multiplex PCR Dotaz Zobrazit nápovědu
This work was aimed to develop a multiplex PCR assay to detect infectious agents such as Clavibacter michiganensis subsp. michiganensis, Fusarium sp, Leveillula taurica, and begomoviruses in tomato (Solanum lycopersicum) plants. Specific primer sets of each pathogen were designed based on intergenic ribosomal RNA sequences for the first three, whereas for begomoviruses, primers were designed based on conserved regions. The design also considered that the length (200-800 bp) of the PCR products was resolvable by electrophoresis; thus 296, 380, 457, and 731 bp fragments for Clavibacter, Fusarium, Leveillula, and begomoviruses, respectively, were considered. PCR conditions were optimized to amplify all the products in a single tube from genomic DNA and circumvent PCR inhibitors from infected plants. Finally, when the multiplex PCR assay was tested with tomato plants infected with any of the four pathogens, specific PCR products confirmed the presence of the pathogens. Optimized PCR multiplex allowed for the accurate and simultaneous detection of Clavibacter, Fusarium, Leveillula, and begomoviruses in infected plants or seeds from tomato.
- MeSH
- Actinomycetales genetika izolace a purifikace MeSH
- Ascomycota genetika izolace a purifikace MeSH
- Begomovirus genetika izolace a purifikace MeSH
- DNA primery genetika MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- Fusarium genetika izolace a purifikace MeSH
- multiplexová polymerázová řetězová reakce metody MeSH
- nemoci rostlin mikrobiologie virologie MeSH
- Solanum lycopersicum mikrobiologie virologie MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- Názvy látek
- DNA primery MeSH
The application of rapid, specific, and sensitive methods for pathogen detection and quantification is very advantageous in diagnosis of human pathogens in several applications, including food analysis. The aim of this study was the evaluation of a method for the multiplexed detection and quantification of three significant foodborne pathogenic species (Escherichia coli O157, Salmonella spp., and Listeria monocytogenes). The assay combines specific DNA extraction by multiplex magnetic capture hybridization (mMCH) with multiplex real-time PCR. The amplification assay showed linearity in the range 106-10 genomic units (GU)/PCR for each co-amplified species. The sensitivity corresponded to 1 GU/PCR for E. coli O157 and L. monocytogenes, and 10 GU/PCR for Salmonella spp. The immobilization process and the hybrid capture of the MCH showed good efficiency and reproducibility for all targets, allowing the combination in equal amounts of the different nanoparticle types in mMCH. MCH and mMCH efficiencies were similar. The detection limit of the method was 10 CFU in samples with individual pathogens and 102 CFU in samples with combination of the three pathogens in unequal amounts (amount's differences of 2 or 3 log). In conclusion, this multiplex molecular platform can be applied to determine the presence of target species in food samples after culture enrichment. In this way, this method could be a time-saving and sensitive tool to be used in routine diagnosis.
- Klíčová slova
- Escherichia coli O157, Listeria monocytogenes, Multiplex magnetic capture hybridization, Multiplex real-time PCR, Quantitative detection, Salmonella spp.,
- MeSH
- Escherichia coli O157 klasifikace genetika MeSH
- hybridizace nukleových kyselin MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé MeSH
- Listeria monocytogenes klasifikace genetika MeSH
- multiplexová polymerázová řetězová reakce MeSH
- Salmonella klasifikace genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: The diagnosis of gastrointestinal nematode (GIN) infections in ruminants is routinely based on morphological/morphometric analysis of parasite specimens recovered by coprological methods, followed by larval culture (LC) techniques. Such an approach is laborious, time-consuming, requires a skilled expert, and moreover suffers from certain limitations. Molecular tools are able to overcome the majority of these issues, providing accurate identification of nematode species and, therefore, may be valuable in sustainable parasite control strategies. METHODS: Two multiplex real-time polymerase chain reaction (PCR) assays for specific detection of five main and one invasive GIN species, including an internal amplification control to avoid false-negative results, were designed targeting SSU rRNA and COI genetic markers, as well as established ITS1/2 sequences. The assays were optimized for analysis of DNA extracted directly from sheep faeces and verified for Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus colubriformis, Nematodirus battus, Chabertia ovina, and Ashworthius sidemi. Semi-quantitative evaluation of infection intensity was enabled using a plasmid construct and a dilution series of sheep faeces with a known number of nematode eggs. Assays were tested on 44 individually collected faecal samples from three farms, and results were compared to those from faecal egg counts (FEC) using the concentration McMaster technique and LC. RESULTS: Multiplex real-time PCR assays showed great specificity to target nematodes. During the analysis of faecal samples, the assays proved to have higher sensitivity in strongylid-type egg detection over FEC by revealing three false-negative samples, while showing moderate agreement in evaluation of infection intensity. The multiplex assays further clarified GIN species identification compared to LC, which had confused determination of Teladorsagia spp. for Trichostrongylus spp. CONCLUSIONS: Our multiplex assays proved to be a rapid and accurate approach enabling simultaneous and reliable GIN species identification from faeces and semi-quantitative estimation of the number of eggs present. This approach increases diagnostic value and may add a high degree of precision to evaluation of anthelmintic efficacy, where it is important to identify species surviving after treatment.
