Psittacine beak and feather disease (PBFD) is one of the most significant viral diseases in psittacine birds. The aim of the presented study was to develop a highly specific and sensitive TaqMan real-time PCR assay for universal detection of beak and feather disease virus (BFDV). Primers and a hydrolysis probe were selected on the highly conserved regions belonging to the ORF1 of the BFDV genome which were identified by aligning 814 genomic sequences downloaded from the GenBank database. The evaluation of the reaction parameters suggested a reaction efficiency of 97.1%, with consistent detection of 101 virus copies/μl of nucleic acid extract. The low values of standard deviation and coefficient of variation indicate a high degree of reproducibility and repeatability. The diagnostic applicability of the assay was proven on 36 BFDV positive and 107 negative specimens of psittacine origin representing 28 species. The assay showed a 100% ability to detect distinct genetic variants of the virus. Our data suggest that the presented TaqMan real-time PCR represents a specific, sensitive and reliable assay facilitating the molecular detection of BFDV.

• MeSH
• Circovirus genetika   izolace a purifikace   MeSH
• diagnostické techniky molekulární metody   MeSH
• DNA primery genetika   MeSH
• infekce viry čeledi Circoviridae diagnóza   veterinární   virologie   MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• nemoci ptáků diagnóza   virologie   MeSH
• oligonukleotidové sondy genetika   MeSH
• ptáci MeSH
• reprodukovatelnost výsledků MeSH
• senzitivita a specificita MeSH
• veterinární lékařství metody   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH

We applied quantitative TaqMan minor groove binder real-time polymerase chain reaction (PCR) on DNA isolates from soft abdominal cuticle of 460 North American crayfish Orconectes limosus and Pacifastacus leniusculus, previously tested for Aphanomyces astaci presence by conventional semi-nested PCR. Both approaches target the internal transcribed spacers of the pathogen nuclear ribosomal DNA, but apply different specific sequence motifs and technologies. The real-time PCR approach seems to provide higher sensitivity; the number of crayfish that tested positive increased from 23 to 32%, and 10 additional crayfish populations were indicated as hosting the disease agent. However, the vast majority of newly recorded positives contained very low agent levels, from 5 to 50 PCR-forming units. An isolate producing a false positive result by the semi-nested PCR (apparently undescribed Aphanomyces related to A. astaci) remained negative using the real-time PCR. The present study shows that previous results based on the semi-nested PCR were not substantially influenced by false positives but might have suffered from some false negatives at low agent levels. Combining alternative methods may therefore provide more reliable conclusions on the pathogen's presence. Further, we found positive correlation between the prevalence of infection carriers in American crayfish populations and the average amounts of A. astaci DNA detected in infected local crayfish individuals.

• MeSH
• Aphanomyces genetika   izolace a purifikace   fyziologie   MeSH
• intergenová DNA genetika   MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• severní raci parazitologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Evropa MeSH

OBJECTIVES: Lung cancer with the ALK rearrangement constitutes only a small fraction of patients with non-small cell lung cancer (NSCLC). However, in the era of molecular-targeted therapy, efficient patient selection is crucial for successful treatment. In this context, an effective method for EML4-ALK detection is necessary. We developed a new highly sensitive variant specific TaqMan based real time PCR assay applicable to RNA from formalin-fixed paraffin-embedded tissue (FFPE). MATERIALS AND METHODS: This assay was used to analyze the EML4-ALK gene in 96 non-selected NSCLC specimens and compared with two other methods (end-point PCR and break-apart FISH). RESULTS: EML4-ALK was detected in 33/96 (34%) specimens using variant specific real time PCR, whereas in only 23/96 (24%) using end-point PCR. All real time PCR positive samples were confirmed with direct sequencing. A total of 46 specimens were subsequently analyzed by all three detection methods. Using variant specific real time PCR we identified EML4-ALK transcript in 17/46 (37%) specimens, using end-point PCR in 13/46 (28%) specimens and positive ALK rearrangement by FISH was detected in 8/46 (17.4%) specimens. Moreover, using variant specific real time PCR, 5 specimens showed more than one EML4-ALK variant simultaneously (in 2 cases the variants 1+3a+3b, in 2 specimens the variants 1+3a and in 1 specimen the variant 1+3b). In one case of 96 EML4-ALK fusion gene and EGFR mutation were detected. All simultaneous genetic variants were confirmed using end-point PCR and direct sequencing. CONCLUSION: Our variant specific real time PCR assay is highly sensitive, fast, financially acceptable, applicable to FFPE and seems to be a valuable tool for the rapid prescreening of NSCLC patients in clinical practice, so, that most patients able to benefit from targeted therapy could be identified.

• MeSH
• adenokarcinom diagnóza   genetika   MeSH
• diagnostické techniky molekulární MeSH
• DNA sondy chemie   MeSH
• fúzní onkogenní proteiny genetika   metabolismus   MeSH
• hybridizace in situ fluorescenční MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé MeSH
• messenger RNA genetika   MeSH
• nádorové buněčné linie MeSH
• nádory plic diagnóza   genetika   MeSH
• nemalobuněčný karcinom plic diagnóza   genetika   MeSH
• spinocelulární karcinom diagnóza   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

A multiplex real-time PCR method based on fluorescent TaqMan® probes was developed for the simultaneous detection of the tomato pathogenic bacteria Clavibacter michiganensis subsp. michiganensis, Pseudomonas syringae pv. tomato and bacterial spot-causing xanthomonads. The specificity of the multiplex assay was validated on 44 bacterial strains, including 32 target pathogen strains as well as closely related species and nontarget tomato pathogenic bacteria. The designed multiplex real-time PCR showed high sensitivity when positive amplification was observed for one pg of bacterial DNA in the cases of Clavibacter michiganensis subsp. michiganensis and Pseudomonas syringae pv. tomato bacteria and 100 pg for bacterial spot-causing xanthomonads. The reliability of the developed multiplex real-time PCR assay for in planta detection was verified by recognition of the target pathogens in 18 tomato plants artificially inoculated by each of the target bacteria and tomato samples from production greenhouses.

• MeSH
• Actinobacteria genetika   izolace a purifikace   fyziologie   MeSH
• kvantitativní polymerázová řetězová reakce * MeSH
• prostředí kontrolované MeSH
• Pseudomonas syringae genetika   izolace a purifikace   fyziologie   MeSH
• Solanum lycopersicum růst a vývoj   mikrobiologie   MeSH
• Xanthomonas genetika   izolace a purifikace   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Mycobacterium avium subsp. paratuberculosis (MAP) is a vigorous microorganism which causes incurable chronic enteritis, Johne's disease (JD) in cattle. A target of control programmes for JD is to accurately detect MAP-infected cattle early to reduce disease transmission. The present study evaluated the efficacy of two different cultural procedures and a TaqMan real-time PCR assay for detection of subclinical paratuberculosis in dairy herds. Therefore, sixty-one faecal samples were collected from two Dutch dairy herds (n = 40 and n = 21, respectively) which were known to be MAP-ELISA positive. All individual samples were assessed using two different cultural protocols in two different laboratories. The first cultural protocol (first laboratory) included a decontamination step with 0.75% hexadecylpyridinium chloride (HPC) followed by inoculation on Herrold's egg yolk media (HEYM). The second protocol (second laboratory) comprised of a decontamination step using 4% NaOH and malachite green-oxalic acid followed by inoculation on two media, HEYM and in parallel on modified Löwenstein-Jensen media (mLJ). For the TaqMan real-time PCR assay, all faecal samples were tested in two different laboratories using TaqMan® MAP (Johne's) reagents (Life Technologies). The cultural procedures revealed positive reactions in 1.64% of the samples for cultivation protocol 1 and 6.56 and 8.20% of the samples for cultivation protocol 2, respectively. The results of the TaqMan real-time PCR performed in two different laboratories yielded 13.11 and 19.76% positive reaction. The kappa test showed proportional agreement 0.54 between the mLJ media (second laboratory) and TaqMan® real-time PCR method (second laboratory). In conclusion, the TaqMan real-time PCR could be a strongly useful and efficient assay for the detection of subclinical paratuberculosis in dairy cattle leading to an improvement in the efficiency of MAP control strategies.

• MeSH
• asymptomatické infekce * MeSH
• bakteriologické techniky metody   MeSH
• diagnostické techniky molekulární metody   MeSH
• feces mikrobiologie   MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• Mycobacterium avium subsp. paratuberculosis klasifikace   genetika   růst a vývoj   izolace a purifikace   MeSH
• nemoci skotu diagnóza   MeSH
• odběr biologického vzorku metody   MeSH
• paratuberkulóza diagnóza   MeSH
• senzitivita a specificita MeSH
• skot MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• srovnávací studie MeSH
• Geografické názvy
• Nizozemsko MeSH

Bacillus anthracis is a spore-forming, Gram-positive microorganism. It is a causative agent of anthrax, a highly infectious disease. It belongs to the "Bacillus cereus group", which contains other closely related species, including Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, Bacillus weihenstephanensis, and Bacillus pseudomycoides. B. anthracis naturally occurs in soil environments. The BA5345 genetic marker was used for highly specific detection of B. anthracis with TaqMan probes. The detection limit of a real-time PCR assay was estimated at the level of 16.9 copies (CI95% - 37.4 to 37.86, SD = 0.2; SE = 0.118). Oligonucleotides designed for the targeted sequences (within the tested locus) revealed 100 % homology to B. anthracis strain reference sequences deposited in the database (NCBI) and high specificity to all tested B. anthracis strains. Additional in silico analysis of plasmid markers pag and cap genes with B. anthracis strains included in the database was carried out. Our study clearly indicates that the BA5345 marker can be used with success as a chromosomal marker in routine identification of B. anthracis; moreover, detection of plasmid markers indicates virulence of the examined strains.

• MeSH
• antigeny bakteriální genetika   MeSH
• Bacillus anthracis genetika   izolace a purifikace   patogenita   MeSH
• Bacillus cereus genetika   patogenita   MeSH
• bakteriální proteiny genetika   MeSH
• bakteriální toxiny genetika   MeSH
• DNA bakterií analýza   MeSH
• faktory virulence genetika   MeSH
• genetické markery MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• plazmidy genetika   MeSH
• senzitivita a specificita MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Flour beetles of the genus Tribolium Macleay (Coleoptera: Tenebrionidae) are important stored product pests in China and worldwide. They are often found or are intercepted in grain depots, flour mills, and entry-exit ports, etc. Traditionally, Tribolium species are identified according to the morphological characteristics of the adult. However, it is almost impossible to rapidly identify adult fragments and non-adult stages based on external morphological characteristics. Molecular techniques for the rapid and accurate identification of Tribolium species are required, particularly for pest monitoring and the quarantine of stored products pests. Here, we establish DNA barcoding, species-specific PCR, and real-time PCR techniques for the identification of six stored-product pest Tribolium species including T. castaneum, T. confusum, T. destructor, T. madens, T. freemani and T. brevicornis. We detected the mitochondrial DNA cytochrome oxidase subunit I (COI) barcodes for Tribolium from 18 geographic populations and 101 individuals, built a Tribolium DNA barcode library, and designed species-specific primers and TaqMan probes for the above six Tribolium species. The three techniques were applied to identify Tribolium collected from stored samples and samples captured from quarantine ports. The results demonstrated that three techniques were all able to identify the six species of Tribolium both rapidly and accurately.

• MeSH
• DNA primery metabolismus   MeSH
• DNA chemie   izolace a purifikace   metabolismus   MeSH
• druhová specificita MeSH
• fylogeneze MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• respirační komplex IV chemie   genetika   MeSH
• sekvence nukleotidů MeSH
• sekvenční seřazení MeSH
• taxonomické DNA čárové kódování * MeSH
• Tribolium klasifikace   genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Due to limitations in commercial diagnostic methods, this study aimed to develop a reliable real-time polymerase chain reaction (Rt-PCR) assay for early diagnosis of brucellosis. Optimization of the Rt-PCR method was performed on serum samples spiked by Brucella melitensis with different densities ranging from 101 to 108 colony-forming units (cfu)/mL; each density was prepared in ten samples. The limit of detection was investigated by using Thermo DNA extraction kit with Maxima SYBR Green Rt-PCR and two TaqMan probe-based Rt-PCR protocols performed by QuantiTect and TEMPase multiplex PCR master mixes in two thermal cyclers, which were Rotor-Gene and Bio-Rad. The validation of the optimized protocol was carried on 20 brucellosis-negative samples and 20 samples spiked with B. melitensis by using a combination of Thermo DNA extraction kit with TEMPase PCR master mix. SYBR Green Rt-PCR yielded positive results on all samples having ≥ 104 cfu/mL of B. melitensis in both thermal cyclers. Its limit of detection was 112 DNA copies per reaction. The positivity of both probe-based Rt-PCR protocols was 100% and 80% on the samples having 103 cfu/mL and 102 cfu/mL of B. melitensis, respectively. The limit of detection of probe-based protocols was defined as 4 DNA copies per reaction. The optimized Rt-PCR protocol showed high-level accuracy, precision, specificity, and sensitivity, each having a rate of 100%. The current study indicated that the TaqMan probe-based Rt-PCR protocol optimized and validated with serum samples can be reliably used for early diagnosis of brucellosis.

