The performance of a new method developed in 2021 by the European Union Reference Laboratory (EURL) for Listeria monocytogenes based on 12 multiplex real-time PCR, allowing the identification of the molecular serotype and the 30 major L. monocytogenes multilocus sequence typing clonal complexes (CC), was assessed through a European interlaboratory validation trial (ILVT). This ILVT was adapted from ISO standard 16140 part 6. Overall, 98 blinded pure strains of Listeria (monocytogenes or spp.), previously characterized by the EURL, were sent to 15 laboratories distributed in 11 countries. The molecular serotype had to be identified for 20 strains of the ILVT panel, while CC identification had to be performed for the whole panel. The results of the 12 multiplex real-time PCR were reproducible between the participating laboratories with high individual concordance values for molecular serotyping (100%) and CC identification (90.8%-100%) irrespective of DNA extraction protocols, PCR master mixes, and thermocycler diversity. Master mixes identified as incompatible with some of the multiplex real-time PCR were excluded from the method. The overall concordance of the results was sufficient for the method to be confidently applied in other laboratories involved in L. monocytogenes typing.IMPORTANCEThis interlaboratory validation trial, coordinated by the European Union Reference Laboratory for Listeria monocytogenes, was the final step to assess the performance of the multiplex real-time PCR method developed and published by B. Félix, K. Capitaine, S. Te, A. Felten, et al. (Microbiol Spectr 11:e0395422, 2023, https://doi.org/10.1128/spectrum.03954-22). Different combinations of parameter settings were applied in 15 French and European laboratories involved in L. monocytogenes typing. It was a prerequisite to establish this new real-time PCR method as a standard for rapid molecular serotyping and clonal complex identification. The accuracy and reproducibility of the results obtained on the panel of 98 strains of L. monocytogenes sent to the participants proved that the real-time PCR was suitable for use in their conditions. Rapid screening of strains is therefore now possible, and the method provides a valuable tool for epidemiological investigations to identify food-associated strains during listeriosis outbreaks.
- Klíčová slova
- Europe, Listeria monocytogenes, MLST, clonal complex, interlaboratory validation trial, molecular serotype, real-time PCR,
- MeSH
- kvantitativní polymerázová řetězová reakce * metody MeSH
- lidé MeSH
- Listeria monocytogenes * genetika klasifikace izolace a purifikace MeSH
- listeriové infekce mikrobiologie MeSH
- multilokusová sekvenční typizace metody MeSH
- multiplexová polymerázová řetězová reakce * metody MeSH
- potravinářská mikrobiologie * metody MeSH
- reprodukovatelnost výsledků MeSH
- séroskupina MeSH
- sérotypizace * metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- validační studie MeSH
- Geografické názvy
- Evropa MeSH
BackgroundMost Toxoplasma gondii infections in humans are considered foodborne, but the relative importance of the various routes of infection is largely unknown. Consumption of green produce contaminated with T. gondii oocysts has been identified as a possible source.AimWe aimed to estimate the occurrence and prevalence of T. gondii oocysts in commercially available ready-to-eat (RTE) salad mixes in 10 European countries.MethodsA real-time PCR-based method for oocyst detection was developed and optimised by two laboratories and validated in an interlaboratory test. This detection method and a harmonised sampling strategy were applied in a multi-country study. Multivariable logistic regression was used to investigate risk factors for oocyst contamination of RTE salad.ResultsThe real-time PCR method had a detection limit of 10 oocysts per 30 g of salad. We collected 3,329 RTE salad samples (baby leaf and cut leaf mixes) from October 2021 to September 2022. The prevalence of T. gondii oocyst contamination was 4.1% (95% confidence interval (CI): 3.4-4.8%; n = 3,293). In multivariable regression analysis, winter season, sampling and packaging of salad in Northern Europe and production of salad in Western Europe were associated with detection of T. gondii, with no statistically significant differences between salad types.ConclusionWe estimated the prevalence of T. gondii oocysts in RTE leafy green salads using a validated and standardised procedure to assess the potential risk for human infection; highlighting the need to address this risk at each critical point of the salad production chain.
- Klíčová slova
- Europe, Leafy greens, Toxoplasma gondii, harmonisation, oocysts, ready-to-eat salads, ring-trial,
- MeSH
- kontaminace potravin * analýza MeSH
- kvantitativní polymerázová řetězová reakce * metody MeSH
- lidé MeSH
- oocysty izolace a purifikace MeSH
- potravinářská parazitologie * metody MeSH
- prevalence MeSH
- Toxoplasma * izolace a purifikace genetika MeSH
- toxoplazmóza * epidemiologie MeSH
- zelenina * parazitologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Evropa epidemiologie MeSH
BACKGROUND: Cercarial dermatitis (CD), or swimmer's itch, is a water-borne allergic skin reaction caused by the penetration of the larval stages of bird schistosomes (cercariae) into the skin. Members of the genus Trichobilharzia are the primary causative agents of CD worldwide. Due to the increasing number of cases, CD is regarded as a (re)emerging disease. Outbreaks in recreational waters can significantly impact public health and local economies. Environmental monitoring of Trichobilharzia is crucial for outbreak prediction and public health management. However, conventional methods, such as cercarial shedding and snail dissections, are labour-intensive and lack sensitivity. To overcome these limitations, we present a molecular toolkit that combines loop-mediated isothermal amplification (LAMP), quantitative polymerase chain reaction (qPCR), and multiplex PCR for rapid, sensitive, and accurate detection and identification of Trichobilharzia spp. from various biological samples. METHODS: Tricho-LAMP and Tricho-qPCR were designed and optimised for Trichobilharzia DNA detection. A multiplex PCR assay was also developed and optimised to identify the three main species causing CD in Europe (Trichobilharzia franki, T. szidati, and T. regenti). RESULTS: Tricho-LAMP specifically detected T. regenti and T. franki at 10-3 ng, and T. szidati at 10-2 ng per reaction with genomic DNA. Using gBlocks synthetic DNA, Tricho-LAMP achieved 100% amplification at 10,000 copies and 85% amplification at 1000 copies, with decreasing success at lower concentrations. Tricho-qPCR showed the highest sensitivity, detecting all species down to 10-4 ng per reaction and showing a limit of detection at 10 copies of synthetic DNA in the reaction. Multiplex PCR allowed reliable species differentiation via gel electrophoresis of the PCR products, but the assay had the lowest sensitivity. CONCLUSIONS: We provide a molecular toolkit consisting of LAMP, qPCR, and multiplex PCR. By exhibiting high sensitivity, Tricho-LAMP and Tricho-qPCR assays are potentially suitable for environmental DNA (eDNA)-based environmental monitoring of bird schistosomes, by both researchers and public health authorities. Multiplex PCR can be used for species determination without the need for further sequencing.
- Klíčová slova
- Trichobilharzia, Bird schistosomes, Cercarial dermatitis, Detection, LAMP, Monitoring, Multiplex PCR, qPCR,
- MeSH
- diagnostické techniky molekulární * metody MeSH
- DNA helmintů genetika MeSH
- hlemýždi parazitologie MeSH
- infekce červy třídy Trematoda * diagnóza parazitologie veterinární MeSH
- kvantitativní polymerázová řetězová reakce * metody MeSH
- multiplexová polymerázová řetězová reakce * metody MeSH
- ptáci parazitologie MeSH
- Schistosomatidae * genetika izolace a purifikace klasifikace MeSH
- senzitivita a specificita MeSH
- techniky amplifikace nukleových kyselin * metody MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA helmintů MeSH
Applications like drug development need simple and streamlined methods to process samples from 96-well cell culture plates for gene expression measurements. Unfortunately, current options are expensive for such processing. Therefore, our aim was to develop a method that would allow streamlined analysis of mRNA from 96-well cell culture plates while being relatively cheap and simple. We developed a method based on the qPCR 'Cells-to-cDNA' approach and validated it against commercially available kits using the same approach or spin columns-based RNA purification. For this purpose, we conducted a series of comparisons of gene expression from peripheral blood mononuclear cells, SK-HEP-1 and U-87 cell cultures in 96-well plates. Our final method involved lysing cells with 25-100 µl solution of 0.5% SDS, 10 mM DTT, 1 mg ml-1 proteinase K dissolved in water, 1 h incubation at 50°C, followed by proteinase K inactivation at 90°C for 5 min and lysate neutralization with 1 : 1 dilution by 20% Tween 20 solution. Reverse transcription and qPCR were carried out using standard methods. This method showed a mean reduction of Ct ± s.d. value by 2.4 ± 1.3 compared with the 'Cells-to-cDNA' kit and by 1.4 ± 0.5 compared with the RNA purification kit with lower variability.
