Species-specific qPCR Assays Research Articles

Molecular detection of Rickettsia species in ectoparasites collected from two southern provinces of Cambodia.

Arthropod-borne rickettsioses comprise a wide variety of subtypes that are endemic in Cambodia, but there remains very little data on the geographic distribution of the pathogens or their vectors. Surveys were conducted in Koh Kong and Preah Sihanouk Provinces between September 2017 and June 2018 to collect ectoparasites from peridomestic animals and the environment using dragging and flagging methods. Collected ectoparasites were sorted and identified morphologically, then pooled by species, host, and location for molecular detection using Rickettsia genus- and species-specific qPCR and/or multilocus sequence typing (MLST) assays. A total of 14,254 ectoparasites were collected including seven new locality records. Rickettsia species were detected in 35.5% (174/505) of the pools screened representing 3,149 randomly selected ectoparasites from the total collected. Rickettsia asembonensis was detected in 89.6% (147/164) of Rickettsia-positive flea pools and 3.6% (6/164) of the flea pools were positive for both R. asembonensis and Rickettsia felis. Candidatus Rickettsia senegalensis from Ctenocephalides orientis fleas and Rickettsia sp. close to Rickettsia japonica and Rickettsia heilongjiangensis from Haemaphysalis ticks were identified by MLST. This appears to be the first report of these new ectoparasite records and rickettsial species in southern Cambodia, suggesting a potential health risk to military and civilians in this region.

QPCR assay optimisation for a clinical study comparing oral health risk in Rett syndrome

PurposeThis study aimed to validate qPCR assays for specific microbiota, for use on dental plaque samples stored on Whatman FTA cards to compare relative oral health risk in Rett syndrome.MethodsSupragingival dental plaque samples were collected, using a sterile swab, (COPAN FLOQswab™) swabbed onto Whatman FTA™ cards. DNA extraction was performed using a modified Powersoil™ protocol. Where published assays were unsuitable, species-specific qPCR assays for caries-associated, gingivitis-associated and oral-health-associated bacteria were designed using multiple sequence alignment, Primer3Plus and PrimerQuest. Assays were run using absolute quantification. Limit of detection (LOD) and limit of quantification (LOQ) were calculated, and PCR products verified by Sanger sequencing.ResultsMost assays allowed detection using real-time qPCR with high specificity on samples collected on FTA cards. Several assays showed low or even single gene copy numbers on the test samples.ConclusionAssays were optimised for detection and evaluation of oral health risk in dental plaque samples stored on FTA cards when cold storage is not feasible, except for F. nucleatum. Several assays showed gene copy numbers less than the LOQ or outside the range of the standard curve, so there is merit in optimising these assays using digital droplet PCR.

Who bit the boat? New DNA collection and genomic methods enable species identification in suspected shark-related incidents

Species identification following shark-related incidents is critical for effective incident management and for collecting data to inform shark-bite mitigation strategies. Witness statements are not always reliable, and species identification is often ambiguous or missing. Alternative methods for species identification include morphological assessments of bite marks, analysis of collected teeth at the scene of the incident, and genetic approaches. However, access to appropriate collection media and robust genetic assays have limited the use of genetic technologies. Here, we present a case study that facilitated a unique opportunity to compare the effectiveness of medical gauze readily available in first-aid kits, and forensic-grade swabs in collecting genetic material for shark-species identification. Sterile medical gauze and forensic-grade swabs were used to collect transfer DNA from the bite margins on a bitten surf ski which were compared to a piece of shark tissue embedded along the bite margin. Witness accounts and the characteristics of the bite mark impressions inferred the involvement of a Carcharodon carcharias (white shark). The morphology of a tooth found on the boat that picked up the surf ski, however, suggested it belonged to an Orectolobus spp. (wobbegong). Genetic analysis of DNA transferred from the shark to the surf ski included the application of a broad-target nested PCR assay followed by Sanger sequencing, with white shark contribution to the ‘total sample DNA’ determined with a species-specific qPCR assay. The results of the genetic analyses were congruent between sampling methods with respect to species identification and the level of activity inferred by the donor-specific DNA contribution. These data also supported the inferences drawn from the bite mark morphology. DNA from the recovered tooth was PCR amplified with a wobbegong-specific primer pair designed for this study to corroborate the tooth’s morphological identification. Following the confirmation of gauze used for sampling in the case study event, two additional isolated incidents occurred and were sampled in situ using gauze, as typically found in a first-aid kit, by external personnel. DNA extracted from these gauze samples resulted in the identification of a white shark as the donor of the DNA collected from the bite marks in both instances. This study, involving three incidents separated by time and location, represents the seminal application of gauze as a sampling media after critical human-shark interactions and strongly supports the practical implementation of these methods in the field.

