Search Results (73)

Singleplex and multiplex real-time PCR-HRM (polymerase chain reaction with high resolution melting), both with specific and non-specific amplicon detection, are used for a wide range of applications, from clinical diagnostics to food authentication. However, their results can be influenced by the quality of the template DNA and composition of the reaction mixture. The methods used for the analysis of these results then influence the conclusions drawn. In this work we present an example from our laboratory practice, where the results of singleplex and multiplex real-time PCR differed, despite using the same reaction conditions, primers and analyzed plant material. We show the influence of a singleplex and multiplex PCR setup on the results, as well as the influence of template contamination on the melting behaviour of amplicons. We also discuss the usefulness of cluster analysis for the clarification of real-time PCR-HRM results which appear unclear when only melting and difference curves or similarity scores are used for the analysis of these results. We provide a discussion of problems which we encountered during an analysis of commercial teas and which should be considered by researchers new to PCR-based analysis of plant material, especially if the studied material is rich in various contaminants. Full article

In this study, a multiplex PCR assay was developed for the rapid and accurate identification of three Chionoecetes species (Chionoecetes bairdi, C. opilio, and C. japonicus) available in global seafood markets. The morphological similarity between imported female C. bairdi from Japan and native C. opilio in Korea complicates visual discrimination and raises concerns over potential mislabeling. To address this issue, mitochondrial cytochrome c oxidase subunit I (COI) gene sequences were analyzed to identify species-specific SNPs, and primers were designed accordingly. Singleplex PCR confirmed species-specific amplification among the three target species, and the optimal annealing temperature was determined. The multiplex PCR simultaneously amplified distinct fragments of 598 bp (C. bairdi), 401 bp (C. opilio), and 194 bp (C. japonicus), with no nonspecific amplification or primer–dimer formation. Sensitivity testing revealed a detection limit of 0.01 ng/µL for all three species, defined as the lowest DNA concentration at which species-specific bands were consistently observed in at least two out of three replicates. These results demonstrate that the developed multiplex PCR is a reliable, rapid, and cost-effective tool for accurate species identification, supporting sustainable resource management, preventing seafood fraud, and ensuring safe distribution in both Korea and global seafood markets. Full article

Tick-borne pathogens pose a significant threat to human health. In this study, a multiple droplet digital PCR (ddPCR) assay was developed to detect four tick-borne pathogens: Borrelia burgdorferi sensu lato (Bbsl), Coxiella burnetii (C. burnetii), spotted fever group Rickettsia (SFGR), and Borrelia miyamotoi (B. miyamotoi). Based on the singleplex ddPCR reaction system of Bbsl, the primer probes of the other three species were incorporated to develop a multiplex ddPCR reaction system. The annealing temperature and the final concentration of the primer probes were then optimized for multiplex ddPCR. The multiplex ddPCR assay was assessed for its sensitivity, specificity, repeatability, and ability to detect simulated and actual samples. The developed multiplex ddPCR approach enables the simultaneous detection of Bbsl, C. burnetii, SFGR, and B. miyamotoi. The positive target microtitre clusters are closely grouped and distinctly separated from each other, with the multiplex ddPCR assay demonstrating a dynamic range of five orders of magnitude. The limits of detection (LOD) for the multiplex ddPCR assay were 4 copies/20 µL for Bbsl, 3 copies/20 µL for C. burnetii, 3 copies/20 µL for SFGR, and 2 copies/20 µL for B. miyamotoi. The assay demonstrated high specificity, with no observed cross-reactivity against non-target pathogens. Performance was validated using both spiked samples and field-collected clinical specimens. In the evaluation of 30 ticks and 30 serum samples, the ddPCR method (in both singleplex and multiplex formats) achieved higher positive detection rates for all four target pathogens compared to quantitative real-time PCR (qPCR). In addition, the detection proportions of multiplex and singleplex ddPCR were consistent. Multiplex ddPCR can detect low DNA concentrations in samples and enables the absolute quantification of Bbsl, C. burnetii, SFGR, and B. miyamotoi, providing a novel detection approach for the clinical diagnosis of tick-borne diseases. Full article

