Abstract
BackgroundQuantitative real-time PCR (qPCR) is now commonly used as a method to confirm diagnosis of malaria and to differentiate recrudescence from re-infection, especially in clinical trials and in reference laboratories where precise quantification is critical. Although anti-malarial drug discovery is based on in vivo murine efficacy models, use of molecular analysis has been limited. The aim of this study was to develop qPCR as a valid methodology to support pre-clinical anti-malarial models by using filter papers to maintain material for qPCR and to compare this with traditional methods.MethodsFTA technology (Whatman) is a rapid and safe method for extracting nucleic acids from blood. Peripheral blood samples from mice infected with Plasmodium berghei, P. yoelii, or P. falciparum were kept as frozen samples or as spots on FTA cards. The extracted genetic material from both types of samples was assessed for quantification by qPCR using sets of specific primers specifically designed for Plasmodium 18S rRNA, LDH, and CytB genes.ResultsThe optimal conditions for nucleic acid extraction from FTA cards and qPCR amplification were set up, and were confirmed to be suitable for parasite quantification using DNA as template after storage at room temperature for as long as 26 months in the case of P. berghei samples and 52 months for P. falciparum and P. yoelii. The quality of DNA extracted from the FTA cards for gene sequencing and microsatellite amplification was also assessed.ConclusionsThis is the first study to report the suitability of FTA cards and qPCR assay to quantify parasite load in samples from in vivo efficacy models to support the drug discovery process.
Highlights
Quantitative real-time PCR is commonly used as a method to confirm diagnosis of malaria and to differentiate recrudescence from re-infection, especially in clinical trials and in reference laboratories where precise quantification is critical
Biological material Plasmodium falciparum-infected blood samples were taken from the tail vein of the NOD-scid IL2R gammanull engrafted mice, and blood samples infected with P. berghei or P. yoelii were taken from CD1 mice
To establish the limit of detection (LOD), P. falciparum blood samples were serially diluted tenfold using fresh AB+ human erythrocyte concentrates generously donated by the Spanish Red Cross blood bank, and P. berghei and P. yoelii blood samples were diluted with uninfected blood from a donor CD1 mouse
Summary
Quantitative real-time PCR (qPCR) is commonly used as a method to confirm diagnosis of malaria and to differentiate recrudescence from re-infection, especially in clinical trials and in reference laboratories where precise quantification is critical. Quantitative real-time PCR (qPCR) has demonstrated a high sensitivity for detection of low parasite burdens, and is Bahamontes‐Rosa et al Malar J (2016) 15:147 commonly used as a method to confirm diagnosis of malaria, especially in clinical trials and in reference laboratories where precise quantification is critical [8,9,10]. This is the first study to report parasite identification and quantification using FTA blood spots to support pre-clinical animal models. It was designed to identify the adequate molecular settings that may be encountered across laboratories to support anti-malarial in vivo assays
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