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.

Rapid Detection and Identification of Vancomycin-Sensitive Bacteria Using an Electrochemical Apta-Sensor.

In order to combat the complex and diverse infections caused by bacteria, it is essential to develop efficient diagnostic tools. Current techniques for bacterial detection rely on laborious multistep procedures, with high costs and extended time of analysis. To overcome these limitations, we propose here a novel portable electrochemical biosensor for the rapid detection and identification of Gram-positive bacteria that leverages the recognition capabilities of vancomycin and aptamers. A vancomycin-modified screen-printed carbon electrode was used to selectively capture Gram-positive bacteria susceptible to this antibiotic. Electrochemical impedance spectroscopy and scanning electron microscopy demonstrated that capture was achieved in 10 min, with a limit of detection of only 2 CFU/mL. We then tested the device's potential for aptamer-based bacterial identification using Staphylococcus aureus and Bacillus cereus as the test strains. Specifically, electrodes with captured bacteria were exposed to species-specific aptamers, and the resulting changes in current intensity were analyzed using differential pulse voltammetry. When used directly in untreated milk or serum, the system was able to successfully identify a small amount of S. aureus and B. cereus (100 CFU/mL) in less than 45 min. This novel biosensor has the potential to serve as an invaluable tool that could be used, even by inexperienced staff, in a broad range of settings including clinical diagnostics, food safety analysis, environmental monitoring, and security applications.

Root-knot nematode assessment: species identification, distribution, and new host records in Portugal.

Considered one of the most devastating plant parasitic nematodes worldwide, Meloidogyne spp. (commonly known as the root-knot nematodes (RKNs)) are obligate sedentary endoparasites that establish in the roots, causing hyperplasia and hypertrophy of surrounding cells, triggering the formation of galls. These galls will affect root development and physiology, leading to substantial yield losses. During 2017-2022, an extensive survey of Meloidogyne species was undertaken in Portugal (mainland and islands). A total of 1,071 samples were collected by the National Plant Protection Organization (DGAV) and private farmers from different regions of the country and were analysed at the Laboratory of Nematology (NemaINIAV). Samples in which the presence of Meloidogyne sp. was detected were used to perform bioassays to obtain females and juveniles for further studies. Since the accurate identification of RKNs is an important aspect of crop management, morphological and biochemical characterisation was performed. The most common morphological features were observed, showing consistency with previous descriptions of the genus. The biochemical identification using the esterase (EST) phenotype revealed the phenotypes of Meloidogyne arenaria, M enterolobi, M. hispanica, M. hapla, M. incognita, M javanica, and M. luci. Meloidogyne incognita and M. javanica were found to be the most prevalent species in the different regions followed by M. arenaria and M. hapla. This is the first distribution report performed in Portugal on RKNs, contributing to the development of management strategies and to updated information on the status of these pests in Europe.

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.

Development and Validation of a Loop-Mediated Isothermal Amplification Diagnostic Method to Detect the Quarantine Potato Pale Cyst Nematode, Globodera pallida.

The potato cyst nematode (PCN) Globodera pallida has acquired significant importance throughout Europe due to its nefarious effects on potato production. Rapid and reliable diagnosis of PCN is critical during the surveillance programs and for the implementation of control measures. Molecular DNA-based methods are available, but they require expensive laboratory facilities, equipment and trained technicians. Moreover, there is an additional need of time for sample shipment and testing. In this work, we have developed a new and simple assay which reliably discriminates G. pallida from other cyst nematodes in less than 40 min. This assay may be applied either on cysts or juveniles with the ability to detect a single juvenile of G. pallida in a sample of at least 40 juveniles of the non-target species G. rostochiensis. This test should be a tool to improve the performance of the laboratory and has the potential to be performed on-site.

Integrative Morphometric and Molecular Approach to Update the Impact and Distribution of Potato Cyst Nematodes Globodera rostochiensis and Globodera pallida (Tylenchida: Heteroderidae) in Algeria.

Morphological and molecular studies were conducted to characterize the specific identity of 36 isolates of potato cyst nematodes (PCNs) recovered from soil samples collected in several potato producing areas of Algeria. Morphometric data revealed that 44% of isolates contained Globodera pallida alone, 28% contained Globodera rostochiensis alone and 28% mixtures of the two species. Morphometric values of cysts and second-stage juveniles were generally distributed with slight differences in the expected ranges for both Globodera species. Inter- and intraspecific morphometric variability in nematode isolates was noted. Molecular analysis using conventional multiplex PCR with species-specific primers and TaqMan real-time PCR confirmed the morphological identification. In addition, the distribution of both potato cyst nematode species throughout various parts of the country was investigated. In the central areas, the isolates of G. pallida alone dominate, whereas isolates of G. rostochiensis alone are more frequent in the southern areas. In the eastern regions, mixed isolates are more representative. Most isolates examined in the western areas are mixtures of the two species or G. rostochiensis alone. Comparatively, G. pallida remains the most widely distributed species in its geographic range. This study confirms the presence of two PCN species, G. pallida and G. rostochiensis, in Algeria and provides additional information on their biogeographic distribution.

Potato Cyst Nematodes: Geographical Distribution, Phylogenetic Relationships and Integrated Pest Management Outcomes in Portugal.

The identification and phylogenetic relationships of potato cyst nematodes (PCN) were studied to assess the potential value of geographical distribution information for integrated pest management of potato production in Portugal. This research focused on PCN species, Globodera pallida and Globodera rostochiensis. From 2013 until 2019, 748 soil samples from the rhizosphere of different potato cultivars were surveyed in the Portuguese mainland to detect and identify both species and track their location. PCN are widespread invasive species throughout Portugal. In fact, during the survey period an incidence of 22.5% was estimated for the tested samples. The patterns of infestation vary among regions, increasing from south to north, where PCN were first detected. Currently, both species are present in all potato producing regions of the country, with a greater incidence of G. pallida. Phytosanitary control measures are influencing to the observed results. The use of potato cultivars resistant to G. rostochiensis led to a decrease of this species but had no influence on G. pallida detections, which continues its reproduction freely since there are no effective resistant cultivars for this species. The relationship between the presence, infestation rate, spread and geographical distribution of PCN is discussed in terms of behavioral responses of the potato cultivars and the implications for developing new integrated crop protection measures.