Experimental Scheme Sharing: Dissection of Haplotype-Specific Drug Response Phenotypes in Multiclonal Plasmodium falciparum Strains

Multiplex Probe qPCR Mix Plus U Experimental Scheme 1

Dissection of Haplotype-Specific Drug Response Phenotypes in Multiclonal Plasmodium falciparum Strains:

1. Preparation of Experimental Materials and Reagents

- Select 3 suspected multiclonal P. falciparum strains, such as: IPC_3445 (MRA-1236), IPC_5202 (MRA-1240), IPC_6403 (MRA-1285).

- Prepare O+ human red blood cells and RPMI 1640 culture medium, as well as the necessary supplements and antibiotics.

- Prepare stock solutions of chloroquine, mefloquine, and piperaquine drugs.

- Prepare reagents for DNA extraction, genotyping, and PCR reactions.

2. In Vitro Culturing of Plasmodium Strains

- Culture the Plasmodium strains under standard in vitro conditions at 37°C, 5% CO2, and 90% N2 atmosphere.

3. Confirmation of Multiclonal Nature

- Extract DNA from each Plasmodium strain and genotype at 24 highly polymorphic SNP sites.

4. Isolation of Single Parasite Lineages

- Dilute the Plasmodium strains in 96-well plates using the limiting dilution method to isolate individual infected red blood cells.

- Use SYBR® Green I fluorescent dye to detect Plasmodium DNA and identify positive wells.

5. Genotyping and Phylogenetic Analysis

- Genotype the parasites in each positive well using the 24-SNP molecular barcode method.

- Construct a phylogenetic tree to analyze the relationships between different parasite lineages.

6. In Vitro Antimalarial Drug Susceptibility Testing

- Use the SYBR® Green Antimalarial Assay to test the susceptibility to chloroquine, mefloquine, and piperaquine.

- Determine the half-maximal inhibitory concentration (IC50) for each drug.

7. Parasite Survival Assays

- For CQ and PPQ, use the Ring-stage Survival Assay (RSA) and Piperaquine Survival Assay (PSA) to determine the survival rate of parasites in response to drugs.

8. Sequencing and Genotyping of Antimalarial Resistance Loci

8.1 Selection of Target Genes

Select genes known to be associated with antimalarial drug resistance, including but not limited to:

- Chloroquine resistance-related gene: pfcrt (Plasmodium falciparum chloroquine resistance transporter)

- Mefloquine and piperaquine resistance-related gene: pfmdr-1 (Plasmodium falciparum multidrug resistance 1 gene)

- Artemisinin resistance-related gene: K13 gene

8.2 DNA Extraction and PCR Amplification

- Extract genomic DNA from Plasmodium cultures using a DNA extraction kit.

- Design specific primers for PCR amplification of the aforementioned genes. For example, use five sets of primers to cover the entire pfcrt gene.

8.3 Detection of SNPs

- Use Sanger sequencing technology to sequence the PCR products to detect single nucleotide polymorphisms (SNPs) in the pfcrt, pfmdr-1, and K13 genes.

- Use sequencing result analysis software (such as DNASTAR Lasergene) for sequence data analysis and visualization.

8.4 Detection of Copy Number Variations

- Use the ΔΔCT method to determine the copy number of pfmdr-1 and pfmp-2 (Plasmodium falciparum merozoite surface protein 2 gene), which are related to mefloquine and piperaquine resistance.

- Use real-time quantitative PCR (qPCR) technology to amplify the target gene and reference gene (such as β-tubulin gene or serine tRNA ligase gene) in the same well.

8.5 qPCR Experimental Operation

- Prepare the master mix, including Multiplex Probe qPCR Mix Plus U (GDSBio, P2701), sterile water, and pre-formulated Taqman SNP detection for the target and reference genes.

- Dispense the master mix into a 96-well PCR plate, with 75 ng of parasite DNA per sample.

- Use the CFX96 real-time PCR instrument for sample amplification and analyze the results using the gene expression module.

8.6 Data Analysis

- Compare the copy number of the target gene with that of the reference gene to determine the relative copy number.

- Calibrate using standard samples with known copy numbers (such as the 3D7 strain).

- For pfmdr-1 and pfmp-2 genes, analyze whether there is gene amplification, which may be related to the development of drug resistance.

8.7 Result Recording and Interpretation

- Record all sequencing and qPCR results, including genotypes and copy numbers.

- Interpret the sequencing and copy number variation data, analyzing their potential connection with in vitro drug resistance phenotypes.

8.8 Quality Control

- Include the use of laboratory control strains with known genotypes as positive and negative controls.

- Repeat each experimental step to ensure the accuracy and reproducibility of the data.

The above steps provide a detailed experimental procedure for detecting and analyzing genetic variations related to antimalarial drug resistance. Experimental operations should strictly follow laboratory protocols and biosafety guidelines to ensure the accuracy and reliability of the data.

9. Data Analysis

- Use GraphPad Prism software to analyze IC50 values and survival rate data.

- Apply statistical methods to compare differences between different parasite lineages.

10. Result Interpretation and Reporting

- Interpret the experimental results and discuss the drug resistance phenotypes of different parasite lineages.

- Write an experimental report, including methods, results, and discussion.

11. Safety and Quality Control

- Ensure that experimental operations comply with biosafety standards.

- Use known laboratory control strains as positive and negative controls.

12. Preservation and Recording of Experimental Materials

- Record all experimental steps and results.

- Save all raw data and experimental materials for subsequent analysis and verification.

This experimental scheme provides a comprehensive framework for studying the multiclonality and drug resistance phenotypes of P. falciparum strains. Experimental operations should strictly adhere to laboratory protocols and biosafety guidelines.

Multiplex Probe qPCR Mix with UDG

Product Introduction

Multiplex Probe qPCR Mix Plus U is a 2× concentrated premix for real-time quantitative PCR with probe method. In use, just add the DNA template, primer and probe to react. This product contains antibody technology modified Hotstart Taq DNA polymerase. Combined with GDSBio's special real-time PCR Buffer, it can not only effectively inhibit primers dimer and other non-specific amplification, but also improve the amplification efficiency, allowing multi-probe qPCR reaction. This reagent introduced dUTP/UDG anti-contamination system, which can remove PCR products containing dUTP before PCR reaction, effectively avoid the influence of cross contamination of amplification products on quantification. This product is convenient for preparation of IVD molecular diagnostic kit. The target gene quantification is accurate, reliable and reproducible. This product can be used with TaqMan and other fluorescent probes, and is perfectly compatible with common quantitative PCR instruments, such as ABI, Roche, Bio-rad, etc.

Application

• Probe gene expression analysis

• Multiple Low-copy gene detection

• Probe microarray validation

• Probe gene knockdown validation

Features

• This kit is suitable for Multiplex fluorescence quantification by probe method

• This kit is compatible with many real-time systems

• Hot-start technology brings high specificity and reproducible amplification

• dUTP/UDG system, effectively prevent PCR product contamination

Experimental Cases

Using human genome DNA as template, it has good multiplex amplification performance.