Dot-blot immunoassays are widely used for the user-friendly detection of clinical biomarkers. However, the majority of dot-blot assays have only limited sensitivity and are only used for qualitative or semiquantitative analysis. To overcome this limitation, we have employed labels based on photon-upconversion nanoparticles (UCNPs) that exhibit anti-Stokes luminescence and can be detected without optical background interference. First, the dot-blot immunoassay on a nitrocellulose membrane was optimized for the quantitative analysis of human serum albumin (HSA), resulting in a limit of detection (LOD) of 0.19 ng/mL and a signal-to-background ratio (S/B) of 722. Commercial quantum dots were used as a reference label, reaching the LOD of 4.32 ng/mL and the S/B of 3, clearly indicating the advantages of UCNPs. In addition, the potential of UCNP-based dot-blot for real sample analysis was confirmed by analyzing spiked urine samples, reaching the LOD of 0.24 ng/mL and recovery rates from 79 to 123%. Furthermore, we demonstrated the versatility and robustness of the assay by adapting it to the detection of two other clinically relevant biomarkers, prostate-specific antigen (PSA) and cardiac troponin (cTn), reaching the LODs in spiked serum of 9.4 pg/mL and 0.62 ng/mL for PSA and cTn, respectively. Finally, clinical samples of patients examined for prostate cancer were analyzed, achieving a strong correlation with the reference electrochemiluminescence immunoassay (recovery rates from 89 to 117%). The achieved results demonstrate that UCNPs are highly sensitive labels that enable the development of dot-blot immunoassays for quantitative analysis of low-abundance biomarkers.

• MeSH
• biologické markery * krev   moč   analýza   MeSH
• imunoanalýza metody   MeSH
• kvantové tečky chemie   MeSH
• lidé MeSH
• lidský sérový albumin analýza   moč   MeSH
• limita detekce * MeSH
• nanočástice * chemie   MeSH
• prostatický specifický antigen * krev   analýza   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biologické markery * MeSH
• lidský sérový albumin MeSH
• prostatický specifický antigen * MeSH

Quantitative Real-Time Polymerase Chain Reaction, better known as qPCR, is the most sensitive and specific technique we have for the detection of nucleic acids. Even though it has been around for more than 30 years and is preferred in research applications, it has yet to win broad acceptance in routine practice. This requires a means to unambiguously assess the performance of specific qPCR analyses. Here we present methods to determine the limit of detection (LoD) and the limit of quantification (LoQ) as applicable to qPCR. These are based on standard statistical methods as recommended by regulatory bodies adapted to qPCR and complemented with a novel approach to estimate the precision of LoD.

• Klíčová slova
• Data analysis, GenEx software, Limit of detection, Limit of quantification, LoD, LoQ, MIQE, Quality control, Real-time PCR, Replicates, Standardization, qPCR,
• Publikační typ
• časopisecké články 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

One third of the world's population is latently infected with Mycobacterium tuberculosis (Mtb) and up to 10% of infected individuals develop active tuberculosis (TB) in their lifetime. Among the major challenges in the control of TB is the implementation of sensitive methods for detection of latent tuberculosis infection (LTBI). Currently, in vitro interferon gamma release assays, yielding single value readout, are used as an alternative to the traditional tuberculin skin test for the diagnosis of LTBI. More complex characterization of immune status of LTBI individuals, however, is desirable for indication of LTBI subjects for preventative chemotherapy. Here we describe a quantitative polymerase chain reaction (qPCR) for determination of expression levels of 14 genes, additional to interferon gamma, which was applied for comparison of the specific Mtb-antigen immune response of blood cells from healthy, latently infected, and TB individuals. With the use of principal component analysis and discriminant analysis, a pattern of mRNA levels of 6 genes was identified, allowing discrimination of healthy individuals from active TB and LTBI subjects. These results open the way to development of multimarker qPCR for the detection of LTBI.

