A Colorimetric LAMP Detection of Xylella fastidiosa in Crude Alkaline Sap of Olive Trees in Apulia as a Field-Based Tool for Disease Containment

Abstract

Xylella fastidiosa subsp. pauca (Xfp) infects olive trees and other hosts in Southern Apulia (Italy), devastating agriculture and landscape. A containment strategy of the disease requires quick and sensitive detection tools. Therefore, a colorimetric LAMP protocol was developed using as a template a crude alkaline sap obtained from incubation of 50–60 mg of thin slices of olive twigs in a NaOH-containing buffer. This rapid molecular assay can be performed directly in the field, as it needs only a portable isothermal block. Tissues of the same olive trees analysed by this technique were also compared to qPCR (using purified total plant DNA as template) as well as digital droplet PCR (on the same crude alkaline extracts used in cLAMP). A titration of the cLAMP reaction with healthy olive sap, spiked with dilutions of in vitro cultivated Xfp cells and plasmid DNA containing the target sequence, gave positive detection results as low as 102 CFU/mL and up to 169.2 target copies/µL, equivalent to about 0.9 pg of the genomic DNA. A portable, sensitive and target-specific Xfp field test was developed, which has a 40 min sample-to-answer time and does not require any DNA isolation procedure or laboratory equipment. The application of this detection assay could help the monitoring and containment of the disease spread.

A decade of monitoring surveys for Xylella fastidiosa subsp. pauca in olive groves in Apulia (Italy) reveals a low incidence of the bacterium in the demarcated areas

“…that were not immediately uprooted due to pending legislative procedures and updating of demarcation areas are currently in an optimal vegetative and productive status many years after the detection of Xfp (Figures 6 and 7). (Amoia et al, 2023;D'Onghia et al, 2022;Faino et al, 2021;Mastin et al, 2022;Passera et al, 2022). This proposal is in line with findings from other scientific studies with regard to measures taken to limit the spread of X. fastidiosa.…”

“…This should provide that advantage of saving potentially healthy plants.” The EFSA also stated the possibility that the detection techniques do not intercept the bacterium, which can occur at a low concentration within the tree (EFSA, 2019). It should be said that in recent years the techniques for detecting Xfp in olive trees have been greatly improved, now requiring only very few cells of the bacterium in the sample (Amoia et al., 2023; D'Onghia et al., 2022; Faino et al., 2021; Mastin et al., 2022; Passera et al., 2022). This proposal is in line with findings from other scientific studies with regard to measures taken to limit the spread of X. fastidiosa .…”

Update of the Xylella spp. host plant database – systematic literature search up to 30 June 2023

“…The collected references were downloaded and imported into an EndNote X9 library (Clarivate Analytics). Duplicates and references already included in the update published in June 2023 (EFSA, 2023) were removed using EndNote X9 and the remaining references were uploaded on DistillerSR online 3 together with the full texts in portable document format (pdf).…”

Meat Authenticity Made Easy: DNA Extraction-Free Rapid Onsite Detection of Duck and Pork Ingredients in Beef and Lamb Using Dual-Recombinase-Aided Amplification and Multiplex Lateral Flow Strips

“…However, cLAMP has limitations in detecting low-concentration target genes compared to qPCR. The threshold for reliable detection of crude extracts is above 10 2 copies/reaction system, restricting its application for samples with low levels of the target gene. , …”

“…The threshold for reliable detection of crude extracts is above 10 2 copies/reaction system, restricting its application for samples with low levels of the target gene. 18,19 To address this challenge, alternative approaches are being explored like a hand-held microfluidic visual LAMP chip system with simple microneedle DNA extraction, capable of detecting 1% simulated adulteration within 60 min. 1 Recombinse-aided amplification (RAA) and related nucleic acid amplification technologies, such as recombinase polymerase amplification (RPA), have gained popularity in meat identification.…”