Comparative assessment of three methods (Chelex-based method, commercial kit, and CTAB-based method) for extracting DNA from Whatman filter paper on which urine samples were filtered and stored for molecular diagnosis of urinary schistosomiasis

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Introduction Storage of urine samples and the need for DNA extract free from PCR inhibitors remains serious challenges for the molecular diagnosis of urinary schistosomiasis. This study was designed to address these challenges by assessing the efficiency of three DNA extraction methods using Whatman filter papers on which urine samples were filtered and stored for molecular diagnosis of urinary schistosomiasis. Methods Urine samples were collected from school-aged children in the Matta health area and analyzed using the urine filtration test. For this study, 120 urine samples with S. haematobium eggs and 54 without eggs were filtered through punched Whatman filter papers and stored for up to 20 weeks. DNA was extracted from these filter papers at different time points using three DNA extraction methods. The performance of these extraction methods and the capacity of the filter papers to store S. haematobium DNA for several weeks were assessed by amplifying a specific fragment of S. haematobium . Results All DNA extracts obtained using the Chelex-based method yielded successful amplification up to 12 weeks of urine preservation on Whatman filter papers, whereas the amplification rate for the cetyltrimethylammonium bromide (CTAB)-based method decreased over time from 99.2% to 96.7%. When comparing amplification rates according to storage time, the amplification rate of extracts obtained using the Chelex-based method was significantly higher than that of extracts obtained using the other extraction methods after 12 weeks (P = 0.0007) and 20 weeks (P < 0.0001) of urine preservation on Whatman filter papers. Using the Chelex-based method, the sensitivity and specificity of PCR ranged from 99.2% to 100% and from 77.8% to 83.3%, respectively. Conclusion This study demonstrated the performance of the Chelex-based method for extracting S. haematobium DNA preserved on these papers. The extraction cost for the Chelex-based method (€0.24) appeared reliable and was at least 2 times more cost-effective than the CTAB-based method and the commercial kit. Combining this method with urine storage on Whatman filter paper could improve the diagnosis, surveillance, and elimination monitoring of urinary schistosomiasis.

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