Biology subjects

Malpartida-Cardenas, K.

Publications and source records attributed to Malpartida-Cardenas, K..

3 recordsLinked to original sources

bioRxiv2022

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

BackgroundNucleic 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). MethodWe 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. ResultsThe 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. ConclusionsThe developed method is a fast, electricity-free, and low-cost solution for NAE from DBSFP enabling molecular testing in virtually any POC setting.

molecular biology↗

bioRxiv2019

Quantitative and rapid Plasmodium falciparum malaria diagnosis and artemisinin-resistance detection using a CMOS Lab-on-Chip platform

Early and accurate diagnosis of malaria and drug-resistance is essential to effective disease management. Available rapid malaria diagnostic tests present limitations in analytical sensitivity, drug-resistant testing and/or quantification. Conversely, diagnostic methods based on nucleic acid amplification stepped forwards owing to their high sensitivity, specificity and robustness. Nevertheless, these methods commonly rely on optical measurements and complex instrumentation which limit their applicability in resource-poor, point-of-care settings. This paper reports the specific, quantitative and fully-electronic detection of Plas-modium falciparum, the predominant malaria-causing parasite worldwide, using a Lab-on-Chip platform developed in-house. Furthermore, we demonstrate on-chip detection of C580Y, the most prevalent single-nucleotide polymorphism associated to artemisinin-resistant malaria. Real-time non-optical DNA sensing is facilitated using Ion-Sensitive Field-Effect Transistors, fabricated in unmodified complementary metal-oxide-semiconductor technology, coupled with loop-mediated isothermal amplification. This work holds significant potential for the development of a fully portable and quantitative malaria diagnostic that can be used as a rapid point-of-care test.

bioengineering↗

bioRxiv2018

Simultaneous single-channel multiplex and quantification of carbapenem-resistant genes using multidimensional standard curves

Multiplexing and absolute quantification of nucleic acids, both have, in their own right, significant and extensive use in biomedical related fields, especially in point-of-care applications. Currently, the ability to detect several nucleic acid targets in a single-reaction scales linearly with the number of targets; an expensive and time-consuming feat. Here, we propose a new methodology based on multidimensional standard curves that extends the use of real-time PCR data obtained by common qPCR instruments. By applying this novel methodology, we achieve simultaneous single-channel multiplexing and enhanced quantification of multiple targets using only real-time amplification data. This is obtained without the need of fluorescent probes, agarose gels, melting curves or sequencing analysis. Given the importance and demand for tackling challenges in antimicrobial resistance, the proposed method is applied to the four most prominent carbapenem-resistant genes: blaOXA-48, blaNDM, blaVIM and blaKPC, which account for 97% of the UKs reported carbapenemase-producing Enterobacteriaceae.

microbiology↗