• MeSH
• Brucella melitensis genetika   izolace a purifikace   MeSH
• brucelóza diagnóza   mikrobiologie   MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• lidé MeSH
• senzitivita a specificita MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH

Cíl studie: Cílem této práce bylo objasnit příčinu diskrepantních výsledků mezi dvěma PCR metodami pro detekci lidského cytomegaloviru (CMV) v klinických vzorcích pacientů Interní hematoonkologické kliniky FN Brno. Materiál a metody: Přítomnost CMV v krvi jsme prokazovali paralelně dvěma metodami, které detekují části major immediate-early (MIE) genu: metodou kvalitativní PCR, která amplifikuje úsek mezi exony 2 a 4, a metodou kvantitativní real-time PCR, která amplifikuje část exonu 4. V období od ledna 2004 do ledna 2005 jsme testovali vzorky od 363 pacientů, 64 z nich bylo alespoň jednou pozitivni citlivější real-time PCR metodou. U dalších 20 pacientů, u nichž byla opakovaně pozitivní jen méně citlivá kvalitativní PCR, jsme provedli sekvenční analýzu sledovaného úseku exonu 4 (nt 2719-2919, GenBank M21295) a stanovení genotypu glykoproteinu B (gB). Výsledky: U těchto vzorků se v místě nasedáni TaqMan sondy a reverse primeru nacházej četné změny oproti sekvenci laboratorního kmene AD 169. Nejsou náhodné a odrážejí reaktivaci kmene CMV, příbuzného spíše s kmenem Towne. Korelace mezi sekvencí exonu 4, genotypem gB a klinickými daty nebyla prokázána. Závěr. Vzhledem k velké sekvenční variabilitě je tento úsek MIE genu zcela nevhodný pro rutinní PCR diagnostiku. Genetická variabilita patogenů může velmi ovlivnit správnou diagnostiku oportunních infekcí.

Aim of the study: The main goal was to explain the discrepancies between two PCR methods used for detection of cytomegalovirus (CMV) in peripheral blood samples of patients of Department of Internal Medicine - Hematooncology, University Hospital, Brno. Materials and methods: In past we used exon 4 of major immediate-early (MIE) gene as the target for quantitative detection of the CMV in clinical samples, but sometimes this method failed to detect the viral load in samples that were positively tested using less sensitive qualitative method targeting another region (exon 2-4) of the same gene. From January 2004 to January 2005 we totally tested samples from 363 patients. 64 patients were at least once CMV positive using quantitative method, but 20 patients were repeatedly false negative.To find the cause of this discrepancy we performed partial sequence analysis of this region (nt positions 2719-2919, GenBank M21295) and glycoprotein B (gB) genotyping. We sequenced samples from 35 patients - 15 giving true positive (both in qualitative and quantitative method) and 20 giving false negative (negative in quantitative but positive in qualitative method) results in several consecutive blood samples. Results: The 15 true positive samples were 100% homological, whereas all 20 false negative samples showed high degree of variation from the laboratory strain AD 169. These changes are not random and indicate that the two groups of patients were infected by different CMV genotypes. Moreover, sequence alignment showed similarity to laboratory strains Toledo and Towne. No preferential concordance was observed between clinical context, MIE exon 4 sequence and gB groups. Conclusions: Because of high sequence variability exon 4 of MIE gene can not be used for routine diagnostics. Genetic varibility among the pathogenic strains may seriously affect its proper diagnostics.

• MeSH
• cytomegalovirové infekce epidemiologie   etiologie   imunologie   MeSH
• exony genetika   imunologie   MeSH
• imunosupresivní léčba škodlivé účinky   MeSH
• klinické laboratorní techniky metody   normy   přístrojové vybavení   MeSH
• lidé MeSH
• polymerázová řetězová reakce metody   přístrojové vybavení   využití   MeSH
• transplantace škodlivé účinky   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• srovnávací studie MeSH

A rapid method for detection, discrimination and quantification of wheat and barley strains of wheat dwarf virus (WDV) was successfully developed. The sensitivity of quantification of the wheat and barley strains of WDV ranged from an average of 1.2 × 10(7)-1.2 × 10(2) and from an average of 1.4 × 10(7)-1.4 × 10(4) copies of viral genome, respectively. These standard serial dilutions were applied to plant and vector tissues for virus titer calculations. Both strains of WDV were clearly discriminated by specific probes and melting curve analysis. Both TaqMan(®) and SYBR(®) Green technologies provided accurate and reliable methods for monitoring, detection, discrimination, and quantification of WDV.

• MeSH
• Geminiviridae klasifikace   genetika   izolace a purifikace   MeSH
• ječmen (rod) virologie   MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• nemoci rostlin virologie   MeSH
• pšenice virologie   MeSH
• senzitivita a specificita MeSH
• virová nálož metody   MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH