mortality in children under five.
- Streptococcus pneumoniae and Mycoplasma pneumoniae are primary pathogens in hospitalized CAP cases.
- There is a critical need for rapid, sensitive, and cost-effective diagnostic methods for CAP.
Purpose of the Study:
- To develop and validate a versatile microfluidic chip platform for simultaneous detection of S. pneumoniae and M. pneumoniae.
- To enable point-of-care (POC) testing for CAP pathogens.
- To provide a low-cost, highly sensitive diagnostic solution for early CAP diagnosis.
Main Methods:
- Development of a polymer/paper microfluidic chip integrated with loop-mediated isothermal amplification (LAMP).
- On-chip magnetic particle-based nucleic acid extraction for pathogen DNA concentration.
- A portable device for chip heating, image capture, and smartphone data transmission.
- Clinical evaluation using oropharyngeal swabs and bronchoalveolar lavage fluid from 63 children.
Main Results:
- The microfluidic chip achieved DNA extraction in ~15 minutes, significantly faster than traditional methods (>1.5 hours).
- Analytical sensitivity was as low as 20 fg of target DNA.
- The IPµchip platform demonstrated high clinical performance: 96.9% positive and 100% negative predictive values for M. pneumoniae.
- Compared to PCR and culture, the IPµchip showed superior clinical sensitivity for S. pneumoniae (100% vs. 60% and 40%).
Conclusions:
- The integrated microfluidic chip platform (IPµchip) offers a versatile and efficient solution for simultaneous POC detection of key CAP pathogens.
- The assay provides rapid, sensitive, and specific results, outperforming conventional methods in certain aspects.
- This technology holds significant potential for improving CAP diagnosis and management, particularly in developing nations.
Abstract:
Community-acquired pneumonia (CAP) is the leading cause of mortality in children under five years of age, globally. Given that Streptococcus pneumoniae (S. pneumoniae) and Mycoplasma pneumoniae (M. pneumoniae) are the most common pathogens associated with CAP requiring hospital admission, a simple, low cost, highly sensitive method is in great need for immediate and early diagnosis of CAP. Herein, we report a versatile microfluidic chip platform integrated with loop-mediated isothermal amplification (LAMP) for simultaneous S. pneumoniae and M. pneumoniae testing at the point of care. The platform includes a polymer/paper microfluidic chip and a portable device. On-chip magnetic particle-based nucleic acid extraction is used for concentration of pathogens' genomic DNA and is followed by LAMP. The portable device has the function of heating the microfluidic chip, and photographing and transmitting the result to a smartphone. Complete extraction of the DNA using the microfluidic chip took ~15 min versus >1.5 h with a phenol-chloroform method. The analytical sensitivity of the assay was determined to be 20 fg by testing serial dilutions of target DNA ranging from 2 ng to 2 fg per reaction. We evaluated the clinical sensitivity and specificity of the IPµchip assay using 63 randomly selected oropharyngeal swabs and bronchoalveolar lavage fluid specimens from children. For comparison, these specimens were also tested against real-time PCR assay (M. pneumoniae), conventional PCR assay (S. pneumoniae), and culture tests (S. pneumoniae). These results yielded positive and negative predictive values for M. pneumoniae testing with the IPµchip platform of 96.9% and 100%, respectively. Compared with S. pneumoniae IPµchip, the clinical sensitivity of S. pneumoniae PCR and culture tests was 60% and 40%, respectively, while clinical specificity of the two tests was 100%. This versatile IPµchip platform has great potential for point of care testing of different kinds of pathogens, especially for developing nations.
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