Development of an Environmental DNA Assay for Prohibited Matter Weed Amazon Frogbit (Limnobium laevigatum)

Environmental DNA (eDNA) is widely used for detecting target species, including monitoring endangered species and detecting the presence of invasive species. Detecting targeted species using the eDNA approach is typically carried out with species-specific qPCR assays. Amazon frogbit (Limnobium laevigatum) is classified as a State-Prohibited Matter Weed in NSW, Australia. It is a fast-growing perennial aquatic weed that outcompetes native aquatic plants, leading to a reduction in the habitats of aquatic animals. Early detection is crucial for the effective management of this species. In this study, we developed a qPCR assay for L. laevigatum based on the rpoB gene sequence. This assay was validated against 25 non-target aquatic and terrestrial species. It was found to be species-specific, with the positive signal exclusively detected in L. laevigatum. The assay was highly sensitive with the modelled detection limits of 3.66 copies of DNA/µL. Furthermore, our assay was validated using environmental samples collected from field sites with and without the presence of L. laevigatum. Our assay is an effective tool for targeted eDNA detection of L. laevigatum, which will enhance efforts to monitor and control this invasive aquatic weed.

Immunostimulation of Asian elephant (Elephas maximus) blood cells by parapoxvirus ovis and CpG motif-containing bacterial plasmid DNA upregulates innate immune gene expression.

The immune system of Asian elephants (Elephas maximus) is poorly studied, compared to that of livestock, rodents or humans. The innate immune response has become a focus of interest in relation to Elephant endotheliotropic herpesviruses (EEHVs). EEHVs cause a fatal hemorrhagic disease (EEHV-HD) and are a significant threat to captive Asian elephant populations worldwide. Similar to other herpesvirus infections, nearly all animals become infected, but only some develop disease. As progression to EEHV-HD is often acute, a robust innate immune response is crucial to control EEHV infections. This is invariably true of the host in the first instance, but it can also potentially be modulated by intervention strategies. Here, two immunostimulant veterinary medicinal products, authorized for use in domestic species, were tested for their ability to induce innate anti-viral immune responses in Asian elephant blood cells. Sequence data were obtained for a range of previously unidentified Asian elephant immune genes, including C-X-C motif chemokine ligand 10 (CXCL10), interferon stimulated gene 15 (ISG15) and myxovirus GTPase 1 (Mx1), and were employed in the design of species-specific qPCR assays. These assays were subsequently used in analyses to determine fold changes in gene expression over a period of 24 hours. This study demonstrates that both immunostimulant medications are capable of inducing significant innate anti-viral immune responses which suggests that both could be beneficial in controlling EEHV infections in Asian elephants.

Consistent detection of Trypanosoma brucei but not T. congolense DNA in faeces of experimentally infected cattle

Animal African trypanosomiasis (AAT) is a significant food security and economic burden in sub-Saharan Africa. Current AAT empirical and immunodiagnostic surveillance tools suffer from poor sensitivity and specificity, with blood sampling requiring animal restraint and trained personnel. Faecal sampling could increase sampling accessibility, scale, and species range. Therefore, this study assessed feasibility of detecting Trypanosoma DNA in the faeces of experimentally-infected cattle. Holstein–Friesian calves were inoculated with Trypanosoma brucei brucei AnTat 1.1 (n = 5) or T. congolense Savannah IL3000 (n = 6) in separate studies. Faecal and blood samples were collected concurrently over 10 weeks and screened using species-specific PCR and qPCR assays. T. brucei DNA was detected in 85% of post-inoculation (PI) faecal samples (n = 114/134) by qPCR and 50% by PCR between 4 and 66 days PI. However, T. congolense DNA was detected in just 3.4% (n = 5/145) of PI faecal samples by qPCR, and none by PCR. These results confirm the ability to consistently detect T. brucei DNA, but not T. congolense DNA, in infected cattle faeces. This disparity may derive from the differences in Trypanosoma species tissue distribution and/or extravasation. Therefore, whilst faeces are a promising substrate to screen for T. brucei infection, blood sampling is required to detect T. congolense in cattle.

Development and validation of quantitative PCR assays for HIV-associated cryptococcal meningitis in sub-Saharan Africa: a diagnostic accuracy study

HIV-associated cryptococcal meningitis is the second leading cause of AIDS-related deaths, with a 10-week mortality rate of 25-30%. Fungal load assessed by colony-forming unit (CFU) counts is used as a prognostic marker andto monitor response to treatment in research studies. PCR-based assessment of fungal load could be quicker andless labour-intensive. We sought to design, optimise, and validate quantitative PCR (qPCR) assays for the detection, identification, and quantification of Cryptococcus infections in patients with cryptococcal meningitis in sub-Saharan Africa. We developed and validated species-specific qPCR assays based on DNA amplification of QSP1 (QSP1A specific to Cryptococcus neoformans, QSP1B/C specific to Cryptococcus deneoformans, and QSP1D specific to Cryptococcus gattii species) and a pan-Cryptococcus assay based on a multicopy 28S rRNA gene. This was a longitudinal study that validated the designed assays on cerebrospinal fluid (CSF) of 209 patients with cryptococcal meningitis at baseline (day 0) and during anti-fungal therapy (day 7 and day 14), from the AMBITION-cm trial in Botswana and Malawi (2018-21). Eligible patients were aged 18 years or older and presenting with a first case of cryptococcal meningitis. When compared with quantitative cryptococcal culture as the reference, the sensitivity of the 28S rRNA was 98·2% (95% CI 95·1-99·5) and of the QSP1 assay was 90·4% (85·2-94·0) in CSF at day 0. Quantification of the fungal load with QSP1 and 28S rRNA qPCR correlated with quantitative cryptococcal culture (R2=0·73 and R2=0·78, respectively). Both Botswana and Malawi had a predominant C neoformans prevalence of 67% (95% CI 55-75) and 68% (57-73), respectively, and lower C gattii rates of 21% (14-31) and 8% (4-14), respectively. We identified ten patients that, after 14 days of treatment, harboured viable but non-culturable yeasts based on QSP1 RNA detection (without any positive CFU in CSF culture). QSP1 and 28S rRNA assays are useful in identifying Cryptococcus species. qPCR results correlate well with baseline quantitative cryptococcal culture and show a similar decline in fungal load during induction therapy. These assays could be a faster alternative to quantitative cryptococcal culture to determine fungal load clearance. The clinical implications of the possible detection of viable but non-culturable cells in CSF during induction therapy remain unclear. European and Developing Countries Clinical Trials Partnership; Swedish International Development Cooperation Agency; Wellcome Trust/UK Medical Research Council/UKAID Joint Global Health Trials; and UKNational Institute for Health Research.

A novel eDNA approach for rare species monitoring: Application of long-read shotgun sequencing to Lynx rufus soil pawprints

Environmental DNA (eDNA) sampling is a relatively new technique that has been employed in biodiversity surveys around the world. Environmental DNA can be an effective, non-invasive method of identifying the presence of target host species. Furthermore, advances in DNA deep sequencing technologies are enabling in-depth information to be recovered from eDNA. Here we report the development of a bobcat (Lynx rufus) species-specific probe-based qPCR assay for eDNA studies, which was validated on wild samples and those from a bobcat housed at the Jacksonville Zoo in Florida. Furthermore, we show that long-read shotgun sequencing of eDNA extracted from pawprint soil samples, using an Oxford Nanopore Technologies MinION, could successfully detect bobcat DNA, including enabling correct species level identification and phylogenetic placement within regionally distanced bobcats. This was all achieved without utilizing any DNA enrichment approaches. The long read shotgun sequencing simultaneously recovered genetic information from microbes known to be part of the bobcat microbiome, indicating that potential health-status-related microbial information can be obtained alongside wildlife eDNA. This study revealed that non-targeted (no metabarcoding or enrichment) long-read shotgun sequencing of eDNA samples can be sufficient for species identification, phylogenetic analyses, and population genetics applications. This paves the way for the rapid deployment of eDNA approaches for any species of interest, without requiring laborious development of targeted approaches, thereby increasing the ease and utility of eDNA research for endangered species conservation and management.

Distribution and habitat preference of polar cod (Boreogadus saida) in the Bering and Chukchi Seas inferred from species-specific detection of environmental DNA

Ongoing warming and sea-ice reductions in the Arctic can seriously impact cold-water species, such as polar cod (Boreogadus saida), necessitating biomonitoring to reveal the ecological consequences. Recent methodological advancements in environmental DNA (eDNA) techniques have increased our ability to conduct ecological monitoring at various locations, including the Arctic. This study aimed to provide an overview of the distribution of polar cod across the Bering and Chukchi Seas by employing species-specific detection of eDNA. First, we successfully developed novel species-specific qPCR assay targeting the mitochondrial D-loop region, which exclusively amplifies eDNA derived from polar cod. Subsequently, polar cod eDNA was detected using the assay from the samples that we collected latitudinally across the study area during the open-water season. Polar cod eDNA was primarily detected in the surface water from the central Chukchi Sea shelf and the northernmost observation line (75°N), which was located on the shelf slope, off the Point Barrow, and in the marginal ice zone. In contrast, only trace amounts of eDNA were detected in the Bering Sea. This pattern corresponded well with the distribution of water masses classified based on environmental conditions. The detection of eDNA in surface water was clearly limited to cold (-1 to 5°C) and low salinity (25–32) water, whereas it was detected in a higher salinity range (32–34) in the middle and bottom layers. These findings are consistent with current knowledge about the distribution and habitat of the polar cod, suggesting that eDNA can be regarded as a reliable tool to replace or supplement conventional methods. Incorporating eDNA techniques into large-scale oceanographic surveys can improve the spatial and temporal resolution of fish species detection with a reasonable sampling effort and will facilitate the continuous monitoring of Arctic ecosystems.

Malaria mitochondrial diagnosis: challenges and pitfalls

BackgroundHigh-copy genomic sequences could be used as PCR targets for the detection of Plasmodium infections, providing increased sensitivity over single- or low-copy genes. Mitochondrial genomes of malaria parasites are present in multiple copies in a single mitochondrion, and each parasite has many mitochondria. Here, we describe the development of seven species-specific qPCR assays for the diagnosis of Plasmodium vivax and Plasmodium falciparum, targeting coding and non-coding mitochondrial genomic regions.MethodsThe optimization of the qPCR protocols involved a gradient of annealing temperatures and concentrations of primers and probes, as well as the inclusion of PCR additives/enhancers [e.g., dimethyl sulfoxide (DMSO), glycerol, bovine serum albumin (BSA)] to improve the specificity of qPCR amplification.ResultsNon-specific amplification of other Plasmodium species and of human targets was observed in different levels for all assays. Regardless of the late Cq values for most non-specific amplifications, the application of a cutoff value did not completely exclude false-positive amplification, compromising the specificity and also the sensitivity of the assays.ConclusionsTherefore, although mitochondrial targets have higher sensitivity, they frequently lose specificity due to their high levels of sequence conservation. A screening to evaluate the cross-reaction between Plasmodium species and the non-specific amplification of human malaria-free samples must be performed for Plasmodium mitochondrial assays.

Validation of a species-specific probe-based qPCR assay for the threatened meltwater stonefly, Lednia tumana, in environmental samples

Validation of a species-specific probe-based qPCR assay for the threatened meltwater stonefly, Lednia tumana, in environmental samples

Development of a real-time PCR (qPCR) method for the identification of the invasive paddle crab Charybdis japonica (Crustacea, Portunidae).

Crabs can be transported beyond their native range via anthropogenic-mediated means such as aquarium trade, live seafood trade and shipping. Once introduced into new locations, they can establish persisting populations and become invasive, often leading to negative impacts on the recipient environment and native species. Molecular techniques are increasingly being used as complementary tools in biosecurity surveillance and monitoring plans for invasive species. Molecular tools can be particularly useful for early detection, rapid identification and discrimination of closely related species, including when diagnostic morphological characters are absent or challenging, such as early life stages, or when only part of the animal is available. In this study, we developed a species-specific qPCR assay, which targets the cytochrome c oxidase subunit 1 (CO1) region of the Asian paddle crab Charybdis japonica. In Australia, as well as many parts of the world, this species is considered invasive and routine biosecurity surveillance is conducted to reduce the risk of establishment. Through rigorous testing of tissue from target and non-target species we demonstrate that this assay is sensitive enough to detect as little as two copies per reaction and does not cross amplify with other closely related species. Field samples and environmental samples spiked with C. japonica DNA in high and low concentrations indicate that this assay is also a promising tool for detecting trace amounts of C. japonica eDNA in complex substrates, making it a useful complementary tool in marine biosecurity assessments.

Whole-mitochondrial genomes of Nannizziopsis provide insights in evolution and detection.

Infectious fungal diseases can have devastating effects on wildlife health and a detailed understanding of the evolution of related emerging fungal pathogen along with the ability to detect them in the wild is considered indispensable for effective management strategies. Several fungi from the genera Nannizziopsis and Paranannizziopsis are emerging pathogens of reptiles and have been observed to cause disease in a wide range of taxa. Nannizziopsis barbatae has become a particularly important pathogen of Australian reptiles with an increasing number of herpetofauna being reported with cases of infection from across the country. Here, we present the mitochondrial genome sequences and phylogenetic analysis for seven species in this group of fungi uncovering new information on the evolutionary relationship of these emerging pathogens. From this analysis, we designed a species-specific qPCR assay for the rapid detection of N. barbatae and demonstrate its application in a wild urban population of a dragon lizard.

Playing “hide and seek” with the Mediterranean monk seal: a citizen science dataset reveals its distribution from molecular traces (eDNA)

Animal conservation relies on assessing the distribution and habitat use of species, but for endangered/elusive animals this can prove difficult. The Monk Seal, Monachus monachus, is one of the world's most endangered species of pinniped, and the only one endemic to the Mediterranean Sea. During recent decades, direct observations have been few and scattered, making it difficult to determine its distribution away from the Aegean Sea (core distribution area of the post-decline relict population). This study relies on environmental DNA (eDNA) analysis to detect the presence of the Monk Seal in 135 samples collected in 120 locations of the central/western Mediterranean Sea, spanning about 1500 km longitudinally and 1000 km latitudinally. A recently described species-specific qPCR assay was used on marine-water samples, mostly collected during 2021 by a Citizen Science (CS) project. Positive detections occurred throughout the longitudinal range, including the westernmost surveyed area (Balearic archipelago). The distribution of the positive detections indicated six “hotspots”, mostly overlapping with historical Monk Seal sites, suggesting that habitat-specific characteristics play a fundamental role. We applied single-season occupancy models to correct for detection probability and to assess the importance of site-specific characteristics. The distance from small islets and protected (or access-restricted) areas was correlated negatively with the detection probability. This novel molecular approach, applied here for the first time in an extensive CS study, proved its potential as a tool for monitoring the distribution of this endangered/elusive species.

Development of a species-specific qPCR assay for the detection of invasive African sharptooth catfish (Clarias gariepinus) using environmental DNA

Detection and monitoring of target species is the primary strategy in the management and control of biological invasions. Traditional methods to detect invasive species are time consuming and cumbersome, with a requisite for trained taxonomists for identification of aquatic species. Environmental DNA (eDNA)-based molecular methods offer an alternative, as they are quick, cost-effective and require minimal manpower. In this study, we design and optimize a reliable eDNA-based quantitative PCR assay to detect the African Sharptooth catfish, a highly invasive and banned species in India. Here, we delineate the step-by-step processes involved in the design and optimization of the assay, and show its performance through field-testing in selected water bodies in and around the city of Hyderabad. The present workflow can be used to design assays to detect a wide range of aquatic species.

Probe-based qPCR as an alternative to modified Knott’s test when screening dogs for heartworm (Dirofilaria immitis) infection in combination with antigen detection tests

BackgroundCurrent recommendations for diagnosis of Dirofilaria immitis infection in dogs rely on the detection of antigen produced largely by adult females coupled with the visualization of microfilariae (mf) in the circulation via a microfilaria detection test (MFDT). It is hypothesized that qPCR assays used in parallel with antigen detection tests will perform better in detecting mf than modified Knott’s test (MK), when combined with antigen detection. This study compares probe-based qPCR and MK techniques for mf detection used in parallel with the DiroCHEK® antigen test to screen for heartworm infection in shelter dogs.MethodsMatching blood and serum samples were collected from 300 shelter dogs in Brazos and Harris counties, Texas, USA. Blood was assessed for the presence of mf via MK and the presence of D. immitis DNA by a species-specific probe-based qPCR assay. Serum samples were tested for the presence of heartworm antigen using DiroCHEK® before and after immune complex dissociation (ICD) via heat treatment. In addition, the performance of each diagnostic test was evaluated via Chi-square test, Cochran’s Q test, and post hoc analysis.ResultsQualitatively, MK detected mf in 22.0% (66/300) of samples, 55 of which were morphologically identified as D. immitis and 11 as Acanthocheilonema reconditum. The range of heartworm mf was 28 to 88,803 mf/ml (median: 6627.5). Real-time PCR detected D. immitis DNA in 20.7% (62/300) of samples. Heartworm antigen was detected in 24.7% (74/300) of samples pre-ICD, and in 29.3% (88/300) post-ICD. When comparing tests, the Chi-square and McNemar’s tests showed that the difference between positive and negative proportions was statistically significant. The Cochran test showed the difference in the distributions of cases and non-cases was significant when individual tests were combined (χ2 = 62.3, df = 3, P < 0.0001) and when parallel methods were combined (χ2 = 43.1, df = 4, P < 0.0001).ConclusionConsidering individual and combined test performances, practicality, and efficient use of bench time, this heartworm-specific probe-based qPCR method is a viable option as a mf detection test to be used in parallel with antigen tests for canine heartworm infection in diagnostic and research settings.Graphical

The Native Hymenoscyphus albidus and the Invasive Hymenoscyphus fraxineus Are Similar in Their Necrotrophic Growth Phase in Ash Leaves.

The populations of European ash and its harmless fungal associate Hymenoscyphus albidus are in decline owing to ash dieback caused by the invasive Hymenoscyphus fraxineus, a fungus that in its native range in Asia is a harmless leaf endophyte of local ash species. To clarify the behavior of H. albidus and its spatial and temporal niche overlap with the invasive relative, we used light microscopy, fungal species-specific qPCR assays, and PacBio long-read amplicon sequencing of the ITS1-5.8S-ITS2 region to examine fungal growth and species composition in attached leaves of European ash. The plant material was collected from a healthy stand in central Norway, where ash saplings in late autumn showed leaflet vein necrosis like that commonly related to H. fraxineus. For reference, leaflet samples were analyzed from stands with epidemic level of ash dieback in southeastern Norway and Estonia. While H. albidus was predominant in the necrotic veins in the healthy stand, H. fraxineus was predominant in the diseased stands. Otherwise, endophytes with pathogenic potential in the genera Venturia (anamorph Fusicladium), Mycosphaerella (anamorph Ramularia), and Phoma, and basidiomycetous yeasts formed the core leaflet mycobiome both in the healthy and diseased stands. In necrotic leaf areas with high levels of either H. albidus or H. fraxineus DNA, one common feature was the high colonization of sclerenchyma and phloem, a region from which the ascomata of both species arise. Our data suggest that H. albidus can induce necrosis in ash leaves, but that owing to low infection pressure, this first takes place in tissues weakened by autumn senescence, 1–2 months later in the season than what is characteristic of H. fraxineus at an epidemic phase of ash dieback. The most striking difference between these fungi would appear to be the high fecundity of H. fraxineus. The adaptation to a host that is phylogenetically closely related to European ash, a tree species with high occurrence frequency in Europe, and the presence of environmental conditions favorable to H. fraxineus life cycle completion in most years may enable the build-up of high infection pressure and challenge of leaf defense prior to autumn senescence.

