Paenibacillus larvae and Melissococcus plutonius represent the most threatening bacterial diseases of honeybee (Apis mellifera)-American and European foulbrood, respectively. For efficient control of those diseases, rapid and accurate detection of the pathogens is crucial. Therefore, we developed a novel multiplex PCR method simultaneously detecting both pathogens. To design and optimize multiplex PCR reaction, four strains of P. larvae representing four ERIC genotypes I-IV (strain DSM 7030-ERIC I, DSM 25430-ERIC II, LMG 16252-ERIC III, DSM 3615-ERIC IV) were selected. Those strains were fully sequenced using long-read sequencing (Sequel I, Pacific Biosciences). For P. larvae, the multicopy insertion sequence IS256 identified in all genotypes of P. larvae was selected to provide high sensitivity. M. plutonius was detected by plasmid pMP1 sequence and the virulence verified by following detection of ETX/MTX2 toxin responsible for pore formation in the cell membrane. As an internal control, a gene encoding for major royal jelly protein 1 specific for honeybees was selected. The method was validated on 36 clinical specimens collected from the colonies suffering from American and European foulbrood in the Czech Republic. Based on the results, sensitivity of PCR was calculated to 93.75% and specificity to 100% for P. larvae diagnosed from hive debris and 100% sensitivity and specificity for honeybee workers and larval scales as well as for diseased brood infected by M. plutonius.
- MeSH
- Enterococcaceae * MeSH
- larva mikrobiologie MeSH
- multiplexová polymerázová řetězová reakce metody MeSH
- Paenibacillus larvae * genetika MeSH
- Paenibacillus * genetika MeSH
- plazmidy genetika MeSH
- transpozibilní elementy DNA MeSH
- včely genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Ciele: Analýza prenatálnych vzoriek za obdobie 2015–2020. Porovnanie miery detekcie klinicky relevantných variant cytogenetickou analýzou karyotypu a cytogenomickými metódami MLPA (Multiplex Ligation-Depent Probe Amplification) a mikročipmi (CMA – chromosomal microarray). Súbor a metodika: Analyzovaných bolo 1 029 prenatálnych vzoriek cytogenetickým hodnotením karyotypu (n = 1 029), cytogenomickými metódami MLPA (n = 144) a CMA (n = 111). Všetky nebalansované zmeny boli potvrdené metódou MLPA alebo CMA. Výsledky: Z analyzovaného súboru plodov, po odčítaní aneuploidií – 107 (10,40 %, n = 1 029), bolo analýzou karyotypu zachytených 22 štruktúrnych aberácií (2,39 %, n = 922) – deväť nebalansovaných zmien (0,98 %), 10 balansovaných zmien (1,08 %), jeden prípad nejasnej mozaiky (0,09 %), jeden prípad prítomnosti marker chromozómu (0,09 %) a jeden prípad diskordancie pohlavia (0,09 %). U 255 vzoriek s fyziologickým karyotypom indikovaných k cytogenomickému vyšetreniu bolo zachytených celkom osem (7,21 %, n = 111) patologických variant metódou CMA. Metódou MLPA bolo z týchto ôsmich patogénnych variant zachytených päť (3,47 %, n = 144). Celkový záchyt patogénnych variant metódami MLPA a CMA vrátane konfirmačných vyšetrení patologického karyotypu je 14 (5,14 %) a 17 (6,25 %) (n = 272). Záchyt patologických variant v skupine s izolovanými poruchami bol nižší než v skupine s mnohopočetnými poruchami (5,08 vs. 21,42 %). Záver: Potvrdila sa vyššia úspešnosť záchytu patologických variant so zmenou v počte kópií, metódou CMA než MLPA.
Objective: Analysis of prenatal samples from 2015 to 2020. Comparison detection rates of clinically relevant variants by cytogenetic karyotype analysis and cytogenomic MLPA (Multiplex Ligation-Depent Probe Amplification) and microarray methods (CMA – chromosomal microarray). Material and method: 1,029 prenatal samples were analyzed by cytogenetic karyotyping (N = 1,029), cytogenomic methods – MLPA (N = 144) and CMA (N = 111). All unbalanced changes were confirmed by MLPA or CMA. Results: From the analyzed set of fetuses, after subtraction of aneuploidies – 107 (10.40%, N = 1,029), 22 structural aberrations (2.39%, N = 922) – nine unbalanced changes (0.98%), 10 balanced changes (1.08%), one case of unclear mosaicism (0.09%), one case of presence of a marker chromosome (0.09%) and one case of sex discordance (0.09%) – were detected by karyotype analysis. A total of eight (7.21%, N = 111) pathological variants were detected by CMA in 255 samples with physiological karyotype indicated for cytogenomic examination. Five (3.47%, N = 144) of eight pathogenic variants were detected by MLPA method. The total capture of pathogenic variants by MLPA and CMA methods was 14 (5.14%) and 17 (6.25%) (N = 272), including confirmatory pathological karyotype testing. Detection of pathological variants in the isolated disorders group was lower than in the multiple disorders group (5.08 vs. 21.42%). Conclusion: A higher success rate for the detection of pathological copy number variation variants by the microarray method than by the MLPA method was confirmed.
- MeSH
- klinická studie jako téma MeSH
- lidé MeSH
- mikročipová analýza metody MeSH
- mozaicismus MeSH
- multiplexová polymerázová řetězová reakce metody MeSH
- plod MeSH
- prenatální diagnóza * MeSH
- těhotenství MeSH
- variabilita počtu kopií segmentů DNA MeSH
- vrozené vady * diagnóza genetika MeSH
- Check Tag
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
Ig gene (IG) clonality analysis has an important role in the distinction of benign and malignant B-cell lymphoid proliferations and is mostly performed with the conventional EuroClonality/BIOMED-2 multiplex PCR protocol and GeneScan fragment size analysis. Recently, the EuroClonality-NGS Working Group developed a method for next-generation sequencing (NGS)-based IG clonality analysis. Herein, we report the results of an international multicenter biological validation of this novel method compared with the gold standard EuroClonality/BIOMED-2 protocol, based on 209 specimens of reactive and neoplastic lymphoproliferations. NGS-based IG clonality analysis showed a high interlaboratory concordance (99%) and high concordance with conventional clonality analysis (98%) for the molecular conclusion. Detailed analysis of the individual IG heavy chain and kappa light chain targets showed that NGS-based clonality analysis was more often able to detect a clonal rearrangement or yield an interpretable result. NGS-based and conventional clonality analysis detected a clone in 96% and 95% of B-cell neoplasms, respectively, and all but one of the reactive cases were scored polyclonal. We conclude that NGS-based IG clonality analysis performs comparable to conventional clonality analysis. We provide critical parameters for interpretation and discuss a first step toward a quantitative scoring approach for NGS clonality results. Considering the advantages of NGS-based clonality analysis, including its high sensitivity and possibilities for accurate clonal comparison, this supports implementation in diagnostic practice.
- MeSH
- B-buněčný lymfom genetika MeSH
- B-lymfocyty imunologie MeSH
- buněčné klony imunologie MeSH
- fenotyp MeSH
- folikulární lymfom genetika MeSH
- genová přestavba * MeSH
- geny pro imunoglobuliny * MeSH
- imunoglobuliny - kappa-řetězce genetika MeSH
- lidé MeSH
- multiplexová polymerázová řetězová reakce metody MeSH
- senzitivita a specificita MeSH
- správnost dat MeSH
- těžké řetězce imunoglobulinů genetika MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- multicentrická studie MeSH
- práce podpořená grantem MeSH
- validační studie MeSH
Animal or human protothecosis belongs to rather rare, endemic, pro-inflammatory infections. It is caused by achlorophyllous algae of the genus Prototheca. Especially, P. bovis (formerly P. zopfii genotype 2) is often inflected as a non-bacterial causative agent of dairy cattle mastitis. In this study, we present a multiplex real-time PCR (qPCR) system for rapid and exact Prototheca spp. detection and quantification. Limit of detection, diagnostic sensitivity, and specificity were determined. For the first time, specific sequences of AccD (encoding acetyl CoA reductase) for P. bovis, cox1 (encoding cytochrome C oxidase subunit 1) for P. wickerhamii, cytB (encoding cytochrome B) for P. blashkeae and atp6 (encoding transporting ATPase F0 subunit 6) for P. ciferrii (formerly P. zopfii genotype 1) were used for species identification and quantification together with 28S rRNA sequence detecting genus Prototheca. The developed qPCR assay was applied to 55 individual cow milk samples from a herd suspected of protothecosis, 41 bulk milk samples from different Czech farms, 16 boxed milk samples purchased in supermarkets and 21 environmental samples originating from a farm suspected of protothecosis. Our work thus offers the possibility to diagnose protothecosis in the samples, where bacterial mastitis is the most commonly presumed and thereby assisting adequate corrective measures to be taken.
- MeSH
- DNA chemie izolace a purifikace MeSH
- farmy MeSH
- klonování DNA MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- limita detekce MeSH
- mikrobiologie životního prostředí MeSH
- mlékárenství MeSH
- mléko mikrobiologie MeSH
- multiplexová polymerázová řetězová reakce metody MeSH
- plazmidy genetika MeSH
- Prototheca genetika růst a vývoj izolace a purifikace MeSH
- senzitivita a specificita MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
Novorozenecká pneumonie je ve většině případů bakteriálního původu, jiná etiologie je méně častá. Prezentujeme případ novorozence s adnátní pneumonií, která se navzdory agresivnímu ventilačnímu režimu a empirické antibiotické terapii nezlepšovala. Vyšetřením PCR ze vzorku z dýchacích cest byla potvrzena Trichomonas vaginalis. Antibiotická terapie byla rozšířena o metronidazol. Tato cílená antibiotická terapie, která trvala 28 dní, vedla ke klinickému zlepšení a pacient byl propuštěn ve stabilizovaném stavu do domácí péče 44. den života. V případě infekcí, které nereagují na konvenční terapii, je potřeba zvážit přítomnost atypických patogenů. K detekci DNA patogenů je možno použít techniku multiplexní PCR. Výsledkem identifikace patogenu je cílená antibiotická terapie.
Neonatal pneumonia is mostly bacterial and other etiology is considered less frequently. We report a case of newborn whose neonatal pneumonia has not improved, despite the aggressive ventilation regime and empiric antibiotic therapy. A special sample from the respiratory tract was collected for PCR examination. The test confirmed the presence of Trichomonas vaginalis. Antibiotic therapy was extended to include metronidazole. Targeted antibiotic therapy, which lasted for 28 days, improved the condition and the patient was discharged in a stabilized condition to home care on the 44th day of life. We demonstrate the need to consider atypical pathogens in the case of infections that do not respond to conventional therapy. The multiplex real-time PCR technique was used to detect the DNA of the pathogen. Targeted antibiotic therapy is the result of pathogen identification.
- MeSH
- klinické laboratorní techniky MeSH
- lidé MeSH
- metronidazol terapeutické užití MeSH
- multiplexová polymerázová řetězová reakce metody MeSH
- novorozenec MeSH
- pneumonie * diagnóza patologie terapie MeSH
- Trichomonas vaginalis MeSH
- výsledek terapie MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- novorozenec MeSH
- Publikační typ
- kazuistiky MeSH
Oxacillinases (OXA) have been mostly described in Enterobacteriaceae, Acinetobacter, and Pseudomonas species. Recent years have witnessed an increased prevalence of intrinsic and/or acquired β-lactamase-producing Acinetobacter in food-producing animals. This study was conducted to assess the prevalence of OXA among selected bacterial species and to characterize these enzymes by in silico analysis. Screening of OXA was performed by PCR amplification using specific pairs of oligonucleotides. Overall, 40 pairs of primers were designed, of which 6 were experimentally tested in vitro. Among 49 bacterial isolates examined, the presence of blaOXA-1-like genes was confirmed in 20 cases (41%; 19 times in Klebsiella pneumoniae and once in Enterobacter cloacae). No OXA were found in animal isolates. The study results confirmed the specificity of the designed oligonucleotide pairs. Furthermore, the designed primers were found to possess the ability to specifically detect 90.2% of all OXA. These facts suggest that the in silico and in vitro tested primers could be used for single or multiplex PCR to screen for the presence of OXA in various bacteria, as well as to monitor their spread. At the same time, the presence of conserved characteristic amino acids and motifs was confirmed by in silico analysis of sequences of representative members of OXA.
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální proteiny genetika metabolismus MeSH
- beta-laktamasy genetika metabolismus MeSH
- DNA primery chemická syntéza metabolismus MeSH
- Enterobacter cloacae klasifikace účinky léků enzymologie genetika MeSH
- Escherichia coli klasifikace účinky léků enzymologie genetika MeSH
- exprese genu MeSH
- fylogeneze MeSH
- gramnegativní bakteriální infekce diagnóza epidemiologie mikrobiologie veterinární MeSH
- Klebsiella pneumoniae klasifikace účinky léků enzymologie genetika MeSH
- kur domácí mikrobiologie MeSH
- lidé MeSH
- maso mikrobiologie MeSH
- mikrobiální testy citlivosti MeSH
- multiplexová polymerázová řetězová reakce metody MeSH
- peniciliny farmakologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
Multiplex oligonucleotide ligation-PCR (MOL-PCR) is a rapid method for simultaneous detection of multiple molecular markers within a single reaction. MOL-PCR is increasingly employed in microbial detection assays, where its ability to facilitate identification and further characterization via simple analysis is of great benefit and significantly simplifies routine diagnostics. When adapted to microsphere suspension arrays on a MAGPIX reader, MOL-PCR has the potential to outperform standard nucleic acid-based diagnostic assays. This study represents the guideline towards in-house MOL-PCR assay optimization using the example of foodborne pathogens (bacteria and parasites) with an emphasis on the appropriate choice of crucial parameters. The optimized protocol focused on specific sequence detection utilizes the fluorescent reporter BODIPY-TMRX and self-coupled magnetic microspheres and allows for a smooth and brisk workflow which should serve as a guide for the development of MOL-PCR assays intended for pathogen detection.
- MeSH
- infekce yersiniemi diagnóza mikrobiologie MeSH
- lidé MeSH
- multiplexová polymerázová řetězová reakce metody MeSH
- nemoci přenášené potravou diagnóza mikrobiologie parazitologie MeSH
- Toxoplasma genetika izolace a purifikace MeSH
- toxoplazmóza diagnóza parazitologie MeSH
- Yersinia enterocolitica genetika izolace a purifikace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The increase of mixed chimerism (MC) after allogeneic hematopoietic stem cell transplantation has been associated with a high risk of relapse. A variety of techniques that use polymorphic markers have been established to survey hematopoietic chimerism status. The highest sensitivity is achieved using real-time quantitative polymerase chain reaction (RQ-PCR) analysis of insertion/deletion polymorphism, which allows the detection of disease recurrence and subsequently the earlier initiation of therapeutic intervention. The purpose of this study is the evaluation of multiplex RQ-PCR for MC assessment (six biallelic genetic systems and Y-specific locus), allowing the amplification and detection of target gene of interest and glyceraldehyde-3-phosphate dehydrogenase reference housekeeping gene in a single microtube. With optimized amounts of primers and probe, the quantification of target DNA was shown to be linear throughout the tested range (100%-0.05%). The efficiencies of multiplex RQ-PCR were in a range of 0.89 to 1.07. The sensitivity of individual systems ranged 0.02% to 0.04% with an average of 0.034%. A high degree of linear correlation between the chimerism results obtained by multiplex RQ-PCR vs singleplex RQ-PCR was observed (P < 0.0001, Spearman's coefficient = 0.9927), while correlation between multiplex RQ-PCR vs short tandem repeat analysis was also statistically significant (P < 0.0001, Spearman's coefficient = 0.9769). This new multiplex RQ-PCR assay is a quick, sensitive, reproducible, and cost-effective method for accurate MC assessment.
- MeSH
- alely MeSH
- chimérismus * MeSH
- DNA primery MeSH
- leukemie terapie MeSH
- lidé MeSH
- lokální recidiva nádoru diagnóza etiologie prevence a kontrola MeSH
- multiplexová polymerázová řetězová reakce metody MeSH
- polymerázová řetězová reakce s reverzní transkripcí metody MeSH
- polymorfismus genetický * MeSH
- transplantace hematopoetických kmenových buněk škodlivé účinky MeSH
- transplantační chiméra krev genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Problems with serological cross-reactivity have led to development of a number of PCRs (individual and multiplex) for molecular typing of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia. Most of these assays were developed for detection of specific amplicons within capsule biosynthetic genes before the availability of complete sequences for the different serovars. Here we describe comparative analysis of the complete capsular loci for all 18 serovars of A. pleuropneumoniae, and development of two multiplex PCRs for comprehensive capsule typing of this important pig pathogen.
- MeSH
- Actinobacillus pleuropneumoniae klasifikace genetika patogenita MeSH
- bakteriální polysacharidy genetika MeSH
- bakteriální pouzdra chemie klasifikace genetika MeSH
- infekce bakteriemi rodu Actinobacillus diagnóza mikrobiologie MeSH
- multiplexová polymerázová řetězová reakce metody MeSH
- nemoci prasat diagnóza mikrobiologie MeSH
- prasata MeSH
- sekvenční analýza * MeSH
- séroskupina MeSH
- sérotypizace MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
