An electrochemical genosensor for the detection and quantification of Karlodinium armiger is presented. The genosensor exploits tailed primers and ferrocene labelled dATP analogue to produce PCR products that can be directly hybridised on a gold electrode array and quantitatively measured using square wave voltammetry. Tailed primers consist of a sequence specific for the target, followed by a carbon spacer and a sequence specifically designed not to bind to genomic DNA, resulting in a duplex flanked by single stranded binding primers. The incorporation of the 7-(ferrocenylethynyl)-7-deaza-2'-deoxyadenosine triphosphate was optimised in terms of a compromise between maximum PCR efficiency and the limit of detection and sensitivity attainable using electrochemical detection via hybridisation of the tailed, ferrocene labelled PCR product. A limit of detection of 277aM with a linear range from 315aM to 10 fM starting DNA concentration and a sensitivity of 122 nA decade-1 was achieved. The system was successfully applied to the detection of genomic DNA in real seawater samples.

• Klíčová slova
• Ferrocene, Karlodinium armiger, PCR, Redox labelled nucleotides, Square wave voltammetry, Tailed primers,
• MeSH
• biosenzitivní techniky přístrojové vybavení   MeSH
• deoxyadeninnukleotidy chemie   MeSH
• design vybavení MeSH
• DNA analýza   MeSH
• elektrochemické techniky přístrojové vybavení   MeSH
• limita detekce MeSH
• metaloceny chemie   MeSH
• mikroelektrody MeSH
• mořská voda analýza   MeSH
• oxidace-redukce MeSH
• polymerázová řetězová reakce přístrojové vybavení   MeSH
• železnaté sloučeniny chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 2'-deoxyadenosine triphosphate MeSH Prohlížeč
• deoxyadeninnukleotidy MeSH
• DNA MeSH
• ferrocene MeSH Prohlížeč
• metaloceny MeSH
• železnaté sloučeniny MeSH

Viruses are common causes of food- and waterborne diseases worldwide. Conventional identification of these agents is based on cultivation, antigen detection, electron microscopy, or real-time PCR. Because recent technological advancements in detection methods are focused on fast and robust analysis, a rapid multiplexing technology, which can detect a broad spectrum of pathogenic viruses connected to food or water contamination, was utilized. A new semiquantitative magnetic bead-based multiplex system has been designed for simultaneous detection of several targets in one reaction. The system includes adenoviruses 40/41 (AdV), rotavirus A (RVA), norovirus (NoV), hepatitis E virus (HEV), hepatitis A virus (HAV), and a target for external control of the system. To evaluate the detection system, interlaboratory ring tests were performed in four independent laboratories. Analytical specificity of the tool was tested on a cohort of pathogenic agents and biological samples with quantitative PCR as a reference method. Limit of detection (analytical sensitivity) of 5 × 100 (AdV, HEV, and RVA) and 5 × 101 (HAV and NoV) genome equivalents per reaction was reached. This robust, senstivie, and rapid multiplexing technology may be used to routinely monitor and manage viruses in food and water to prevent food and waterborne diseases.

• MeSH
• lidé MeSH
• limita detekce MeSH
• mikrobiologie vody * MeSH
• polymerázová řetězová reakce metody   MeSH
• potravinářská mikrobiologie * MeSH
• viry izolace a purifikace   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

PCR detection of fungal pathogens in clinical samples has been discussed in journals for more than two decades. However, its use for diagnosing invasive aspergillosis is still controversial, despite the fact that molecular methods are routinely used in various fields of modern microbiology. These are e. g. genotyping of bacterial strains resistant to antibiotics, molecular epidemiology or routine detection of viral infections in clinical material. PCR methods have made the diagnostic applications faster, simpler and more accurate. This review deals with issues related to molecular methods for diagnosing invasive fungal infections and the main factors limiting their use in everyday clinical practice.

• MeSH
• Aspergillus izolace a purifikace   MeSH
• aspergilóza diagnóza   MeSH
• DNA fungální analýza   MeSH
• lidé MeSH
• polymerázová řetězová reakce * MeSH
• senzitivita a specificita MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• anglický abstrakt MeSH
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• DNA fungální MeSH

We developed a PCR assay for the detection of Babesia odocoilei based on the 18S rRNA gene. Multiple specimens of B. odocoilei were examined, and the assay consistently produced a small specific PCR product of 306 bp. The PCR assay was also challenged with DNA from 13 other Babesia species and 2 Theileria species, originating from 10 different host species; however, nonspecific DNA amplification and multiple banding patterns were observed, and the amplicon banding patterns varied between different isolates of the same species. Sensitivity was determined to be 6.4 pg of DNA, and an estimated 0.0001% parasitism. This assay can be utilized for species-specific differential detection of B. odocoilei.

• Klíčová slova
• Babesia odocoilei, PCR, cervids, specificity,
• MeSH
• Babesia * genetika   MeSH
• babezióza * diagnóza   MeSH
• polymerázová řetězová reakce veterinární   MeSH
• protozoální DNA genetika   MeSH
• RNA ribozomální 18S genetika   MeSH
• Theileria * genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• protozoální DNA MeSH
• RNA ribozomální 18S MeSH

Increasing antimicrobial resistance of nosocomial pathogens is becoming a serious threat to public health. To control the spread of this resistance, it is necessary to detect β-lactamase-producing organisms in the clinical setting. The aims of the study were to design a PCR assay for rapid detection of clinically encountered β-lactamase genes described in Enterobacteriaceae and Gram-negative non-fermenting bacteria. The functionality of proposed primers was verified using eight reference strains and 17 strains from our collection, which contained 29 different β-lactamase genes. PCR products of the test strains were confirmed by Sanger sequencing. Sequence analysis was performed using bioinformatics software Geneious. Overall, 67 pairs of primers for detecting 12 members of the class C β-lactamase family, 15 members of class A β-lactamases, six gene families of subclass B1, one member each of subclasses B2, B3 and class D β-lactamases were designed, of which 43 pairs were experimentally tested in vitro. All 29 β-lactamase genes, including 10 oxacillinase subgroups, were correctly identified by PCR. The proposed set of primers should be able to specifically detect 99.7% of analyzed β-lactamase subtypes and more than 79.8% of all described β-lactamase genes.

• Klíčová slova
• β-lactamase, PCR, antibiotic resistance, bacteria, primer,
• MeSH
• Bacteria enzymologie   genetika   izolace a purifikace   MeSH
• bakteriologické techniky * MeSH
• beta-laktamasy genetika   metabolismus   MeSH
• beta-laktamová rezistence genetika   MeSH
• DNA bakterií genetika   MeSH
• DNA primery MeSH
• lidé MeSH
• polymerázová řetězová reakce * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• beta-laktamasy MeSH
• DNA bakterií MeSH
• DNA primery MeSH

The majority of carcinomas that were developed due to the infection with human papillomavirus (HPV) are caused by high-risk HPV types, HPV16 and HPV18. These HPV types contain the E6 and E7 oncogenes, so the fast detection of these oncogenes is an important point to avoid the development of cancer. Many different HPV tests are available to detect the presence of HPV in biological samples. The aim of this study was to design a fast and low cost method for HPV identification employing magnetic isolation, polymerase chain reaction (PCR) and electrochemical detection. These assays were developed to detect the interactions between E6-HPV16 oncogene and magnetizable particles (MPs) using commercial Dynabeads M-280 Streptavidin particles and laboratory-synthesized "homemade" particles called MANs (MAN-37, MAN-127 and MAN-164). The yields of PCR amplification of E6-HPV16 oncogene bound on the particles and after the elution from the particles were compared. A highest yield of E6-HPV16 DNA isolation was obtained with both MPs particles commercial M-280 Streptavidin and MAN-37 due to reducing of the interferents compared with the standard PCR method. A biosensor employing the isolation of E6-HPV16 oncogene with MPs particles followed by its electrochemical detection can be a very effective technique for HPV identification, providing simple, sensitive and cost-effective analysis.

• Klíčová slova
• PCR, electrochemistry, human papillomavirus, magnetic isolation, magnetizable particles, nucleic acid detection,
• MeSH
• diagnostické techniky molekulární metody   MeSH
• lidský papilomavirus 16 chemie   genetika   izolace a purifikace   MeSH
• magnetické nanočástice chemie   MeSH
• onkogenní proteiny virové chemie   genetika   MeSH
• polymerázová řetězová reakce metody   MeSH
• represorové proteiny chemie   genetika   MeSH
• streptavidin chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• E6 protein, Human papillomavirus type 16 MeSH Prohlížeč
• magnetické nanočástice MeSH
• onkogenní proteiny virové MeSH
• represorové proteiny MeSH
• streptavidin 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

