FTA-LAMP based biosensor for a rapid in-field detection of Globodera pallida-the pale potato cyst nematode.

The combination of a sensitive and specific magnetoresistive sensing device with an easy DNA extraction method and a rapid isothermal amplification is presented here targeting the on-site detection of Globodera pallida, a potato endoparasitic nematode. FTA-cards were used for DNA extraction, LAMP was the method developed for DNA amplification and a nanoparticle functionalized magnetic-biosensor was used for the detection. The combinatorial effect of these three emerging technologies has the capacity to detect G. pallida with a detection limit of one juvenile, even when mixed with other related species. This combined system is far more interesting than what a single technology can provide. Magnetic biosensors can be combined with any DNA extraction protocol and LAMP forming a new solution to target G. pallida. The probe designed in this study consistently distinguished G. pallida (∆Vac binding/Vac sensor above 1%) from other cyst nematodes (∆Vac binding/Vac sensor below 1%). It was confirmed that DNA either extracted with FTA-cards or Lab extraction Kit was of enough quantity and quality to detect G. pallida whenever present (alone or in mixed samples), ensuring probe specificity and sensitivity. This work provides insights for a new strategy to construct advanced devices for pathogens in-field diagnostics. LAMP runs separately but can be easily integrated into a single device.

A Lab-on-a-Chip Approach for the Detection of the Quarantine Potato Cyst Nematode Globodera pallida.

The potato cyst nematode (PCN), Globodera pallida, has acquired significant importance throughout Europe due to its widespread prevalence and negative effects on potato production. Thus, rapid and reliable diagnosis of PCN is critical during surveillance programs and for the implementation of control measures. The development of innovative technologies to overcome the limitations of current methodologies in achieving early detection is needed. Lab-on-a-chip devices can swiftly and accurately detect the presence of certain nucleotide sequences with high sensitivity and convert the presence of biological components into an understandable electrical signal by combining biosensors with microfluidics-based biochemical analysis. In this study, a specific DNA-probe sequence and PCR primers were designed to be used in a magnetoresistive biosensing platform to amplify the internal transcribed spacer region of the ribosomal DNA of G. pallida. Magnetic nanoparticles were used as the labelling agents of asymmetric PCR product through biotin−streptavidin interaction. Upon target hybridization to sensor immobilized oligo probes, the fringe field created by the magnetic nanoparticles produces a variation in the sensor's electrical resistance. The detection signal corresponds to the concentration of target molecules present in the sample. The results demonstrate the suitability of the magnetic biosensor to detect PCR target product and the specificity of the probe, which consistently distinguishes G. pallida (DV/V > 1%) from other cyst nematodes (DV/V < 1%), even when DNA mixtures were tested at different concentrations. This shows the magnetic biosensor's potential as a bioanalytical device for field applications and border phytosanitary inspections.

Combined detection of molecular and serological signatures of viral infections: The dual assay concept.

The recent worldwide spread of viral infections has highlighted the need for accurate, fast, and inexpensive disease diagnosis and monitorization methods. Current diagnostics tend to focus either on molecular or serological testing. In this work we propose a dual detection assay approach for viral diseases, where both serological and molecular assays are combined in a single analysis performed on a magnetoresistive system. This type of assay guarantees an accurate assessment of the infection phase, saving time and costs associated with multiple independent tests. Zika and dengue viruses were used as model diseases for the validation of the system. Human IgG anti-zika and anti-dengue antibodies were successfully detected in infected patients' serum, using a novel approach combining competitive and sandwich strategies in a magnetoresistive portable platform. Specificity and sensitivity values of 100% were obtained. Calibration curves with dynamic ranges between 10 ng/mL and 1 µg/mL were established achieving LODs of 1.26 and 1.38 nM for IgG anti-ZIKV and anti-DENV antibodies, respectively. Viral RNA detection down to a few hundreds of pM was also successfully carried out after the design of specific oligo probes and primers for RT-PCR amplification. Dual assays were performed for both viruses, where viral RNA and anti-virus antibodies in serum samples were simultaneously detected. The results obtained for the detection of the molecular and serological targets in the dual assay format show no significant difference between the ones obtained individually, proving the feasibility and accuracy of the dual detection assay. This assay format represents a new paradigm in viral infections diagnostics.