• MeSH
• antigeny bakteriální imunologie   MeSH
• diagnostické techniky molekulární metody   MeSH
• dospělí MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• latentní tuberkulóza diagnóza   MeSH
• leukocyty mononukleární imunologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• Mycobacterium tuberculosis imunologie   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• stanovení celkové genové exprese metody   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny bakteriální MeSH

This study focused on the development of a reliable and cost-efficient DNA isolation procedure for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) in faeces by previously developed IS900 and F57 quantitative real time PCR (qPCR) and their comparison with culture. The recovery of MAP DNA from the spiking experiments ranged from 29.1 to 102.4% of the input amount of MAP with median 37.9%. The limit of detection was determined to be 1.03 × 10(4) for F57 qPCR and 6.87 × 10(2)MAP cells per gram of faeces for IS900 qPCR, respectively. The developed technique for DNA isolation was coupled with IS900 qPCR and compared to traditional MAP culture using a cohort of 1906 faecal samples examined from 12 dairy cattle farms in our laboratory. From those 1906 original faecal samples, 875 were positive by IS900 qPCR and 169 by culture. None of the culture positive samples was negative by IS900 qPCR. This data facilitated development of a predictive model capable of estimating the probability of being culture positive by estimating the absolute number of MAP per gram of faeces as determined IS900 qPCR without performing the culture.

• MeSH
• DNA bakterií analýza   izolace a purifikace   MeSH
• feces mikrobiologie   MeSH
• limita detekce MeSH
• logistické modely * MeSH
• Mycobacterium avium subsp. paratuberculosis genetika   izolace a purifikace   MeSH
• nemoci skotu diagnóza   mikrobiologie   MeSH
• paratuberkulóza diagnóza   mikrobiologie   MeSH
• polymerázová řetězová reakce metody   veterinární   MeSH
• senzitivita a specificita MeSH
• skot MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• DNA bakterií MeSH

Quantitative PCR (qPCR) has become a frequently employed direct method for the detection and quantification of Mycobacterium avium subsp. paratuberculosis (MAP). The quantity of MAP determined by qPCR, however, may be affected by the type of qPCR quantification standard used (PCR product, plasmid, genomic DNA) and the way in which standard DNA quantity is determined (absorbance, fluorescence). In practice, this can be reflected in the inability to properly compare quantitative data from the same qPCR assays in different laboratories. Thus, the aim of this study was to prepare a prototype of an international MAP reference standard, which could be used to calibrate routinely used qPCR quantification standards in various laboratories to promote clinical data comparability. Considering stability, storage and shipment issues, a lyophilised fecal suspension artificially contaminated with a MAP reference strain was chosen as the most suitable form of the standard. The effect of five types of lyophilisation matrices on standard stability was monitored on 2-weeks interval basis for 4 months by F57 qPCR. The lyophilisation matrix with 10% skimmed milk provided the best recovery and stability in time and was thus selected for subsequent comparative testing of the standard involving six diagnostic and research laboratories, where DNA isolation and qPCR assay procedures were performed with the parallel use of the identical supplied genomic DNA solution. Furthermore, the effect of storage conditions on the standard stability was tested for at least 6 months. The storage at room temperature in the dark and under light, at + 4 °C, - 20 °C and - 80 °C showed no significant changes in the stability, and also no substantial changes in MAP viability were found using phage amplification assay. The prepared MAP quantification standard provided homogeneous and reproducible results demonstrating its suitability for utilisation as an international reference qPCR standard.