Effects of Seasonality and Climate on the Propagule Deposition Patterns of the Chestnut Blight Pathogen Cryphonectria parasitica in Orchards of the Alpine District of North Western Italy

Chestnut blight is the major disease of chestnuts (Castanea spp.) cultivated worldwide for the production of edible nuts. The disease is caused by the pathogenic fungus Cryphonectria parasitica, which infects trees by means of airborne propagules penetrating through fresh wounds on stems and branches. The aims of this study were to (I) assess the temporal propagule deposition patterns of C. parasitica in the Alpine district of North Western Italy, (II) test and model the effects of seasonality and climate on the above patterns, and (III) investigate the spatial distribution of propagule deposition at the within-site scale. A two-year-long spore trapping experiment was conducted in three chestnut orchards. Approximately 1300 samples were collected and processed with a species-specific qPCR assay to quantitatively assess the propagule deposition of C. parasitica. Results showed that C. parasitica can release propagules all over the year, though with significant seasonal peaks in the spring and fall (p &lt; 0.05). Large propagule loads were significantly correlated (p &lt; 0.05) with an increasing number of rainy days of the week (days providing 1–10 mm/day of water). Models predicting periods at high risk of infection based on climate and seasonality were fitted and successfully validated (p &lt; 0.05).

A species-specific qPCR assay provides novel insight into range expansion of the Mediterranean monk seal (Monachus monachus) by means of eDNA analysis

The monk seal is the most endangered pinniped worldwide and the only one found in the Mediterranean, where its distribution and abundance have suffered a drastic decline in the last few decades. The limited understanding of the population demographics and conservation status of this species are due to both its rarity and evasiveness, with records biased towards occasional, mostly coastal, encounters. Current molecular techniques allow us to detect DNA traces released into the environment (eDNA) by any organism. We have developed three species-specific qPCR-assays targeting the 12S/16S rRNA mitogenome regions of the monk seal. The assays have been tested extensively on a comprehensive and diverse set of samples (n = 73), including positive controls from a breeding population in Madeira collected during their peak abundance, and two opportunistic collections of Mediterranean eDNA samples (offshore/coastal) from on-going projects. Monk seal DNA was detected in 47.2% of the samples collected from a ferry platform in the Tyrrhenian (2018–2019) and in 66.7% of those obtained in the Pelagie archipelago in the Strait of Sicily (2020). These findings anticipated subsequent visual observations in the proximity of these sampling areas by up to 2 years. At the Tyrrhenian site, monk seal detection increased between 2018 and 2019. The demonstrated approach provides a non-invasive and highly sensitive tool for defining the monk seal actual distribution and home range -enabling monitoring also in nocturnal hours-, for assessing its recovery rate and pinpointing coastal/offshore sites where prioritizing conservation, research, citizen-science, and educational opportunities.

QPCR Assays for Sensitive and Rapid Detection of Quambalaria Species from Plant Tissues.

Several species from the genus Quambalaria (order Microstromatales) cause diseases on eucalypts (Eucalyptus and related genera) both in plantations and natural ecosystems. We developed real-time quantitative PCR (qPCR) assays to rapidly detect and distinguish five Quambalaria species. The design of the species-specific qPCR assay for each species, Q. pitereka (PIT), Q. coyrecup (COR), Q. cyanescens (CYN), Q. pusilla (PUS), and Q. eucalypti (EUC), was based on the ITS region and was evaluated for specificity and sensitivity. The PIT, COR, and CYN qPCR assays could amplify as little as 10 fg µl-1 from pure cultures, whereas PUS and EUC qPCR assays could amplify 100 fg µl-1 of their target species. The PIT, COR, and CYN qPCR assays were further validated using naturally and artificially infected samples of their plant host Corymbia calophylla. These assays will be used for rapid diagnostics and future experiments on the infection process.