Real-time validation
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vii, 284 s., [4] s. příloh : il., tab. ; 26 cm
- MeSH
- polymerázová řetězová reakce metody trendy MeSH
- Publikační typ
- monografie MeSH
- Konspekt
- Biochemie. Molekulární biologie. Biofyzika
- NLK Obory
- biologie
... An introduction to real-time PCR / N. A. Saunders -- Ch. 2. ... ... An overview of real-time PCR platforms / J. M. J. Logan and K. J. Edwards -- Ch. 3. ... ... Performing real-time PCR / K. J. Edwards -- Ch. 5. ... ... Quantitative real-time PCR / N. A. Saunders -- Ch. 7. ... ... Real-time NASBA / S. Hibbitts and J. D. Fox -- Ch. 11. ...
vi, 346 s. : il. ; 25 cm
- MeSH
- amplifikace genu MeSH
- polymerázová řetězová reakce metody trendy MeSH
- Publikační typ
- monografie MeSH
- Konspekt
- Biochemie. Molekulární biologie. Biofyzika
- NLK Obory
- biologie
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.
Cílem práce bylo zavedení molekulární real-time PCR metodiky doporučené CDC (Centers for Disease Control and Prevention) pro účely detekce bakterií Neisseria meningitidis, Haemophilus influenzae a Streptococcus pneumoniae v klinických (kultivačně negativních) vzorcích při podezření na invazivní bakteriální onemocnění. Klinické vzorky jsou zasílány do NRL pro meningokokové nákazy Oddělení bakteriálních vzdušných nákaz CEM SZÚ z různých regionů České republiky. Jedná se především o likvor, nesrážlivou krev či sérum, výjimečně sekční materiál. NRL má zavedenou molekulární diagnostiku těchto bakteriálních původců menigitid a sepsí z klinických vzorků již od roku 1999. Původně se jednalo o seminested polymerázovou řetězovou reakci s elektroforetickým vyhodnocením. V roce 2014 byla implementována molekulární metoda real-time PCR s kvalitativním hodnocením.
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.
- MeSH
- DNA bakterií analýza MeSH
- Haemophilus influenzae * genetika izolace a purifikace MeSH
- lidé MeSH
- Neisseria meningitidis * genetika izolace a purifikace MeSH
- polymerázová řetězová reakce s reverzní transkripcí využití MeSH
- reprodukovatelnost výsledků MeSH
- statistika jako téma MeSH
- Streptococcus pneumoniae * genetika izolace a purifikace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
BACKGROUND: The aims of this work were to replace the obsolete PCR method for the laboratory diagnosis of the acute form of leptospirosis using the G1, G2 and B64 I, B64 II primers, and to improve the PCR detection time. METHODS: We introduced a real-time PCR method for the detection of the gene encoding the surface lipoprotein LipL32 of pathogenic Leptospira into our laboratory diagnosis of the acute form of leptospirosis. The positive and negative analytical specificities of the real-time PCR method were both equal to 100%; the detection limit was determined to be 1-5 genome copies/1 ml of liquid biological material. The method was further validated on 230 laboratory strains of leptospires. RESULTS: All laboratory strains of pathogenic Leptospira were evaluated as LipL32-positive and all non-pathogenic strains as LipL32-negative. In addition, 455 biological materials (253 plasma, 121 urine, 72 cerebrospinal fluid (CSF), 7 bronchoalveolar lavage, and 2 sputum) from 295 patients with suspected leptospirosis were examined. From this set of patients, 9 were evaluated to be LipL32-positive, from 15 positive biological materials (10 urine, 4 blood plasma, and 1 CSF). CONCLUSIONS: This real-time PCR method for the detection of the gene encoding the surface lipoprotein LipL32 is a reliable, sensitive, and rapid method for the detection of the acute form of leptospirosis.
- MeSH
- bakteriologické techniky metody MeSH
- časové faktory MeSH
- diagnostické techniky molekulární metody MeSH
- dospělí MeSH
- klinické laboratorní techniky metody MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- Leptospira genetika izolace a purifikace MeSH
- leptospiróza diagnóza mikrobiologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- lipoproteiny genetika MeSH
- mladý dospělý MeSH
- proteiny vnější bakteriální membrány genetika MeSH
- senioři MeSH
- senzitivita a specificita MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
The aim of this work was to identify reliable reference genes for expression studies in adult Haemonchus contortus. Eleven candidate genes were identified and the stability of their expression was assessed in adult males and females of two genetically divergent H. contortus isolates: drug-susceptible (ISE) and multi-drug-resistant (WR). Five genes with the most stable expression patterns were further assessed for suitability as reference genes in anthelmintic-treated H. contortus adults versus non-treated controls. We identified important differences in the expression of a number of candidate genes in anthelmintic-treated samples, confirming the need for careful validation of control genes for such experiments. We propose the use of multiple reference genes for expression studies in this species and found gpd, ama and far most suitable for adult H. contortus.
- MeSH
- anthelmintika farmakologie MeSH
- Haemonchus účinky léků genetika MeSH
- kvantitativní polymerázová řetězová reakce metody normy MeSH
- referenční standardy * MeSH
- stanovení celkové genové exprese metody normy MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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
... HTA in “Real-Time” -- CONTENT -- Bouřek A., Suchý M., Forýtková L., Vlk D. ... ... Pavliš The importance of valid parametric data in haematology documented on functional clinical registry ...
1st ed. 82 s. : tab., grafy, mapy ; 24 cm
- MeSH
- metody pro podporu rozhodování MeSH
- nádory MeSH
- poskytování zdravotní péče trendy MeSH
- věda trendy MeSH
- výzkum MeSH
- výzkumný projekt MeSH
- Publikační typ
- kongresy MeSH
- sborníky MeSH
- Konspekt
- Lékařské vědy. Lékařství
- NLK Obory
- lékařská informatika
- onkologie
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
