Search Results (41)

The European honey bee (Apis mellifera) significantly contributes to Australian agriculture, especially in honey production and the pollination of key crops. However, managed bee populations are declining due to pathogens, agrochemicals, poor forage, climate change, and habitat loss. Major threats include bacteria, fungi, mites, and pests. With the increasing demand for pollination and the movement of bee colonies, monitoring these threats is essential. It has been demonstrated that honey constitutes an easily accessible source of environmental DNA. Environmental DNA in honey comes from all organisms that either directly or indirectly aid in its production and those within the hive environments. In this study, we extracted eDNA from 135 honey samples and tested for the presence of DNA for seven key honey bee pathogens and pests—Paenibacillus larvae, Melissococcus plutonius (bacterial pathogens), Nosema apis, Nosema ceranae (microsporidian fungi), Ascosphaera apis (fungal pathogen), Aethina tumida, and Galleria mellonella (arthropod pests) by using end-point singleplex and multiplex PCR assays. N. ceranae emerged as the most prevalent pathogen, present in 57% of the samples. This was followed by the pests A. tumida (40%) and G. mellonella (37%), and the pathogens P. larvae (21%), N. apis (19%), and M. plutonius (18%). A. apis was detected in a smaller proportion of the samples, with a prevalence of 5%. Additionally, 19% of the samples tested negative for all pathogens and pests analysed. The data outlines essential information about the prevalence of significant arthropod, fungal, and bacterial pathogens and pests affecting honey bees in Australia, which is crucial for protecting the nation’s beekeeping industry. Full article

Swine infectious diseases, often caused by multiple co-infecting agents, pose severe global threats to pig health and industry economics. Conventional single-plex testing assays, whether relying on pathogen antigens or nucleic acids, exhibit limited efficacy in the face of co-infection events. The modern nucleic acid-based multiplex testing (NAMT) methods demonstrate substantial strengths in the simultaneous detection of multiple pathogens involving co-infections owing to their remarkable sensitivity, exceptional specificity, high-throughput, and short turnaround time. The development, commercialization, and application of NAMT assays in swine infectious disease surveillance would be advantageous for early detection and control of pathogens at the onset of an epidemic, prior to community transmission. Such approaches not only contribute to saving the lives of pigs but also aid pig farmers in mitigating or preventing substantial economic losses resulting from infectious disease outbreaks, thereby alleviating unwanted pressure on animal and human health systems. The current literature review provides an overview of some modern NAMT methods, such as multiplex quantitative real-time PCR, multiplex digital PCR, microarrays, microfluidics, next-generation sequencing, and their applications in the diagnosis of swine infectious diseases. Furthermore, the strengths and weaknesses of these methods were discussed, as well as their future development and application trends in swine disease diagnosis. Full article

Background/Objectives: Concurrent outbreaks of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A and B viruses (IAV/IBV), and respiratory syncytial virus (RSV) necessitate rapid and precise differential laboratory diagnostic methods. This study aimed to evaluate the multiplex molecular diagnostic performance of the geneLEAD VIII system (Precision System Science Co., Ltd., Matsudo, Japan), a fully automated sample-to-result precision instrument, in conjunction with the VIASURE SARS-CoV-2, Flu & RSV Real Time PCR Detection Kit (CerTest Biotec, S.L., Zaragoza, Spain). Methods: The specific detection capabilities of SARS-CoV-2, IAV/IBV, and RSV genes were evaluated using virus-spiked saliva and nasal swab samples. Using saliva samples, the viral titer detection limits of geneLEAD/VIASURE and manual referent singleplex RT-qPCR assays were compared. The performance of geneLEAD/VIASURE in analyzing single- and multiple-infection models was scrutinized. The concordance between the geneLEAD/VIASURE and the manual assays was assessed. Results: The geneLEAD/VIASURE successfully detected all the virus genes in the saliva and nasal swab samples despite some differences in the Ct values. The viral titer detection limits in the saliva samples for SARS-CoV-2, IAV, IBV, and RSV using geneLEAD/VIASURE were 10⁰, ≤10⁻², 10⁰, and 10² TCID₅₀/mL, respectively, compared to ≤10⁻¹, ≤10⁰, ≤10⁰, and ≤10⁴ TCID₅₀/mL, respectively, in the manual assays. geneLEAD/VIASURE yielded similar Ct values in the single- and multiple-infection models, with some exceptions noted in the triple-infection models when low titers of RSV were spiked with high titers of the other viruses. The concordance between geneLEAD/VIASURE and the manual assays was high, with Pearson’s R² values of 0.90, 0.85, 0.92, and 0.95 for SARS-CoV-2, IAV, IBV, and RSV, respectively. Conclusions: geneLEAD/VIASURE is a reliable diagnostic tool for detecting SARS-CoV-2, IAV/IBV, and RSV in single- and multiple-infection scenarios. Full article

The diagnosis of allergic diseases and anaphylaxis is complex and encompasses a broad spectrum of in vitro and in vivo diagnostic tests. The choice of diagnostic tests is related to the presumed pathophysiological mechanism of the allergic reaction. In the past decade the implementation of component-resolved diagnostics (CRD) into clinical practice has significantly improved the depicting of sensitization profiles, which has aided in the assessment of clinically relevant allergen components that are associated with true allergy, as well as the levels of risk of severe anaphylactic reactions. Recently, multiplex-specific immunoglobulin E (IgE) platforms have emerged for better selection of patients at risk for anaphylaxis and have improved the selection criteria for patients undergoing allergen immunotherapy, including novel regimes such as oral immunotherapy. This review describes the advantages of the utilization of component-resolved diagnostics and multiplex assays in clinical settings, especially in cases of anaphylaxis when no clear trigger is recognized or where multiple culprits are suspected. As multiplex component-resolved diagnostics becomes more readily available globally and with the use of novel approaches, CRD will certainly be a crucial tool in personalized and individually tailored management plans and reduce the financial burden of anaphylaxis. Full article

Since its emergence in China in 2018, African swine fever virus (ASFV) has posed a severe threat to the pig farming industry due to its high transmissibility and mortality rate. The clinical signs of ASFV infection often overlap with those caused by other swine viruses such as classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), and porcine circovirus type 2 (PCV2), making timely and precise diagnosis a considerable challenge. To address this, we established a TaqMan-based multiplex real-time quantitative PCR (qPCR) assay capable of simultaneously detecting ASFV, CSFV, PRRSV, PRV, and PCV2. Specific primer-probe sets were developed targeting conserved genomic regions: the ASFV P72 gene, CSFV 5’UTR region, PRRSV ORF6, PCV2 cap gene, and PRV gB gene. After thorough optimization, the assay demonstrated robust analytical performance, exhibiting strong target specificity with no cross-detection of non-target pathogens. The detection threshold was determined to be 10 copies/μL per virus, indicating high assay sensitivity. Repeatability analysis revealed low variability, with intra- and inter-assay coefficient of variation values remaining below 2.3%. When applied to 95 clinical samples, the multiplex assay yielded results that were fully consistent with those obtained using commercially available singleplex qPCR kits. In conclusion, the multiplex TaqMan qPCR method developed in this study is characterized by high specificity, sensitivity, and reproducibility. It provides a reliable and efficient diagnostic tool for the simultaneous detection and differential diagnosis of ASFV and other clinically similar viral infections in swine, thereby offering robust technical support for swine disease surveillance and control. Full article

Background/Objectives: Sensitization to the carbohydrate antigen α-Gal is associated with allergic reactions against different types of food that contain α-Gal (e.g., mammalian meat). This form of allergy is termed α-Gal syndrome (AGS), and the diagnosis is challenging due to delayed symptom onset and cross-reactivity with multiple mammalian products. It is estimated that AGS is underdiagnosed, pointing to an unmet need for patient care. Methods: Sera from patients with suspected AGS based on clinical history were analyzed by ImmunoCAP and the IgE cross-reactivity immune profiling (ICRIP) system specifically developed by us. IgE from patient sera against different forms of α-Gal was analyzed using α-Gal-containing analytes and negative controls. Results: Sera from 33 patients with suspected AGS were analyzed. Sera from 22 patients yielded a clearly positive signal (>0.35 kU/L) for IgE against α-Gal in ImmunoCAP. For 7 of the remaining 11 patients with negative or ambiguous (IgE level between 0.1 and 0.35 kU/L) results in ImmunoCAP, ICRIP analyses supported the suspected association of the allergy symptoms with IgE against α-Gal components. This component-resolved analysis helps the allergist to provide an individual diagnosis for each patient. Conclusions: The diagnosis of AGS is challenging. An interplay between clinical history and lab analysis via ImmunoCAP and the specifically developed ICRIP system helps patients and allergists in establishing the correct diagnosis, thereby preventing accidental exposure and recurrent AGS episodes. Full article

