A test performance study (TPS) was conducted in 2020 to evaluate performance of serological and PCR-based tests for the detection of
Xylophilus ampelinus
in homogenised vine samples. In total, 11 ...labs participated, although there were fewer participants for the serological tests than the PCR-based tests. The panel of samples sent to participants included spiked samples containing 10
4
–10
8
cfu per ml (serological tests) or 10
2
–10
6
cfu per ml (PCR-based tests) as well as positive and negative controls. The five PCR-based tests were found to be fit for purpose, with similar performance across a range of metrics (analytical sensitivity, diagnostic sensitivity and specificity, and repeatability and reproducibility assessed in terms of accordance and concordance, respectively). Serological methods (two immunofluorescence tests and two ELISA tests) were found to be less sensitive with regard to both analytical and diagnostic sensitivity. Furthermore, the occurrence of false positives suggests that a positive IF result may not be conclusive when considered in isolation. One of the ELISA tests exhibited much lower analytical and diagnostic sensitivity than the other serological tests and would not be considered suitable for the purpose considered by this TPS.
Huanglongbing disease affects the Rutaceae family and is associated with three phloem-limited bacterium species:
Candidatus
Liberibacter asiaticus, africanus and americanus. These species are ...considered quarantine pathogens in the world, and pose major risks for citrus production and industry.
Due to the low titer and the uneven distribution of the bacteria within its host plant, conventional PCR detection protocols can lead to false negative results, especially for early detection. Herein, three real-time PCR diagnostic methods recommended by the EPPO and FAO for asiaticus and africanus species detection were evaluated for their performance and compared with a conventional duplex PCR. Assessments were done as part of an international cooperative project under the EUPHRESCO guidance. Intra-laboratory assessment of the analytical specificity and analytical sensitivity was performed on 33 target or non-target DNA samples and seven target DNA samples were used to determine the sensitivity. Thereafter, repeatability, reproducibility, and concordance odds ratio were assessed on 20 target or non-target DNA samples through a collaborative test performance study organized among eight international laboratories. Results showed that the Li protocol proved to be the best method for asiaticus and africanus species detection, along with the conventional duplex PCR; whereas the Morgan protocol showed high performance only for asiaticus species. Interlaboratory reproducibility was high, suggesting that these real-time PCR methods can be readily transferred to diagnostic laboratories.
This open access book in the field of plant pest detection shows a constant demand in development and improvement of fast and reliable detection tools, especially for high-priority pests. This open ...access book describes and summarizes the whole process of the organization of test performance study (TPS) for these tools. The outcome of TPS, obtained through the evaluation of the performance of one or more diagnostic tests by several laboratories on defined samples, is the finding of the best performing test/s for particular pest and for specific uses. Nowadays the intensification of worldwide trade and associated controls increases the need for quality assurance accreditation and harmonization of laboratories practices. Therefore, such studies are very important, but, non-existent. Considering those facts, our goal was to develop guidelines, by using the data and experiences of involved partners, for further TPS in the field of plant health. Developed guidelines could be easily transferable to other microbiology fields.
An international test performance study (TPS) was organised to generate validation data for three molecular
Synchytrium endobioticum
tests: van den Boogert et al. (
European Journal of Plant ...Pathology 113,
47–57,
2005
), and van Gent-Pelzer et al. (
European Journal of Plant Pathology, 126,
129-133,
2010
) for the detection of
S. endobioticum
, and the pathotype 1(D1) identification test described by Bonants et al. (
European Journal of Plant Pathology, 143
, 495-506,
2015
). Two TPS rounds were organised focussing on different test matrices, i.e. round 1: warted potato tissue, and round 2: resting spore suspensions. When using the tests for detection and identification of
S. endobioticum
in warted potato tissue, no significant differences were observed for diagnostic sensitivity, diagnostic specificity, overall accuracy, analytical sensitivity and robustness. When using the tests for detection and identification of
S. endobioticum
in resting spore suspensions, the van den Boogert and van Gent-Pelzer tests significantly outperform the Bonants test for diagnostic sensitivity and diagnostic specificity. For overall accuracy and analytical sensitivity, the van Gent-Pelzer significantly outperforms the van den Boogert and Bonants tests and is regarded as the test of choice when identifying
S. endobioticum
from resting spores. Tests regarded fit for purpose for routine testing of wart material and resting spore suspensions are proposed for the update of EPPO standard PM7/28(1)
Synchytrium endobioticum
.