Grass pollen allergy has a high prevalence worldwide, making an accurate diagnosis critical in the framework of multifaceted environmental exposures. Our narrative review provides a comprehensive synopsis of component-resolved diagnosis biomarkers for pollen of Pooideae and Chloridoideae grasses, along with practical approaches in European clinical settings. We present a structured overview of allergen components utilized in singleplex, multiparameter, and multiplex IgE immunoassays. Molecular biomarkers have key roles in distinguishing genuine grass pollen sensitization from cross-reactivity and in assessing the risks associated with various IgE sensitization patterns, thereby enabling precise allergy diagnosis and facilitating targeted allergen immunotherapy. Diagnostic algorithms are provided to assist clinicians in making molecular biomarker-based personalized decisions. This tailored approach supports better management of patients with grass pollen sensitizations and allergies. Full article

Background: Acute otitis media (AOM) is one of the most common pediatric infections. We aimed to investigate the bacterial profile of AOM in children, the serotype distribution, and the main genetic virulence factors involved in adhesion, immune evasion, and tissue spread. Methods: In total, 121 AOM cases involving children aged 0 to 14 years were studied. Middle ear fluids (MEF) (n = 42) and nasopharyngeal samples (n = 79) were collected. All strains were identified using routine microbiological tests, conventional PCRs and real-time PCR methods. Molecular serotyping was performed for S. pneumoniae and H. influenzae isolates. An immunofluorescence serotyping technique was employed for M. catarrhalis. Target genetic factors were determined for all involved bacterial agents using singleplex or multiplex PCRs. Results: We analyzed 148 nasopharyngeal and MEF. Among 121 AOM cases, a total of 127 bacterial agents were identified, including S. aureus (n = 41), S. pneumoniae (n = 28), H. influenzae (n = 23), M. catarrhalis (n = 19), and S. pyogenes (n = 16). The leading three serotypes among S. pneumoniae were: 19A (18.0%), 6A (14.3%), and 15B (14.3%). 91.3% of H. influenzae isolates were non-typeable (lacking a capsule—NTHi). The M. catarrhalis isolates were distributed in serotypes A (57.9%), B (26.3%), and C (15.8%). Presence of pili type 1 was detected in 21.4% pneumococci, and the fimbrial gene hifA was found in 34.8% of the H. influenzae strains. In 73.6% of the M. catarrhalis strains, ompCD was identified, while 84.2% contained ompE. 62.5% of the S. pyogenes isolates harbored the sdc gene, and 56.2% possessed the sdaD gene, predominantly in the MEF isolates. The cna adhesin was found in 28.0% of the S. aureus strains. Conclusions: The monitoring of bacterial pathogens responsible for otitis media, along with their serotype distribution and the prevalence of genetic factors involved in disease pathogenesis, is essential for public health and can help predict disease severity and treatment options. Full article

Salmonella enterica serovar Gallinarum is a non-motile serovar and is the causative agent of fowl typhoid, and poses a major challenge to poultry production, particularly where rapid diagnostics are lacking. Existing methods are either time-consuming or fail to distinguish motile from non-motile serotypes. We developed a dual-target colorimetric LAMP that detects Salmonella spp. via invA and discriminates S. Gallinarum via TRX (a taxon-restricted sequence), using two separate singleplex reactions. Specificity testing confirmed 100% accuracy, with exclusive amplification of S. Gallinarum through TRX. Analytical sensitivity was comparable to real-time PCR, detecting down to 2.41 CFU/µL (invA) and 1.65 CFU/µL (TRX). Applied to cloacal swabs from experimentally infected chickens (n = 12), the assay consistently outperformed bacteriological culture, detecting up to 25% more positives during early infection when bacterial loads were low or cells were non-culturable. This dual-target LAMP provides a rapid, sensitive, and serovar-discriminating diagnostic tool with strong potential for point-of-care use and real-time surveillance in poultry farms, thereby improving sanitary control of fowl typhoid and reducing associated economic losses. Full article

