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Development and Application of a Multiple Cross Displacement Amplification Combined With Nanoparticle-Based Lateral
Yu Wang1,2, Xue Zhao1, Jinzhi Cheng3
1Department of Clinical Laboratory, The First People's Hospital of Guiyang, Guiyang, China.
Abstract:
Candida tropicalis is an increasingly opportunistic pathogen that causes serious invasive candidiasis threatening a patient's life. Traditional methods to detect C. tropicalis infection depends on time-consuming, culture-based gold-standard methods. So, we sought to establish a new method that could detect target pathogens quickly, accurately, and straightforwardly. Herein, a combination of multiple cross displacement amplification (MCDA) and lateral flow biosensors (LFB) was employed to detect C. tropicalis. In the MCDA system, 10 primers were designed to identify the specific genes of C. tropicalis and amplify the genes in an isothermal amplification device. Then, MCDA amplification reaction products could be identified visibly by color change, and all the amplification products would be tested by LFB with no special equipment. The results demonstrated that the optimal reaction condition of C. tropicalis-MCDA assay was 64°C within 30 min, and only 10 fg DNA was required in each reaction. No cross-reaction was found between C. tropicalis strains and non-C. tropicalis strains. For 300 sputum samples, the results showed that MCDA-LFB assay could rapidly and successfully detect all of the C. tropicalis-positive (28/300) samples detected by the gold-standard method. The entire procedure, including specimen processing (40 min), isothermal reaction (30 min) and result reporting (within 2 min), could be completed within 75 min. Briefly, the study results demonstrated that the detection ability of C. tropicalis-MCDA-LFB assay was better than culture methods with more simplicity, rapidity, sensitivity and specificity. Hence, MCDA-LFB strategy is an effective tool to rapidly detect C. tropicalis in clinical samples, especially in resource-poor areas.
Insights
A new multiple cross displacement amplification (MCDA) and lateral flow biosensor (LFB) assay rapidly detects Candida tropicalis. This method offers a faster, more sensitive, and specific alternative to traditional culture methods for diagnosing invasive candidiasis.
Area of Science:
- Medical Microbiology
- Molecular Diagnostics
- Biosensor Technology
Background:
- Candida tropicalis is a significant opportunistic pathogen causing life-threatening invasive candidiasis.
- Conventional diagnostic methods rely on time-consuming, culture-based techniques.
- There is a critical need for rapid, accurate, and straightforward diagnostic tools.
Purpose of the Study:
- To develop and validate a novel diagnostic assay for the rapid detection of Candida tropicalis.
- To combine multiple cross displacement amplification (MCDA) with lateral flow biosensors (LFB) for enhanced detection capabilities.
- To provide a simple and effective diagnostic tool, particularly for resource-limited settings.
Main Methods:
- Design of 10 specific primers for Candida tropicalis gene amplification using MCDA.
- Isothermal amplification of target genes at an optimal temperature of 64°C for 30 minutes.
- Visual detection of MCDA products and confirmation using lateral flow biosensors (LFB) without specialized equipment.
Main Results:
- The optimized MCDA-LFB assay demonstrated high sensitivity, requiring only 10 fg of DNA.
- No cross-reactivity was observed with non-Candida tropicalis strains, ensuring specificity.
- The assay successfully detected all 28 Candida tropicalis-positive sputum samples (out of 300) identified by the gold-standard method.
- The entire diagnostic procedure, from specimen processing to result reporting, was completed within 75 minutes.
Conclusions:
- The developed Candida tropicalis-MCDA-LFB assay is simpler, faster, more sensitive, and more specific than traditional culture methods.
- This MCDA-LFB strategy represents an effective tool for the rapid diagnosis of Candida tropicalis infections in clinical settings.
- The assay holds particular promise for improving diagnostics in resource-poor areas due to its simplicity and minimal equipment requirements.
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