Electricity-free nucleic acid extraction method from dried blood spots on filter paper for point-of-care diagnostics

Abstract

Background: Nucleic acid extraction is a crucial step for molecular biology applications, being a determinant for any diagnostic test procedure. Dried blood spots (DBS) have been used for decades for serology, drug monitoring, environmental investigations, and molecular studies. Nevertheless, nucleic acid extraction from DBS remains one of the main challenges to translate them to the point-of-care (POC). Method: We have developed a fast nucleic acid extraction (NAE) method from DBS which is electricity-free and relies on cellulose filter papers (DBSFP). The performance of NAE was assessed with loop-mediated isothermal amplification (LAMP), targeting the human reference gene beta-actin. The developed method was evaluated against FTA cards and magnetic bead-based purification, using time-to-positive (min) for comparative analysis. We optimised and validated the developed method for elution (eluted disk) and disk directly in the reaction (in-situ disk), RNA and DNA detection, and whole blood stored in anticoagulants (K2EDTA and lithium heparin). Furthermore, the compatibility of DBSFP with colourimetric detection was studied to show the transferability to the POC. Results: The proposed DBSFP is based on grade 3 filter paper pre-treated with 8% (v/v) igepal surfactant, 1 min washing step with PBS 1X and elution in TE 1X buffer after 5 min incubation at room temperature, enabling NAE under 7 min. Obtained results were comparable to gold standard methods across tested matrices, targets and experimental conditions, demonstrating the versatility of the methodology. Lastly, eluted disk colourimetric detection was achieved with a sample-to-result turnaround time under 35 min. Conclusions: The developed method is a fast, electricity-free, and low-cost solution for NAE from DBSFP enabling molecular testing in virtually any POC setting.

Sensitive Detection of Asymptomatic and Symptomatic Malaria with Seven Novel Parasite-Specific LAMP Assays and Translation for Use at Point-of-Care

“…Seventy samples from symptomatic and asymptomatic Ghanaian children were collected in Obom, Ghana. DNA was extracted, and samples were tested with our species-specific LAMP assays, a LAMP assay targeting the human housekeeping gene ACTB (LAMP-ACTB) ( 28 ), and two TaqMan qPCR assays ( 12 ) targeting the 18S rRNA gene of P. falciparum (PCR-Pf) and Plasmodium pan-genus (PCR-Pan). Primer sequences are detailed in Table S3.…”

“…Lastly, to evaluate a truly sample-to-result device at the POC, a low-cost and simple sample preparation module has to be integrated or easily combined with the LoC platform. ( 28 ) Future work will address these limitations, providing a reliable molecular-based test for POC malaria diagnostics.…”

“…All primers were purchased from Integrated DNA Technologies (IDT) and rehydrated in Tris-EDTA (TE) buffer at 100 μM. The LAMP assay specific to the human housekeeping gene ACTB , LAMP-ACTB, was used as reported in reference 28 . Primer sequences are shown in Table S3.…”