- Klíčová slova
- RNA isolation, cell lysis, in vitro, mRNA, peripheral blood mononuclear cells, proteinase k, qPCR,
- MeSH
- analýza nákladů a výnosů MeSH
- buněčné kultury * metody MeSH
- komplementární DNA * genetika biosyntéza MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- leukocyty mononukleární metabolismus MeSH
- lidé MeSH
- messenger RNA genetika MeSH
- stanovení celkové genové exprese metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- komplementární DNA * MeSH
- messenger RNA MeSH
UNLABELLED: The aim of this study was to identify parameters influencing DNA extraction and PCR amplification efficiencies in an attempt to standardize Mucorales qPCR. The Fungal PCR Initiative Mucorales Laboratory Working Group distributed two panels of simulated samples to 26 laboratories: Panel A (six sera spiked with Mucorales DNA and one negative control serum) and Panel B (six Mucorales DNA extracts). Panel A underwent DNA extraction in each laboratory according to the local procedure and were sent to a central laboratory for testing using three different qPCR techniques: one in-house qPCR assay and two commercial assays (MucorGenius and Fungiplex). Panel B DNA extracts were PCR amplified in each laboratory using local procedures: nine in-house qPCR assays and two commercial kits (MucorGenius and MycoGENIE). All data were compiled and anonymously analyzed at the central laboratory. For Panel A, a total of six different automated platforms and five manual extraction methods were used. Positive rates were 64%, 70%, and 89%, for the MucorGenius, Fungiplex, and the in-house qPCR assay, respectively. Using a large volume of serum for DNA extraction provided the highest analytical sensitivity (82.5% for 1 mL compared with 62.7% for smaller volumes, P < 0.01). For Panel B, five in-house qPCR assays and two commercial kits had >78% positivity. Using larger PCR input volumes (≥7 µL) was associated with the highest sensitivity at 95.5% compared to 58.3% when lower input volumes were used (P < 0.01). Using larger sample volumes for nucleic acid extraction and DNA template volumes for PCR amplification significantly improves the performance of Mucorales qPCR when testing serum. IMPORTANCE: Mucormycosis is a life-threatening mold infection affecting immunosuppressed patients but also other patients with diabetes or trauma. Better survival is linked to shorter delays in diagnosis and treatment initiation. Detection of Mucorales-free DNA in serum or plasma using quantitative PCR allows a prompt diagnosis and earlier treatment. Several techniques and protocols of quantitative Mucorales PCR are used in Europe, and improving performance remains a common objective of laboratories participating in the fungal PCR Initiative Working Group. This study, which combined results from 26 laboratories in Europe, showed that the main parameters underpinning sensitivity are the preanalytical variables (volume of serum used for DNA extraction and DNA template volume), irrespective of the extraction platforms and qPCR assay/platform.
- Klíčová slova
- Mucorales PCR, interlaboratory assay, mucormycosis, standardization,
- MeSH
- diagnostické techniky molekulární * normy metody MeSH
- DNA fungální * krev genetika izolace a purifikace MeSH
- kvantitativní polymerázová řetězová reakce * normy metody MeSH
- lidé MeSH
- Mucorales * genetika izolace a purifikace MeSH
- mukormykóza * diagnóza mikrobiologie MeSH
- senzitivita a specificita MeSH
- sérum * mikrobiologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA fungální * MeSH
PREMISE: Despite the high functional importance of endophytes, we still have limited understanding of the biotic and abiotic factors that influence colonization of plant hosts along major ecological gradients and lack quantitative estimates of their colonization extent. In this study, we hypothesized that the developmental stage of the ecosystem will affect the levels of bacterial and fungal endophytic assemblages in the foliar endosphere. METHODS: We quantified levels of bacterial and fungal endophytes in leaves of four plant hosts at four stages of vegetation succession using an optimized qPCR protocol with bacteria-specific 16S and fungi-targeting primers. RESULTS: (1) The ecosystem developmental stage did not have a significant effect on the colonization levels of bacterial or fungal endophytes. (2) Colonization levels by bacterial and fungal endophytes were governed by different mechanisms. (3) Endophytic colonization levels and their relationship to foliar tissue stoichiometry were highly host specific. CONCLUSIONS: Quantifying colonization levels is important in the study of endophytic ecology, and the fast, relatively low-cost qPCR-based method can supply useful ecological information, which can significantly enhance the interpretation potential of descriptive data generated, for example, by next-generation sequencing.
- Klíčová slova
- cell counts, ecological succession, foliar endophyte, fungi‐bacteria ratios, qPCR, soil chronosequence,
- MeSH
- Bacteria * genetika růst a vývoj izolace a purifikace MeSH
- ekosystém MeSH
- endofyty * fyziologie genetika MeSH
- hostitelská specificita * MeSH
- houby * genetika izolace a purifikace fyziologie MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- listy rostlin * mikrobiologie MeSH
- Publikační typ
- časopisecké články MeSH
Fish from the pike (Esox) genus are valued in gastronomy for their superior meat quality. However, they can cause allergic reactions in sensitive consumers. This work aimed to fill the gap in the detection of pike allergens using molecular-biological techniques. New, fast, and accurate loop-mediated isothermal amplification (LAMP) and real-time PCR (qPCR) assays were designed to detect pike DNA using the parvalbumin gene as a marker. LAMP was assessed by electrophoresis, SYBR green optical detection, and real-time fluorescence detection. The latter was the most sensitive, detecting as little as 0.78 ng of pike DNA; the qPCR detection limit was 0.1 ng. The LAMP analysis took 20-70 min, which is significantly faster than qPCR. The study provides reliable detection and quantification of the parvalbumin gene in both fresh and processed samples and further highlights the versatility of the use of the parvalbumin gene for the authentication of food products and consumer protection via refined allergen risk assessment that is independent of the type of tissue or food processing method used.