Groundnut bud necrosis orthotospovirus (GBNV), a tripartite single-stranded RNA virus, poses a significant threat to United States agriculture. GBNV is a quarantine pathogen, and its introduction could lead to severe damage to economically important crops, such as groundnuts, tomatoes, potatoes, peas, and soybeans. For the rapid and accurate detection of GBNV at points of entry, TaqMan reverse transcriptase–quantitative polymerase chain reaction (RT-qPCR) assays were developed and the results validated using conventional reverse transcriptase–polymerase chain reaction (RT-PCR) followed by Sanger sequencing. These assays target highly conserved regions of the nucleocapsid (NP) and movement (MP) proteins within the viral genome. Multiplex GBNV detection assays targeting the NP and MP genes, as well as an internal control plant gene, ACT11, showed efficiency rates between 90% and 100% and R² values of 0.98 to 0.99, indicating high accuracy and precision. Moreover, there was no significant difference in sensitivity between multiplex and singleplex assays, ensuring reliable detection across various plant tissues. This rapid, sensitive, and specific diagnostic assay will provide a valuable tool at ports of entry to prevent the entry of GBNV into the United States. Full article

Pseudostellaria heterophylla (Miq.) Pax, a highly valued Chinese medicinal plant, is experiencing a notable decline in yield and quality due to viral diseases, particularly caused those by TuMV and BBWV2. Currently, the absence of a quantitative detection method for these viruses in P. heterophylla impedes the accurate diagnosis. The development of an accurate quantitative detection method is thus essential for effectively managing viral diseases in this plant. In this study, singleplex and duplex TaqMan qPCR were developed for the detection of the two viruses, based on two viral cloning vectors. Concurrently, the levels of both viruses were examined in the main produced regions of P. heterophylla. Furthermore, the levels of BBWV2 were examined during its infection of P. heterophylla. The optimal singleplex qPCR employed 0.1 μM of hydrolysis probe and 0.1 μM of primer for TuMV, while 0.2 μM of hydrolysis probe and 0.1 μM of primer were utilised for BBWV2. In contrast, the duplex qPCR employed the use of 0.1 μM of the upstream/downstream primer from each primer pair, with 0.2 μM of the respective hydrolysis probes. The 95% limit of detection (LOD) for singleplex qPCR was 734 copies for TuMV and 20 copies for BBWV2, while the 95% LOD for duplex qPCR was 945 copies for TuMV and 47 copies for BBWV2. Furthermore, the intra- and inter-assay coefficients of variation were found to be less than 1.2% for both singleplex and duplex qPCR. Of the P. heterophylla sampled 60 sites, 96% were found to be infected by one of two viruses. The levels of BBWV2 in N. benthamiana and P. heterophylla demonstrated an initial increase, followed by a subsequent decrease. The TaqMan qPCR methods provide a technical foundation for the monitoring of virus infections in P. heterophylla. Full article

Abortion is one of the major causes of economic losses in livestock production worldwide. Because several factors can lead to abortion in cattle, sheep and goats, laboratory diagnosis, including the molecular detection of pathogens causing abortion, is often necessary. Bacterial zoonotic diseases such as brucellosis, coxiellosis, leptospirosis, and listeriosis have been implicated in livestock abortion, but they are under diagnosed and under-reported in most developing countries, including Botswana. This study applied a recently developed multiplex high-resolution melting analysis technique, coupled with singleplex qPCR assays, to investigate abortions in livestock in Botswana, using 152 samples from cattle, sheep, and goat abortion cases. Brucella spp. were the most frequent pathogen detected, with an overall frequency of 21.1%, followed by Coxiella burnetii with 19.1%. Listeria monocytogenes and Leptospira spp. were not detected in any of specimens samples investigated. Mixed infections with Brucella spp. and C. burnetii were observed in 35% specimes examined. There was a good agreement between the multiplex qPCR-HRM and singleplex qPCR for detecting Brucella spp. and C. burnetii. This study is the first report on the syndromic testing of abortion-causing pathogens in Botswana. It shows the importance of molecular methods in the differential diagnosis of abortion-causing diseases in domestic ruminants. Full article