PURPOSE OF THE STUDY: The study was intended to summarize and evaluate the results in patients with a suspected infection by the genus Chlamydia, investigated with an in-house method of nested PCR (polymerase chain reaction). The study worked with data from patients living in eastern Bohemia, who were examined in the years 2001-2003 at the Dept. of Molecular Biology, a research laboratory shared by the Institute of Clinical Microbiology and the Institute of Clinical Biochemistry and Diagnostics. MATERIAL AND METHODS: 291 explorations were done in 2001, in 2002 already 562 and in 2003 their figure reached 760. The total number of samples received for investigation during that period was 1 613. 1 587 were actually investigated, 26 were unsuitable and could not be used. More than 70 % of all investigations were done with three types of material: urine (41.8 % of all the investigated samples), BAL (15.3 % of all the investigated samples) and whole blood (14.9 % of all the investigated samples). The investigations were carried out with the in-house nested PCR method, which uses primers from the MOMP(ompA) area of the genus Chlamydia spp. RESULTS: Total positivity was 5.67 %, in 1.26 % of the samples the resulted was considered uncertain and 93.07 % of the investigated samples were negative. In men PCR positivity was 6.11 %, in women 5.35 %. The major proportion of positive samples was from the age groups 70-79 years (11.67 %), 10-19 years (6.51 %) and 40-49 years (6.45 %). Overall positivity in smears from the urogenital system was 6.48 %, from urine 3.92 %, from BAL 10.70 % and from whole blood 5.51 %. KEYWORDS: nested PCR, Chlamydia spp., detection.

• MeSH
• Chlamydia trachomatis * MeSH
• chlamydiové infekce * MeSH
• lidé MeSH
• polymerázová řetězová reakce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Syphilis is a multistage sexually transmitted disease caused by Treponema pallidum ssp. pallidum (TPA). This study analyzed clinical samples collected from patients with a diagnosed syphilis infection from 2004-2022, isolated in the Czech Republic. Mucocutaneous swab samples (n = 543) from 543 patients were analyzed, and from these samples, 80.11 % (n = 435) were PCR positive, and 19.89 % (n = 108) were PCR negative for TPA DNA. Swabs were more often positive when collected from syphilis patients in the primary and secondary stages, compared to the latent or unknown stage. There was no significant difference in PCR positivity between the primary and secondary stages (p = 0.099). In IgM-positive patients, a statistically significant association with PCR-positivity was found in samples from seropositive (p = 0.033) and serodiscrepant (RPR negative) patients (p = 0.0006). When assessing our laboratory-defined cases of syphilis, the RPR, IgM, and PCR tests were similarly effective (within the range of 80.1-86.1 %). However, parallel testing with these methods was even more effective, i.e., RPR + PCR was 96.1 % effective and RPR + IgM + PCR was 97.8 % effective. A combination of RPR + PCR, or a combination of all three tests (RPR, IgM, and PCR) can therefore be used to reliably detect active syphilis cases, including reinfections. Our findings show that the reverse algorithm for detecting syphilis could be substantially improved by adding IgM and PCR testing.

• Klíčová slova
• Nontreponemal tests, PCR syphilis detection, RPR, Syphilis, Syphilis diagnostics, Syphilis serology, Treponemal tests,
• MeSH
• DNA bakterií genetika   MeSH
• dospělí MeSH
• imunoglobulin M krev   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• polymerázová řetězová reakce * metody   MeSH
• protilátky bakteriální krev   MeSH
• senzitivita a specificita MeSH
• syfilis * diagnóza   mikrobiologie   epidemiologie   MeSH
• Treponema pallidum * izolace a purifikace   genetika   imunologie   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika epidemiologie   MeSH
• Názvy látek
• DNA bakterií MeSH
• imunoglobulin M MeSH
• protilátky bakteriální MeSH

The study aim was to implement a molecular real-time polymerase chain reaction (PCR) assay recommended by the CDC (Centers for Disease Control and Prevention) for the detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in clinical (culture negative) specimens from patients with suspected invasive bacterial disease. Clinical specimens are referred to the National Reference Laboratory (NRL) for Meningococcal Infections, Unit for Airborne Bacterial Infections, Centre for Epidemiology and Microbiology, National Institute of Public Health from various regions of the Czech Republic. Clinical specimens are, in particular, cerebrospinal fluid, anti-coagulated blood or serum and, exceptionally, post-mortem specimens. The NRL has implemented molecular diagnosis of these bacterial pathogens involved in meningitis and sepsis from clinical specimens since 1999. The first diagnostic method was semi-nested PCR followed by electrophoretic analysis. In 2014, a molecular qualitative real-time PCR assay was implemented.

• Klíčová slova
• Haemophilus influenzae, and Streptococcus pneumoniae - rt-PCR -detection of the agents of bacterial meningitis and sepsis., rt-PCR detection of Neisseria meningitidis,
• MeSH
• dospělí MeSH
• Haemophilus influenzae klasifikace   genetika   izolace a purifikace   MeSH
• hemofilové infekce diagnóza   mikrobiologie   MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• lidé středního věku MeSH
• lidé MeSH
• meningitida bakteriální diagnóza   mikrobiologie   MeSH
• Neisseria meningitidis klasifikace   genetika   izolace a purifikace   MeSH
• pneumokokové infekce diagnóza   mikrobiologie   MeSH
• senzitivita a specificita MeSH
• Streptococcus pneumoniae klasifikace   genetika   izolace a purifikace   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• Geografické názvy
• Česká republika MeSH
• Spojené státy americké MeSH