• MeSH
• DNA bakterií klasifikace   genetika   MeSH
• feces chemie   mikrobiologie   MeSH
• kvantitativní polymerázová řetězová reakce normy   MeSH
• lyofilizace MeSH
• Mycobacterium avium subsp. paratuberculosis klasifikace   genetika   izolace a purifikace   MeSH
• nemoci skotu diagnóza   MeSH
• paratuberkulóza diagnóza   mikrobiologie   MeSH
• referenční standardy MeSH
• senzitivita a specificita MeSH
• skot MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA bakterií MeSH

BCR-ABL1 molecular detection using quantitative PCR (qPCR) methods is the golden standard of chronic myeloid leukemia (CML) monitoring. However, due to variable sensitivity of qPCR assays across laboratories, alternative methods are tested. Digital PCR (dPCR) has been suggested as a robust and reproducible option. Here we present a comparison of droplet dPCR with routinely used reverse-transcription qPCR (RT-qPCR) and automated GeneXpert systems. Detection limit of dPCR was above 3 BCR-ABL1 copies, although due to background amplification the resulting sensitivity was 0.01% BCR-ABL1 (MR4.0). Nevertheless, in comparison with GeneXpert, dPCR categorized more than 50% of the patients into different MR groups, showing a potential for improved BCR-ABL1 detection.

• Klíčová slova
• BCR-ABL1 monitoring, CML, chronic myeloid leukemia, Chronic myeloid leukemia, Digital PCR, EAC, Europe Against Cancer, FPR, false positivity rate, GeneXpert BCR-ABL Monitor assay, IS, international scale, LOB, limit of blank, LOD, limit of detection, MR, molecular response, NTC, no template control, RT-qPCR, RT-qPCR, reverse-transcription quantitative PCR, TKI, tyrosine kinase inhibitors, cDNA, complementary DNA, dPCR, digital PCR, pDNA, plasmid DNA, qPCR, quantitative PCR,
• Publikační typ
• časopisecké články MeSH

INTRODUCTION: Hammondia hammondi and Toxoplasma gondii are closely related protozoan parasites, but only T. gondii is zoonotic. Both species use felids as definitive hosts and cannot be differentiated by oocyst morphology. In T. gondii, a 529-base pair (bp) repetitive element (TgREP-529) is of utmost diagnostic importance for polymerase chain reaction (PCR) diagnostic tests. We identified a similar repetitive region in the H. hammondi genome (HhamREP-529). METHODS: Based on reported sequences, primers and probes were selected in silico and optimal primer probe combinations were explored, also by including previously published primers. The analytical sensitivity was tested using serial dilutions of oocyst DNA. For testing analytical specificity, DNA isolated from several related species was used as controls. The newly established TaqMan PCR (Hham-qPCR1) was applied to tissues collected from H. hammondi-infected gamma-interferon gene knockout (GKO) mice at varying time points post-infection. RESULTS: Ten forward and six reverse primers were tested in varying combinations. Four potentially suitable dual-labelled probes were selected. One set based on the primer pair (Hham275F, Hham81R) and the probe (Hham222P) yielded optimal results. In addition to excellent analytic specificity, the assay revealed an analytical sensitivity of genome equivalents of less than one oocyst. Investigation of the tissue distribution in GKO mice revealed the presence of parasite DNA in all examined organs, but to a varying extent, suggesting 100- to 10,000-fold differences in parasitic loads between tissues in the chronic state of infection, 42 days post-infection. DISCUSSION: The use of the 529-bp repeat of H. hammondi is suitable for establishing a quantitative real-time PCR assay, because this repeat probably exists about 200 times in the genome of a single organism, like its counterpart in T. gondii. Although there were enough sequence data available, only a few of the primers predicted in silico revealed sufficient amplification; the identification of a suitable probe was also difficult. This is in accord with our previous observations on considerable variability in the 529-bp repetitive element of H. hammondi. CONCLUSIONS: The H. hammondi real-time PCR represents an important novel diagnostic tool for epidemiological and cell biological studies on H. hammondi and related parasites.

• Klíčová slova
• Faecal examination, Hammondia hammondi, Oocyst, Quantitative polymerase chain reaction, TaqMan polymerase chain reaction,
• MeSH
• diferenciální diagnóza MeSH
• feces parazitologie   MeSH
• kočky parazitologie   MeSH
• kokcidióza veterinární   MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• molekulární patologie metody   MeSH
• myši parazitologie   MeSH
• oocysty genetika   izolace a purifikace   MeSH
• protozoální geny MeSH
• repetitivní sekvence nukleových kyselin MeSH
• Sarcocystidae * genetika   izolace a purifikace   MeSH
• Toxoplasma * genetika   izolace a purifikace   MeSH
• toxoplazmóza zvířat MeSH
• zvířata MeSH
• Check Tag
• kočky parazitologie   MeSH
• myši parazitologie   MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

A sensitive and rapid method for the quantitative detection of bovine-, ovine-, swine-, and chicken-specific mitochondrial DNA sequences based on real-time PCR has been developed. The specificity of the primers and probes for real-time PCR has been tested using DNA samples of other vertebrate species that may also be present in rendered products. The quantitative detection was performed with dual-labeled probes (TaqMan) using absolute quantification with external standards of single species meat-and-bone meals. This method facilitates the detection of 0.01% of the target species-derived material in concentrate feed mixtures and fish meals.

• MeSH
• druhová specificita MeSH
• krmivo pro zvířata analýza   MeSH
• kur domácí genetika   MeSH
• mitochondriální DNA analýza   MeSH
• ovce genetika   MeSH
• polymerázová řetězová reakce metody   MeSH
• prasata genetika   MeSH
• přežvýkavci * MeSH
• prionové nemoci prevence a kontrola   MeSH
• rybí výrobky analýza   MeSH
• senzitivita a specificita MeSH
• skot genetika   MeSH
• zvířata MeSH
• Check Tag
• skot genetika   MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitochondriální DNA MeSH

Lysosomes are the terminal end of catabolic pathways in the cell, as well as signaling centers performing important functions such as the recycling of macromolecules, organelles, and nutrient adaptation. The importance of lysosomes in human health is supported by the fact that the deficiency of most lysosomal genes causes monogenic diseases called as a group Lysosomal Storage Diseases (LSDs). A common phenotypic hallmark of LSDs is the expansion of the lysosomal compartment that can be detected by using conventional imaging methods based on immunofluorescence protocols or overexpression of tagged lysosomal proteins. These methods require the alteration of the cellular architecture (i.e., due to fixation methods), can alter the behavior of cells (i.e., by the overexpression of proteins), and require sample preparation and the accurate selection of compatible fluorescent markers in relation to the type of analysis, therefore limiting the possibility of characterizing cellular status with simplicity. Therefore, a quantitative and label-free methodology, such as Quantitative Phase Imaging through Digital Holographic (QPI-DH), for the microscopic imaging of lysosomes in health and disease conditions may represent an important advance to study and effectively diagnose the presence of lysosomal storage in human disease. Here we proof the effectiveness of the QPI-DH method in accomplishing the detection of the lysosomal compartment using mouse embryonic fibroblasts (MEFs) derived from a Mucopolysaccharidosis type III-A (MSP-IIIA) mouse model, and comparing them with wild-type (WT) MEFs. We found that it is possible to identify label-free biomarkers able to supply a first pre-screening of the two populations, thus showing that QPI-DH can be a suitable candidate to surpass fluorescent drawbacks in the detection of lysosomes dysfunction. An appropriate numerical procedure was developed for detecting and evaluate such cellular substructures from in vitro cells cultures. Results reported in this study are encouraging about the further development of the proposed QPI-DH approach for such type of investigations about LSDs.

• Klíčová slova
• digital holography, intracellular specificity, label‐free imaging, lysosomal storage diseases, lysosomes, quantitative phase imaging,
• MeSH
• fibroblasty metabolismus   patologie   MeSH
• kvantitativní fázové zobrazování MeSH
• lidé MeSH
• lyzozomální nemoci z ukládání metabolismus   patologie   genetika   diagnóza   MeSH
• lyzozomy * metabolismus   MeSH
• mukopolysacharidóza III metabolismus   patologie   genetika   MeSH
• myši MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH