Fast & Highly Sensitive Detection of Chloroquine Resistance in Plasmodium falciparum Using Innovative Biosensing Technology
- The RPA-PfAgo platform detects the Pfcrt mutant haplotype quickly and sensitively.
- A set of RPA primers is designed based on specific guidelines and software.
- Guide DNA (gDNA) is created to help PfAgo recognize specific sequences.
Overall Workflow of the RPA-PfAgo Platform
Table of Contents
- Overall Workflow of the RPA-PfAgo Platform
- PfAgo Protein Expression and Purification
- Primer Design and Screening
- Guide DNA and ssDNA Reporters Design
- Plasmid Construction
- RPA-PfAgo Platform Cleavage Assay
- Optimization of PfAgo Cleavage Reaction
- Urea-Denaturing PAGE
- Nested PCR and Sequencing
- Sensitivity Validation
- Clinical Evaluation
- Data Analysis
The RPA-PfAgo platform detects the Pfcrt mutant haplotype quickly and sensitively. The process begins with collecting blood samples from malaria patients. Genomic DNA is extracted using the Chelex-100 method. The target DNA sequence is amplified through RPA technology. The resulting product is then analyzed with the PfAgo cleavage system.
Key Steps:
- RPA Amplification: The DNA product is amplified.
- Guided Cleavage: The PfAgo protein targets and cleaves a specific strand of the DNA that matches the mutation (gDNA-M sequence).
- Fluorescent Reporter: The cleavage generates a new ssDNA fragment that interacts with a fluorescent probe, confirming the first cleavage.
- Mutant vs. Wild Type: The system can distinguish between the mutant (CVMNK) and wild-type sequences, using different fluorescent reporters for each.
PfAgo Protein Expression and Purification
PfAgo protein is prepared following established protocols. Key steps include:
- Gene Optimization: Codon optimization using JCat software.
- Recombinant Expression: Inserting the PfAgo gene into a plasmid and expressing it in E. coli.
- Protein Purification: Using Ni-affinity chromatography to purify the His-tag fusion protein, which is stored under specific buffer conditions.
Primer Design and Screening
The Pfcrt gene sequence is sourced from PlasmoDB. A set of RPA primers is designed based on specific guidelines and software. Multiple primer combinations are tested for effective amplification of the targeted sequence.
Guide DNA and ssDNA Reporters Design
Guide DNA (gDNA) is created to help PfAgo recognize specific sequences. The design includes:
- Combinations of 5′-phosphorylated gDNA that align with amplification products.
- ssDNA reporters modified with fluorophores to indicate successful cleavage.
Plasmid Construction
Two plasmids representing the wild-type and mutant Pfcrt fragments are created and sequenced for validation.
RPA-PfAgo Platform Cleavage Assay
The assay involves mixing RPA products with specific concentrations of PfAgo and other components, followed by incubation. The resulting fluorescence signals indicate successful detection.
Optimization of PfAgo Cleavage Reaction
Conditions such as probe and gDNA concentrations are adjusted to maximize performance.
Urea-Denaturing PAGE
The cleavage products are verified using urea-PAGE, which separates DNA fragments based on size and composition.
Nested PCR and Sequencing
Nested PCR amplifies the Pfcrt gene for further validation through Sanger sequencing, ensuring the accuracy of results.
Sensitivity Validation
Various dilutions of plasmids are tested to determine the RPA-PfAgo platform’s sensitivity and detection abilities for both mutant and wild-type strains.
Clinical Evaluation
Genomic DNA from Plasmodium falciparum samples undergoes extraction and analysis. The platform is validated by comparing results with traditional sequencing for a selection of patient samples.
Data Analysis
Results from the experiments are processed using SPSS for statistical evaluation.