Early detection of biohazardous bacteria that can be misused as biological weapons is one of the most important measures to prevent the spread and outbreak of biological warfare. For this reason, many instrument platforms need to be introduced into operation in the field of biological warfare detection. Therefore the purpose of this study is to establish a new detection panel for biothreat bacteria (Bacillus anthracis, Yersinia pestis, Francisella tularensis, and Brucella spp.) and confirm it by collaborative validation by using a multiplex oligonucleotide ligation followed by polymerase chain reaction and hybridization to microspheres by MagPix detection platform (MOL-PCR). Appropriate specific sequences in bacterial DNA were selected and tested to assemble the detection panel, and MOLigo probes (short specific oligonucleotides) were designed to show no cross-reactivity when tested between bacteria and to decrease the background signal measurement on the MagPix platform. During testing, sensitivity was assessed for all target bacteria using serially diluted DNA and was determined to be at least 0.5 ng/µL. For use as a diagnostic kit and easier handling, the storage stability of ligation premixes (MOLigo probe mixes) was tested. This highly multiplex method can be used for rapid screening to prevent outbreaks arising from the use of bacterial strains for bioterrorism, because time of analysis take under 4 h.
- Publikační typ
- časopisecké články 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
Toxoplasma gondii is an important ubiquitous protozoan parasite, which can infect almost all warm-blooded vertebrates, including humans. The diagnosis of T. gondii infection is crucial for the prevention, surveillance, and control of its transmission. Here, a triplex real-time PCR assay targeting the B1 gene and 529rep element was used to determine the presence of T. gondii in feathered game (Anas platyrhynchos and Phasianus colchicus) hunted in the Czech Republic. The prevalence of T. gondii was 5.4% in wild ducks (n = 280) and 3.4% in common pheasants (n = 350). Additionally, genotyping of 28 T. gondii-positive samples revealed the presence of archetypal genotypes II and III as well as non-archetypal genotypes combining both type II and III alleles. Our results suggest that consumption of feathered game could pose a risk of T. gondii transmission to humans in the Czech Republic.
- MeSH
- Galliformes parazitologie MeSH
- genotyp MeSH
- kachny parazitologie MeSH
- lidé MeSH
- nemoci ptáků parazitologie MeSH
- protozoální DNA genetika MeSH
- Toxoplasma genetika izolace a purifikace MeSH
- toxoplazmóza zvířat epidemiologie přenos MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
High resolution melting analysis (HRMA) is a single-tube method, which can be carried out rapidly as an additional step following real-time quantitative PCR (qPCR). The method enables the differentiation of genetic variation (down to single nucleotide polymorphisms) in amplified DNA fragments without sequencing. HRMA has previously been adopted to determine variability in the amplified genes of a number of organisms. However, only one work to date has focused on pathogenic parasites-nematodes from the genus Trichinella. In this study, we employed a qPCR-HRMA assay specifically targeting two sequential gene fragments-cytochrome c oxidase subunit I (COI) and expansion segment V (ESV), in order to differentiate 37 single L1 muscle larvae samples of eight Trichinella species. We show that qPCR-HRMA based on the mitochondrial COI gene allows differentiation between the sequences of PCR products of the same length. This simple, rapid and reliable method can be used to identify at the species level single larvae of eight Trichinella taxa.
- MeSH
- denaturace nukleových kyselin MeSH
- genotypizační techniky metody normy MeSH
- polymorfismus genetický MeSH
- respirační komplex IV genetika MeSH
- taxonomické DNA čárové kódování metody normy MeSH
- Trichinella klasifikace genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Toxoplasmosis is a major public health issue, due to the presence of Toxoplasma gondii, mainly in pork. The aim of this study was to determine the occurrence of T. gondii in pigs and wild boars bred in different production systems in the Czech Republic using ELISA and qPCR methods. Our results show that T. gondii infection is widespread in pigs and wild boars bred and slaughtered in the Czech Republic and that there is a higher exposure to T. gondii in backyard slaughter operations and organic pig farming, indicating a potential risk for meat consumption. Additionally, genotyping of amplified loci for Type II suggests the presence of one clonal genotype circulating in these animals.
- MeSH
- bezpečnost potravin MeSH
- červené maso parazitologie MeSH
- ELISA MeSH
- genotyp MeSH
- nemoci prasat epidemiologie MeSH
- prasata MeSH
- protilátky protozoální MeSH
- séroepidemiologické studie MeSH
- Sus scrofa parazitologie MeSH
- Toxoplasma klasifikace genetika MeSH
- toxoplazmóza zvířat epidemiologie parazitologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika epidemiologie MeSH