Muscovy duck reovirus (MDRV) and novel duck reovirus (NDRV) are major pathogens in duck breeding, leading to substantial economic losses in the waterfowl industry. This study aimed to develop a precise detection and differentiation method for both viruses simultaneously. Specific primers and probes targeting the S3 gene were designed, and a duplex TaqMan-based real-time RT-PCR assay was established following optimization of reaction conditions. The assay demonstrated high amplification efficiency (100.1–106.7%), strong linear correlation (R² > 0.999), and low limits of detection (13–25 copies/µL). Intra- and inter-assay coefficients of variation were below 1.5%, confirming excellent repeatability and stability. Applied to 122 clinical duck tissue samples, the assay detected MDRV in 29.5% (36/122) and NDRV in 39.3% (48/122) of samples, with results fully validated by singleplex RT-PCR assays. Our study provides a reliable, specific, and reproducible tool for surveillance and epidemiological studies of MDRV and NDRV. Full article

Background/Objectives: We have previously reported the engineering of a point-of-care (POC) system that fully automates the procedures for nucleic acid extraction and multiplexed real-time RT-PCR, with a major advantage of high-level multiplexing. In this study, we applied and validated the system in a respiratory tract infection setting. Methods: An automatic nested real-time RT-PCR assay was developed (POCm). It was a 40-plex assay that simultaneously detected 39 epidemiologically important respiratory pathogens in 1.5 h in the POC system. The analytical and clinical performance was evaluated. Results: The analytical sensitivities of the POCm assay were comparable to those of its single-plex counterparts performed manually on a bench-top. The minimum detectable concentrations ranged from 53 copies/mL to 5.3 × 10³ copies/mL for all pathogen targets except hCoV-NL63 (5.3 × 10⁴ copies/mL). The quantitative performance was demonstrated by the linear correlations between Ct values and input concentrations for all pathogen targets, with 24 of them demonstrating coefficients of correlation (r) greater than 0.9. The POCm assay was subsequently evaluated in 283 clinical samples. A high level of agreement (98.2–100%) was achieved for pathogen detection results between POCm and standard diagnostic methods. The POCm result was also fully concordant with the result of another commercial POC multiplex platform. For positive clinical samples, pairwise Ct values measured by POCm closely correlated with those of the bench-top reference method (r = 0.70). The feasibility of mutation genotyping of the viral subtype was further demonstrated. Conclusions: This study demonstrated the practicality of POCm for routine testing in clinical laboratories. Further clinical trials are being conducted to evaluate the clinical performance of the system. Full article

Edible insects, particularly Tenebrio molitor (Linnaeus) (mealworm) and Zophobas morio (Fabricius) (superworm), have drawn increasing attention as alternative protein sources. This study aims to develop an accurate molecular detection method for T. molitor, an EU-approved food species, and to differentiate it from Z. morio, which remains unapproved for human consumption in the EU. The process enables precise and sensitive identification methods by optimizing singleplex and duplex PCR techniques targeting 16S rRNA and COI gene regions. The DNA of T. molitor was detected in various food matrices, including pastries, chocolate, and porridge, while avoiding cross-reactivity with Z. morio, Gryllus asimilis, and Locusta migratoria. The detection limit for both singleplex and duplex PCR was 10 pg of DNA, ensuring robustness against inhibitory effects from complex food matrices. The developed approach ensures reliable detection and compliance with EU regulations regarding insect-based foods, providing a critical tool for food authentication and preventing adulteration. The key advancements of this approach lie in its improved specificity and sensitivity, allowing for the ability to detect complex food matrices. An applied perspective was evaluated using real commercial food products. Full article

SMoV and SVBV are two major viruses that pose significant threats to the global strawberry industry. Both are latent viruses, making early detection difficult due to their uneven distribution and low concentration in host tissues. Traditional RT-PCR techniques are insufficient for precise and quantitative detection. In this study, TaqMan RT-qPCR detection systems for SMoV and SVBV were established for application in practical production settings, enabling accurate, rapid, and efficient detection of strawberry viruses. When viral accumulation in plants is low, the highly sensitive TaqMan RT-qPCR technique allows for accurate quantification, facilitating the early identification of infected plants and preventing large-scale outbreaks in cultivation areas. The development of a duplex TaqMan RT-qPCR assay enables simultaneous quantification of SMoV and SVBV in a single reaction, improving detection efficiency and providing technical support for risk assessment and effective control of strawberry viral diseases. Full article

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