Due to the increasing number of authorized events in the European Union, it is crucial for the official laboratories to enforce market control to detect and quantify genetically modified organisms. In this study, an in-house validation of quantitative duplex ddPCR methods was performed involving MON87701, MON87769, MON89788 and CV-127-9 assays with respect to the lectin reference gene. Since the ddPCR methods provide accurate quantification, show less sensitivity to PCR inhibitors and are more suitable for multiplexing compared to the real-time PCR, the optimization of the existing assays was performed with the exception of MON87701, according to the JRC Guidance documents and technical reports. However, some concerns related to practical settings for the quantitative multiplex of ddPCR methods and their validation were encountered; therefore, a general workflow to develop and validate a ddPCR-based method is shown. The obtained data and the validation performance parameters such as specificity, cross-talk, robustness, dynamic range, linearity, the limit of quantification, trueness and precision comply with international recommendations for GMO quantification methods. The duplex ddPCR methods here investigated are equivalent in terms of performance compared to the singleplex real-time PCR methods, showing higher flexibility and cost effectiveness. Full article

More than one-hundred Babesia species that affect animals and humans have been described, eight of which have been associated with emerging and underdiagnosed zoonoses. Most diagnostic studies in humans have used serology or molecular assays based on the 18S rRNA gene. Because the 18S rRNA gene is highly conserved, obtaining an accurate diagnosis at the species level is difficult, particularly when the amplified DNA fragment is small. Also, due to its low copy number, sequencing of the product is often unsuccessful. In contrast, because the Babesia internal transcribed regions (ITS), between 18S rRNA and 5.8S rRNA, and between 5.8S rRNA and 28S rRNA, contain highly variable non-coding regions, the sequences in these regions provide a good option for developing molecular assays that facilitate differentiation at the species level. In this study, the complete ITS1 and ITS2 intergenic regions of different Piroplasmida species were sequenced to add to the existing GenBank database. Subsequently, ITS1 and ITS2 sequences were used to develop species-specific PCR assays and specific single-plex and multiplex conventional (c)PCR, quantitative real-time (q)PCR, and digital (d)PCR assays for four zoonotic Babesia species (Babesia divergens, Babesia odocoilei, Babesia duncani, and Babesia microti). The efficacy of the assay protocols was confirmed by testing DNA samples extracted from human blood or enrichment blood cultures. Primers were first designed based on the 18S rRNA-5.8S rRNA and 5.8S rRNA-28S rRNA regions to obtain the ITS1 and ITS2 sequences derived from different Piroplasmida species (B. odocoilei, Babesia vulpes, Babesia canis, Babesia vogeli, Babesia gibsoni, Babesia lengau, Babesia divergens-like, B. duncani, B. microti, Babesia capreoli, Babesia negevi, Babesia conradae, Theileria bicornis, and Cytauxzoon felis). Subsequently, using these sequences, single-plex or multiplex protocols were optimized targeting the ITS1 region of B. divergens, B. microti, and B. odocoilei. Each protocol proved to be sensitive and specific for the four targeted Babesia sp., detecting 10⁻² (for B. microti and B. odocoilei) and 10⁻¹ (for B. divergens and B. duncani) DNA copies per microliter. There was no cross-amplification among the Babesia species tested. Using 226 DNA extractions from blood or enrichment blood cultures obtained from 82 humans, B. divergens (seven individuals), B. odocoilei (seven individuals), and B. microti (two individuals) were detected and identified as a single infection, whereas co-infection with more than one Babesia sp. was documented by DNA sequencing in six (7.3%) additional individuals (representing a 26.8% overall prevalence). These newly developed protocols proved to be effective in detecting DNA of four Babesia species and facilitated documentation of co-infection with more than one Babesia sp. in the same individual. Full article

IgE-mediated wheat allergy can take on various forms, including childhood food allergy to wheat, wheat-dependent exercise-induced anaphylaxis in young adults, baker’s respiratory allergy/asthma in workers exposed to wheat flour inhalation, and contact urticaria that is caused by hydrolyzed wheat proteins in some cosmetics, and that is sometimes associated with a food allergy. Singleplex and multiplex immunoassays detect specific IgE antibodies to wheat allergenic molecular biomarkers such as omega-5 gliadin Tri a 19, lipid transfer protein Tri a 14, and alpha-amylase inhibitors. The fluorescence enzyme immunoassay with capsulated cellulose polymer solid-phase coupled allergens is a commonly used singleplex assay. Multiplex methods include the ELISA-based macroarray immunoassay using nano-bead technology and a microarray immunoassay on polymer-coated slides. Another promising diagnostic tool is the basophil activation test performed with omega-5 gliadin and other wheat protein types. Detailed comprehension of the structural and immunological features of the numerous wheat allergens significant in clinical settings is imperative for advancing diagnostic biomarkers for IgE-mediated wheat allergies. Full article