- Klíčová slova
- DNA, Esox, LAMP, PCR, food allergy, food fraud,
- MeSH
- alergeny * genetika analýza imunologie MeSH
- biologické markery analýza MeSH
- diagnostické techniky molekulární MeSH
- Esocidae * genetika imunologie MeSH
- hodnocení rizik MeSH
- kontaminace potravin analýza MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- lidé MeSH
- parvalbuminy * genetika imunologie analýza MeSH
- potravinová alergie * imunologie MeSH
- rybí proteiny genetika imunologie MeSH
- techniky amplifikace nukleových kyselin metody MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- Názvy látek
- alergeny * MeSH
- biologické markery MeSH
- parvalbuminy * MeSH
- rybí proteiny MeSH
Minimal/measurable residual disease (MRD) diagnostics using real-time quantitative PCR analysis of rearranged immunoglobulin and T-cell receptor gene rearrangements are nowadays implemented in most treatment protocols for patients with acute lymphoblastic leukemia (ALL). Within the EuroMRD Consortium, we aim to provide comparable, high-quality MRD diagnostics, allowing appropriate risk-group classification for patients and inter-protocol comparisons. To this end, we set up a quality assessment scheme, that was gradually optimized and updated over the last 20 years, and that now includes participants from around 70 laboratories worldwide. We here describe the design and analysis of our quality assessment scheme. In addition, we here report revised data interpretation guidelines, based on our newly generated data and extensive discussions between experts. The main novelty is the partial re-definition of the "positive below quantitative range" category by two new categories, "MRD low positive, below quantitative range" and "MRD of uncertain significance". The quality assessment program and revised guidelines will ensure reproducible and accurate MRD data for ALL patients. Within the Consortium, similar programs and guidelines have been introduced for other lymphoid diseases (e.g., B-cell lymphoma), for new technological platforms (e.g., digital droplet PCR or Next-Generation Sequencing), and for other patient-specific MRD PCR-based targets (e.g., fusion genes).
- MeSH
- akutní lymfatická leukemie genetika diagnóza MeSH
- genová přestavba * MeSH
- geny pro imunoglobuliny MeSH
- kvantitativní polymerázová řetězová reakce metody normy MeSH
- lidé MeSH
- reziduální nádor * genetika diagnóza MeSH
- směrnice pro lékařskou praxi jako téma normy MeSH
- zajištění kvality zdravotní péče MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
AIM: In the past, Pneumocystis jirovecii belonged to the Protozoa group, but is currently taxonomically included in the kingdom Fungi. P. jirovecii is an opportunistic pathogen, responsible for pneumocystis pneumonia with frequent complications of immunocompromised patients. Delayed initiation of appropriate therapy increases the risk of death in immunocompromised patient. The aim of this work was to determine and evaluate the reliability of methods of laboratory diagnosis of pneumocystosis used in routine laboratories as well as the occurrence of this disease in patients from Slovakia during 19 years. MATERIAL AND METHODS: The diagnosis is based on microscopic examination (Giemsa- and Gram-Weigert-staining) and detection of parasite DNA by classical or real-time PCR in bronchoalveolar lavage and sputum. RESULTS: Pneumocysts were detected in 190 persons (5.7%) from the whole group of patients. Cancer patients represented the riskiest group in terms of pneumocystosis, which was confirmed by the highest percentage (57.9%) of individuals infected with P. jirovecii. Compared with the PCR, 33.7% sensitivity and 100% specificity of microscopy was calculated by using a binary classification test. Molecular methods are more sensitive in the detection of P. jirovecii compared to microscopic evidence and currently represent a reliable detection system in the diagnosis of pneumocystosis. CONCLUSION: In view of the increasing number of immunocompromised persons, diagnostics of P. jirovecii in patients with pulmonary complications is essential. This was also confirmed in our study, where the number of examinations and detection of this opportunistic pathogen increased over the years.
- Klíčová slova
- Pneumocystis jirovecii, microscopic evidence, pneumocystosis, polymerase chain reaction (PCR),
- MeSH
- bronchoalveolární lavážní tekutina mikrobiologie MeSH
- imunokompromitovaný pacient MeSH
- incidence MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- lidé MeSH
- Pneumocystis carinii * genetika MeSH
- pneumocystová pneumonie * diagnóza epidemiologie mikrobiologie MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
