Terminology and Guidelines for Diagnostic Assay Development and Validation: Best Practices for Molecular Tests

Detection and quantification of pathogen propagules in the air or other environmental samples is facilitated by culture-independent assays. We developed a quantitative PCR assay for the hop powdery mildew fungus, Podosphaera macularis, for detection of the organism from air samples. The assay utilizes primers and a TaqMan probe designed to target species-specific sequences in the 28S large subunit (LSU) of the nuclear ribosomal rDNA. Analytical sensitivity was not affected by the presence of an exogenous internal control or potential PCR inhibitors associated with DNA extracted from soil. The level of quantification of the assay was between 200 and 350 conidia when DNA was extracted from a fixed number of conidia. The assay amplified all isolates of P. macularis tested and had minimal cross-reactivity with other Podosphaera species when assayed with biologically relevant quantities of DNA. Standard curves generated independently in two other laboratories indicated that assay sensitivity was qualitatively similar and reproducible. All laboratories successfully detected eight unknown isolates of P. macularis and correctly discriminated Pseudoperonospora humuli and a water control. The usefulness of the assay for air sampling for late-season inoculum of P. macularis was demonstrated in field studies in 2019 and 2020. In both years, airborne populations of P. macularis in hop yards were detected consistently and increased during bloom and cone development.

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PCR-Based Detection for the Quarantine Fungus Colletotrichum kahawae, a Biosecurity Threat to the Coffee (Coffea arabica) Industry Worldwide

Ferrucho,

Marín-Ramírez,

Ochoa-Corona

et al. 2024

Plant Disease

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Coffee berry disease (CBD) is caused by Colletotrichum kahawae, a quarantine fungus still absent from most coffee-producing countries. Given the potential adverse effects on coffee berry production, it is a severe worldwide threat to farmers and industry. Current biosecurity management focuses on exclusion by applying quarantine measures, including certification of coffee plants and their products. However, methods for detecting C. kahawae by the NPPO (National Plant Protection Organization) laboratories still need approval. This research aims to functionally demonstrate, standardize, and validate a method for detecting and discriminating C. kahawae from other Colletotrichum species that may be present in coffee plant samples. The method proposes to use an end-point PCR marker for the mating type gene (MAT1-2-1) and a confirmatory test with a qPCR marker developed on the glutamine synthetase (GS) gene. The C. kahawae amplicons for the Cen-CkM10 marker exhibited specific melting temperature (Tm) values that could be readily differentiated from other tested species, including their relatives. Given the fungus’s quarantine status, specificity was tested using artificial mixtures of DNA of C. kahawae with other Colletotrichum species and coffee plant DNA. The described method will enable NPPOs in coffee producing and exporting countries, especially Colombia, to prevent this pathogen's entry, establishment, and spread.

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Terminology and Guidelines for Diagnostic Assay Development and Validation: Best Practices for Molecular Tests

Cited by 10 publications

References 73 publications

High-Throughput Preclinical Models and Pharmacoproteomics

High-Throughput Preclinical Models and Pharmacoproteomics

Detection of Podosphaera macularis in Air Samples by Quantitative PCR

PCR-Based Detection for the Quarantine Fungus Colletotrichum kahawae, a Biosecurity Threat to the Coffee (Coffea arabica) Industry Worldwide

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