- Klíčová slova
- Cell-free DNA, Gastrointestinal nematode, Multiplex detection, Real-time PCR, Sheep,
- MeSH
- feces parazitologie MeSH
- gastrointestinální nemoci diagnóza parazitologie veterinární MeSH
- gastrointestinální trakt parazitologie MeSH
- hlístice klasifikace genetika MeSH
- multiplexová polymerázová řetězová reakce metody MeSH
- nematodózy diagnóza parazitologie veterinární MeSH
- nemoci ovcí diagnóza parazitologie MeSH
- ovce MeSH
- počet parazitárních vajíček MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Group B Streptococcus (GBS) causes severe infections in infants and in immunocompromised adults. GBS pathogenicity varies between and within serotypes, with considerable variation in genetic content between strains. For this reason, it is important to be able to carry out immediate and comprehensive diagnostics of these infections. Seven genes important for screening of GBS infection were detected: cfb gene encoding the CAMP factor presented in every GBS; the cps operon genes such as cps1aH, cps1a/2/3IJ, and cps5O specific for capsular polysaccharide types Ia, III, and V, respectively; macrolide resistance genes ermB and mefA/E; and the gbs2018 S10 region specific for ST17 hypervirulent clone. Standardization of multiplex PCR with the use of seven primer pairs was performed on 81 bacterial strains representing different GBS isolates (n = 75) and other Gram-positive cocci (n = 10). Multiplex PCR can be used as an effective screening method to detect different sequences important for the screening of GBS infection.
- MeSH
- bakteriální geny MeSH
- bakteriologické techniky metody normy MeSH
- DNA bakterií genetika MeSH
- DNA primery genetika MeSH
- dospělí MeSH
- kojenec MeSH
- lidé MeSH
- multiplexová polymerázová řetězová reakce metody normy MeSH
- plošný screening metody normy MeSH
- Streptococcus agalactiae klasifikace genetika izolace a purifikace MeSH
- Check Tag
- dospělí MeSH
- kojenec MeSH
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA bakterií MeSH
- DNA primery MeSH
Since its invention in 1985 the polymerase chain reaction (PCR) has become a well-established method for amplification and detection of segments of double-stranded DNA. Incorporation of fluorogenic probe or DNA intercalating dyes (such as SYBR Green) into the PCR mixture allowed real-time reaction monitoring and extraction of quantitative information (qPCR). Probes with different excitation spectra enable multiplex qPCR of several DNA segments using multi-channel optical detection systems. Here we show multiplex qPCR using an economical EvaGreen-based system with single optical channel detection. Previously reported non quantitative multiplex real-time PCR techniques based on intercalating dyes were conducted once the PCR is completed by performing melting curve analysis (MCA). The technique presented in this paper is both qualitative and quantitative as it provides information about the presence of multiple DNA strands as well as the number of starting copies in the tested sample. Besides important internal control, multiplex qPCR also allows detecting concentrations of more than one DNA strand within the same sample. Detection of the avian influenza virus H7N9 by PCR is a well established method. Multiplex qPCR greatly enhances its specificity as it is capable of distinguishing both haemagglutinin (HA) and neuraminidase (NA) genes as well as their ratio.
- MeSH
- fluorescenční barviva * MeSH
- kvantitativní polymerázová řetězová reakce * MeSH
- multiplexová polymerázová řetězová reakce přístrojové vybavení metody MeSH
- ptačí chřipka u ptáků diagnóza virologie MeSH
- ptáci MeSH
- virus chřipky A klasifikace genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- fluorescenční barviva * MeSH
Alternative pre-mRNA splicing increases transcriptome plasticity by forming naturally-occurring alternative splicing variants (ASVs). Alterations of splicing processes, caused by DNA mutations, result in aberrant splicing and the formation of aberrant mRNA isoforms. Analyses of hereditary cancer predisposition genes reveal many DNA variants with unknown clinical significance (VUS) that potentially affect pre-mRNA splicing. Therefore, a comprehensive description of ASVs is an essential prerequisite for the interpretation of germline VUS in high-risk individuals. To identify ASVs in a gene of interest, we have proposed an approach based on multiplex PCR (mPCR) amplification of all theoretically possible exon-exon junctions and subsequent characterization of size-selected and pooled mPCR products by next-generation sequencing (NGS). The efficiency of this method is illustrated by a comprehensive analysis of BRCA1 ASVs in human leukocytes, normal mammary, and adipose tissues and stable cell lines. We revealed 94 BRCA1 ASVs, including 29 variants present in all tested samples. While differences in the qualitative expression of BRCA1 ASVs among the analyzed human tissues were minor, larger differences were detected between tissue and cell line samples. Compared with other ASV analysis methods, this approach represents a highly sensitive and rapid alternative for the identification of ASVs in any gene of interest.
- Klíčová slova
- Alternative splicing, BRCA1, Multiplex PCR, NGS, mRNA splicing variant,
- MeSH
- alternativní sestřih * MeSH
- izoformy RNA MeSH
- lidé MeSH
- multiplexová polymerázová řetězová reakce metody MeSH
- mutace * MeSH
- nádory prsu genetika MeSH
- protein BRCA1 genetika MeSH
- výpočetní biologie MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- BRCA1 protein, human MeSH Prohlížeč
- izoformy RNA MeSH
- protein BRCA1 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
OBJECTIVE: Rapid detection of most frequent aneuploidies by the multiplex QF PCR method in non-cultured samples of chorial tissue. Summarized results of QF PCR method applied in the management of care of pregnant women in the first trimester of pregnancy. TYPE OF STUDY: An original contribution. SETTING: Institute of Medical Genetics and Fetal Medicine, Faculty Hospital and Medical Faculty, Palacky University Olomouc. METHODS: The samples of chorial tissue were obtained from 101 pregnant women. Non-cultured samples were processed by the multiplex QF PCR method. STR loci of chromosomes 13, 18, 21 and X and Y were analyzed. These markers were amplified in two separate multiplex PCR reactions under the same conditions and subjected to fragmentation analysis in capillary electrophoresis. RESULTS: All 101 analyzed samples of chorial tissue were successfully amplified. In this group, 16 pathologies of the fetuses were detected by the multiplex QF PCR method. Triploidy was detected in two cases, trisomy of chromosome 21--Down syndrome was found in seven cases, and trisomy of chromosome 18--Edwards syndrome was found in six cases and monosomy of gonosome X--the Turner' s syndrome was revealed once. CONCLUSIONS: The multiplex QF PCR method is an indispensable part of the screening of the first trimester and provides a rapidly available and reliable result in the examined patients.
- MeSH
- aneuploidie * MeSH
- chromozomální poruchy diagnóza MeSH
- lidé MeSH
- odběr choriových klků * MeSH
- polymerázová řetězová reakce * MeSH
- první trimestr těhotenství * MeSH
- tandemové repetitivní sekvence MeSH
- těhotenství MeSH
- Check Tag
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- anglický abstrakt MeSH
- časopisecké články MeSH
DNA profiling--inclusive sex determination-- with microsatellite markers is currently a commonly used genetic method of studying humans. An efficient technique of producing the genetic data is amplification of multiple microsatellites in a single PCR reaction. Here we introduce a novel PCR-multiplex system for analysis of four polymorphic Y-STRs. Specifically, these are DYS449, DYS456, DYS458, and DYS464. These loci were chosen because of their reported high diversity in Euroamerican population (10), as well as their absence in the commercial analytical kits at the time of beginning of this study. Our objective was to design this PCR-multiplex for use of fragmentation analysis by electrophoresing samples on a capillary semi-automated genetic analyzer applying only one fluorescent dye. The PCR system we propose, may be notably used in fields such as forensic and human population genetics.
BACKGROUND: A one-step polymerase chain reaction (PCR) method for the simultaneous detection of the major allergens of pecan and Brazil nuts was developed. Primer pairs for the amplification of partial sequences of genes encoding the allergens were designed and tested for their specificity on a range of food components. RESULTS: The targeted amplicon size was 173 bp of Ber e 1 gene of Brazil nuts and 72 bp of vicilin-like seed storage protein gene in pecan nuts. The primer pair detecting the noncoding region of the chloroplast DNA was used as the internal control of amplification. The intrinsic detection limit of the PCR method was 100 pg mL(-1) pecan or Brazil nuts DNA. The practical detection limit was 0.1% w/w (1 g kg(-1)). The method was applied for the investigation of 63 samples with the declaration of pecans, Brazil nuts, other different nut species or nuts generally. In 15 food samples pecans and Brazil nuts allergens were identified in the conformity with the food declaration. CONCLUSION: The presented multiplex PCR method is specific enough and can be used as a fast approach for the detection of major allergens of pecan or Brazil nuts in food.
- MeSH
- 2S albuminy rostlinné škodlivé účinky analýza genetika MeSH
- alergeny analýza genetika MeSH
- alergie na arašídy prevence a kontrola MeSH
- alergie na ořechy prevence a kontrola MeSH
- analýza potravin metody MeSH
- antigeny rostlinné škodlivé účinky analýza genetika MeSH
- Bertholletia škodlivé účinky chemie MeSH
- Carya škodlivé účinky chemie MeSH
- DNA rostlinná metabolismus MeSH
- kontrola potravin metody MeSH
- limita detekce MeSH
- multiplexová polymerázová řetězová reakce MeSH
- ořechy škodlivé účinky chemie MeSH
- zásobní proteiny semen škodlivé účinky analýza genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 2S albumin, brazil nut MeSH Prohlížeč
- 2S albuminy rostlinné MeSH
- alergeny MeSH
- antigeny rostlinné MeSH
- DNA rostlinná MeSH
- vicilin-like protein, plant MeSH Prohlížeč
- zásobní proteiny semen MeSH