In 2022, a test performance study (TPS) assessing the influence of different master mixes on the performance of the tetraplex real-time PCR (TqPCR) assay was organized. TqPCR allows for the specific ...detection and identification of Xylella fastidiosa (Xf) subspecies in a single reaction. Eighteen official laboratories of the Italian National Plant Protection Organization received a panel of 12 blind samples, controls, primers, probes, and different master mixes to participate in the TPS. Furthermore, the Research Centre for Plant Protection and Certification of the Council for Agricultural Research and Economics performed an intra-laboratory study (ITS) on spiked plant matrices to evaluate the analytical sensitivity of TqPCR employing the selected master mixes with the best performance. Naturally infected samples were analyzed for subspecies identification via TqPCR compared with the official multilocus-sequence-typing (MLST) method. The best results in this comparative study were obtained using Fast Universal PCR Master Mix (Applied Biosystems) and Brilliant multiplex QPCR Master Mix (Agilent), and they confirmed that the TqPCR test is reliable, offering the advantage of identifying this subspecies at the same time, thus saving time and resources. The TqPCR assay is suggested among the tests to be used by laboratories performing the official diagnosis of Xf to support the activities of official monitoring.
High-throughput sequencing (HTS) technologies and bioinformatic analyses are of growing interest to be used as a routine diagnostic tool in the field of plant viruses. The reliability of HTS ...workflows from sample preparation to data analysis and results interpretation for plant virus detection and identification must be evaluated (verified and validated) to approve this tool for diagnostics. Many different extraction methods, library preparation protocols, and sequence and bioinformatic pipelines are available for virus sequence detection. To assess the performance of plant virology diagnostic laboratories in using the HTS of ribosomal RNA depleted total RNA (ribodepleted totRNA) as a diagnostic tool, we carried out an interlaboratory comparison study in which eight participants were required to use the same samples, (RNA) extraction kit, ribosomal RNA depletion kit, and commercial sequencing provider, but also their own bioinformatics pipeline, for analysis. The accuracy of virus detection ranged from 65% to 100%. The false-positive detection rate was very low and was related to the misinterpretation of results as well as to possible cross-contaminations in the lab or sequencing provider. The bioinformatic pipeline used by each laboratory influenced the correct detection of the viruses of this study. The main difficulty was the detection of a novel virus as its sequence was not available in a publicly accessible database at the time. The raw data were reanalysed using Virtool to assess its ability for virus detection. All virus sequences were detected using Virtool in the different pools. This study revealed that the ribodepletion target enrichment for sample preparation is a reliable approach for the detection of plant viruses with different genomes. A significant level of virology expertise is needed to correctly interpret the results. It is also important to improve and complete the reference data.
Ceratocystis platani (CP), an ascomycetous fungus, is the agent of canker stain, a lethal vascular disease of Platanus species. Ceratocystis platani has been listed as a quarantine pest (EPPO A2 ...list) due to extensive damage caused in Southern Europe and the Mediterranean region. As traditional diagnostic assays are ineffective, a Real-Time PCR detection method based on EvaGreen, SYBR Green, and Taqman assays was previously developed, validated in-house, and included in the official EPPO standard PM7/14 (2). Here, we describe the results of a test performance study performed by nine European laboratories for the purpose of an interlaboratory validation. Verification of the DNA extracted from biological samples guaranteed the high quality of preparations, and the stability and the homogeneity of the aliquots intended for the laboratories. All of the laboratories reproduced nearly identical standard curves with efficiencies close to 100%. Testing of blind-coded DNA extracted from wood samples revealed that all performance parameters—diagnostic sensitivity, diagnostic specificity, accuracy and reproducibility—were best fit in most cases both at the laboratory and at the assay level. The previously established limit of detection, 3 fg per PCR reaction, was also validated with similar excellent results. The high interlaboratory performance of this Real-Time PCR method confirms its value as a primary tool to safeguard C. platani-free countries by way of an accurate monitoring, and to investigate the resistance level of potentially canker stain-resistant Platanus genotypes.
Ensuring the reliability of diagnostic activities is an essential cornerstone of plant health
strategies to reduce the risk of entry and spread of plant pests in a region and ultimately
their ...impacts. Diagnostic tests should be validated to ensure that they are fit for purpose.
Validation is usually done by diagnostic laboratories, although companies commercializing
diagnostic kits also produce validation data for their products. Due to the high number of
pest,matrix, and method combinations and given the significant resources required to validate
tests, it is essential that validation data are shared with the entire diagnostic community
and produced in a harmonized way to facilitate their use by different stakeholders. Indeed,
the selection of tests to be used in specific contexts is not the sole responsibility of diagnostic
laboratories but also involves national plant protection organizations.The VALITEST
EU project (2018 to 2021) was established to tackle all these issues.New validation data for
tests targeting important pests for the European and Mediterranean Plant Protection Organization
region were produced. Guidelines to improve and harmonize the validation framework
were developed. Sharing of validation data and experience was ensured through the
development of new or existing databases, the organization of training courses, and the
dissemination of the project outputs in scientific publications and standards. Finally, the
involvement of researchers, diagnosticians, policy makers, inspectors, and industries and
the establishment of the European Plant Diagnostic Industry Association were important
actions to strengthen the interactions between plant health stakeholders
PDF