Bovine leukemia virus (BLV) is prevalent worldwide, causing serious problems in the cattle industry. The BLV proviral load (PVL) is a useful index for estimating disease progression and transmission risk. We previously developed a quantitative real-time PCR (qPCR) assay to measure the PVL using the coordination of common motif (CoCoMo) degenerate primers. Here, we constructed a novel duplex BLV-CoCoMo qPCR assay that can amplify two genes simultaneously using a FAM-labeled MGB probe for the BLV LTR gene and a VIC-labeled MGB probe for the BoLA-DRA gene. This liquid duplex assay maintained its original sensitivity and reproducibility in field samples. Furthermore, we developed a dry duplex assay composed of PCR reagents necessary for the optimized liquid duplex assay. We observed a strong positive correlation between the PVLs measured using the dry and liquid duplex assays. Validation analyses showed that the sensitivity of the dry duplex assay was slightly lower than that of the other methods for the detection of a BLV molecular clone, but it showed similar sensitivity to the singleplex assay and slightly higher sensitivity than the liquid duplex assay for the PVL quantification of 82 field samples. Thus, our liquid and dry duplex assays are useful for measuring the BLV PVL in field samples, similar to the original singleplex assay. Full article

G protein-coupled receptors (GPCRs) are relevant targets for health and disease as they regulate various aspects of metabolism, proliferation, differentiation, and immune pathways. They are implicated in several disease areas, including cancer, diabetes, cardiovascular diseases, and mental disorders. It is worth noting that about a third of all marketed drugs target GPCRs, making them prime pharmacological targets for drug discovery. Numerous functional assays have been developed to assess GPCR activity and GPCR signaling in living cells. Here, we review the current literature of genetically encoded cell-based assays to measure GPCR activation and downstream signaling at different hierarchical levels of signaling, from the receptor to transcription, via transducers, effectors, and second messengers. Singleplex assay formats provide one data point per experimental condition. Typical examples are bioluminescence resonance energy transfer (BRET) assays and protease cleavage assays (e.g., Tango or split TEV). By contrast, multiplex assay formats allow for the parallel measurement of multiple receptors and pathways and typically use molecular barcodes as transcriptional reporters in barcoded assays. This enables the efficient identification of desired on-target and on-pathway effects as well as detrimental off-target and off-pathway effects. Multiplex assays are anticipated to accelerate drug discovery for GPCRs as they provide a comprehensive and broad identification of compound effects. Full article

Cryptosporidium spp., Giardia duodenalis and Entamoeba histolytica are species of protozoa- causing diarrhoea that are common worldwide, while Entamoeba dispar, Dientamoeba fragilis and Blastocystis sp. appear to be commensal parasites whose role in pathogenicity remains controversial. We conducted the clinical evaluation of five singleplex and one duplex CerTest VIASURE Real-Time PCR Assays against a large panel of positive DNA samples (n = 358), and specifically to Cryptosporidium spp. (n = 96), G. duodenalis (n = 115), E. histolytica (n = 25) E. dispar (n = 11), Blastocystis sp. (n = 42), D. fragilis (n = 37), and related parasitic phylum species such as Apicomplexa, Euglenozoa, Microsporidia and Nematoda. DNA samples were obtained from clinical stool specimens or cultured isolates in a national reference centre. Estimated diagnostic sensitivity and specificity values were 0.94–1 for Cryptosporidium spp., 0.96–0.99 for G. duodenalis, 0.96–1 for E. histolytica, 1–1 for E. dispar, and 1–0.99 for D. fragilis in the evaluated singleplex assays. In the duplex assay for the simultaneous detection of Blastocystis sp. and D. fragilis these values were 1–0.98 and 1–0.99, respectively. Measures of diagnostic precision for repeatability and reproducibility were found to be under acceptable ranges. The assays identified six Cryptosporidium species (C. hominis, C. parvum, C. canis, C. felis, C. scrofarum, and C. ryanae), four G. duodenalis assemblages (A, B, C, and F), and six Blastocystis subtypes (ST1-ST5, and ST8). The evaluated singleplex and duplex VIASURE Real-Time PCR assays provide sensitive, practical, and cost-effective choices to the molecular diagnosis of the main diarrhoea-causing intestinal protists in clinical microbiology and research laboratories. Full article