Comparative DNA extraction methods to use for LAMP assay as molecular diagnosis of human schistosomes from urine samples.
Author(s): Pulkkila B, Sikasunge C, Mwansa J, Lodh N
Publication: BMC Infect Dis, 2025, Vol. 25, Page 1114
PubMed ID: 40983929 PubMed Review Paper? No
Purpose of Paper
This paper compared the detection of cell-free repeat DNA of two parasites (S. mansoni and S. haematobium) by loop-mediated isothermal amplification (LAMP) using DNA extracted from urine by four different methods.
Conclusion of Paper
When cfDNA extraction was performed with either the QIAamp Blood and Tissue Mini Kit or the LAMP-PURE Extraction Kit, S. mansoni and S. haematobium were detected by LAMP in all PCR-positive specimens and identified additional positives that were missed by PCR. When S. mansoni was detected by LAMP, the specificity was 100% regardless of the extraction method, although sensitivity differed among the extraction kits investigated: QIAamp Blood and Tissue Mini Kit (100%), LAMP-PURE Extraction Kit (100%), Chelex (72.73%), and heating in water (76.19%). When S. haematobium was detected by LAMP, the specificity was 100% regardless of extraction method, but the sensitivity was highest when extraction was with Chelex (100%), followed by heating in water (96.43%), the LAMP-Pure Kit (95.45%), and the QIAamp Blood and Tissue Mini Kit (94.44%). When LAMP detection of the two different parasites were compared, extraction with Chelex or heating in water led to better specificty for S. haematobium (90%, both) than S. mansoni (73.3% and 70%, respectively). In contrast, the sensitivity of PCR detection was comparably lower for both S. mansoni (57.14%) and S. haematobium (83.33%) despite a specificity of 100%. The authors conclude that DNA extraction with LAMP-Pure had the highest overall sensitivity and was the fastest, but also the most expensive method. QIAamp extraction had the second highest overall sensitivity and was less expensive than LAMP-Pure but was more expensive and slower than Chelex and heating methods. The Chelex method had better sensitivity and was faster than heating, but it was slightly more expensive than heating and less sensitive than the other kits.
Studies
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Study Purpose
This study compared the detection of cell-free repeat DNA of two parasites (S. mansoni and S. haematobium) by loop-mediated isothermal amplification (LAMP) using DNA extracted from urine by four different methods. A total of 30 urine specimens were collected from children located in two schistosome-endemic regions of Zambia; based on PCR assays, 8 children were positive for both S. mansoni and S. haematobium , 7 were negative for both bacteria, 8 were positive for S. mansoni and 7 were positive for S. haematobium. Urine specimens were applied to Whatman #3 filter paper and air-dried before shipping at room temperature to the United States and storage at 4°C. Each filter paper was divided into four quadrants for DNA extraction with each of the four methods. DNA was extracted using the QIAamp Blood and Tissue Mini Kit, the LAMP-PURE Extraction Kit, by a Chelex-based extraction (immersed in a Chelex suspension, vortexed, incubated at 95°C for 20 min, and centrifuged), and a heating-based method that included incubation in water at 95°C for 20 min and centrifugation to remove the paper. DNA was quantified by NanoDrop and stored at -20°C. Cell-free DNA repeats of S. mansoni and S. haematobium were LAMP-amplified and visualized by gel electrophoresis.
Summary of Findings:
When cfDNA extraction was performed with either the QIAamp Blood and Tissue Mini Kit or the LAMP-PURE Extraction Kit, S. mansoni and S. haematobium were detected by LAMP in all PCR positive specimens and identified additional positives missed by PCR. When S. mansoni was detected by LAMP, the specificity was 100% regardless of the extraction method, although sensitivity differed among the extraction kits investigated: QIAamp Blood and Tissue Mini Kit (100%), LAMP-PURE Extraction Kit (100%), Chelex (72.73%), and heating in water (76.19%). When S. haematobium was detected by LAMP, the specificity was 100% regardless of extraction method, but the sensitivity was highest when extraction was with Chelex (100%), followed by heating in water (96.43%), the LAMP-Pure Kit (95.45%), and the QIAamp Blood and Tissue Mini Kit (94.44%). When LAMP detection of the two different parasites were compared, extraction with Chelex or heating in water led to better specificty for S. haematobium (90%, both) than S. mansoni (73.3% and 70%, respectively). In contrast, the sensitivity of PCR detection was comparably lower for both S. mansoni (57.14%) and S. haematobium (83.33%) despite a specificity of 100%. The authors conclude that DNA extraction with LAMP-Pure had the highest overall sensitivity and was the fastest, but also the most expensive method. QIAamp extraction had the second highest overall sensitivity and was less expensive than LAMP-Pure but was more expensive and slower than Chelex and heating methods. The Chelex method had better sensitivity and was faster than heating, but it was slightly more expensive than heating and less sensitive than the other kits.
Biospecimens
Preservative Types
- Other Preservative
Diagnoses:
- Other diagnoses
Platform:
Analyte Technology Platform DNA PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method QIAamp Blood and Tissue Mini Kit
LAMP-PURE Extraction Kit
Chelex based extraction (immersed in a Chelex suspension, vortexed, incubated at 95°C for 20 min, and centrifuged)
Heating-based method (incubation in water at 95°C for 20 min and centrifugation)
PCR Specific Technology platform PCR
LAMP
PCR Specific Targeted nucleic acid S. mansoni
S. haematobium