UNLABELLED: The aim of this study was to identify parameters influencing DNA extraction and PCR amplification efficiencies in an attempt to standardize Mucorales qPCR. The Fungal PCR Initiative Mucorales Laboratory Working Group distributed two panels of simulated samples to 26 laboratories: Panel A (six sera spiked with Mucorales DNA and one negative control serum) and Panel B (six Mucorales DNA extracts). Panel A underwent DNA extraction in each laboratory according to the local procedure and were sent to a central laboratory for testing using three different qPCR techniques: one in-house qPCR assay and two commercial assays (MucorGenius and Fungiplex). Panel B DNA extracts were PCR amplified in each laboratory using local procedures: nine in-house qPCR assays and two commercial kits (MucorGenius and MycoGENIE). All data were compiled and anonymously analyzed at the central laboratory. For Panel A, a total of six different automated platforms and five manual extraction methods were used. Positive rates were 64%, 70%, and 89%, for the MucorGenius, Fungiplex, and the in-house qPCR assay, respectively. Using a large volume of serum for DNA extraction provided the highest analytical sensitivity (82.5% for 1 mL compared with 62.7% for smaller volumes, P < 0.01). For Panel B, five in-house qPCR assays and two commercial kits had >78% positivity. Using larger PCR input volumes (≥7 μL) was associated with the highest sensitivity at 95.5% compared to 58.3% when lower input volumes were used (P < 0.01). Using larger sample volumes for nucleic acid extraction and DNA template volumes for PCR amplification significantly improves the performance of Mucorales qPCR when testing serum. IMPORTANCE: Mucormycosis is a life-threatening mold infection affecting immunosuppressed patients but also other patients with diabetes or trauma. Better survival is linked to shorter delays in diagnosis and treatment initiation. Detection of Mucorales-free DNA in serum or plasma using quantitative PCR allows a prompt diagnosis and earlier treatment. Several techniques and protocols of quantitative Mucorales PCR are used in Europe, and improving performance remains a common objective of laboratories participating in the fungal PCR Initiative Working Group. This study, which combined results from 26 laboratories in Europe, showed that the main parameters underpinning sensitivity are the preanalytical variables (volume of serum used for DNA extraction and DNA template volume), irrespective of the extraction platforms and qPCR assay/platform.
- MeSH
- Molecular Diagnostic Techniques standards methods MeSH
- DNA, Fungal * blood genetics MeSH
- Real-Time Polymerase Chain Reaction * standards methods MeSH
- Humans MeSH
- Mucorales * genetics isolation & purification MeSH
- Mucormycosis * diagnosis microbiology blood MeSH
- Sensitivity and Specificity * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Evaluation Study MeSH
Mycoplasma spp. contamination is a major concern in laboratories handling cell cultures, and routine detection methods are usually time-consuming, laborious and lack sensitivity. This study presents a streamlined workflow integrating rapid thermal DNA extraction (99 °C-1 min) with a SYBR Green-based qPCR for Mycoplasma detection. High-coverage primers targeting an 86-bp region of the 16S rDNA were designed using 109 Mycoplasma spp. sequences from GeneBank. In silico analysis confirmed full primer annealing to major cell culture contaminants (M. arginini, M. hominis, M. orale, and M. hyorhinis). Upon thermal lysis and qPCR optimization, the yield of the protocol was equivalent to that of phenol-chloroform extraction plus qPCR, with a detection limit of 64 bacterial cells. Finally, the performance of the protocol was confirmed in cell cultures with known Mycoplasma spp. contamination, accurately reproducing the contamination status. Thus, the developed protocol provides a simple, rapid, cost-effective, and sensitive method for monitoring Mycoplasma spp. in cell cultures.
- MeSH
- Cell Culture Techniques * methods MeSH
- DNA, Bacterial genetics isolation & purification MeSH
- DNA Primers genetics MeSH
- Real-Time Polymerase Chain Reaction * methods MeSH
- Humans MeSH
- Mycoplasma * genetics isolation & purification classification MeSH
- Workflow MeSH
- DNA, Ribosomal genetics MeSH
- RNA, Ribosomal, 16S genetics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Applications like drug development need simple and streamlined methods to process samples from 96-well cell culture plates for gene expression measurements. Unfortunately, current options are expensive for such processing. Therefore, our aim was to develop a method that would allow streamlined analysis of mRNA from 96-well cell culture plates while being relatively cheap and simple. We developed a method based on the qPCR 'Cells-to-cDNA' approach and validated it against commercially available kits using the same approach or spin columns-based RNA purification. For this purpose, we conducted a series of comparisons of gene expression from peripheral blood mononuclear cells, SK-HEP-1 and U-87 cell cultures in 96-well plates. Our final method involved lysing cells with 25-100 μl solution of 0.5% SDS, 10 mM DTT, 1 mg ml-1 proteinase K dissolved in water, 1 h incubation at 50°C, followed by proteinase K inactivation at 90°C for 5 min and lysate neutralization with 1 : 1 dilution by 20% Tween 20 solution. Reverse transcription and qPCR were carried out using standard methods. This method showed a mean reduction of Ct ± s.d. value by 2.4 ± 1.3 compared with the 'Cells-to-cDNA' kit and by 1.4 ± 0.5 compared with the RNA purification kit with lower variability.
- MeSH
- Cost-Benefit Analysis MeSH
- Cell Culture Techniques methods economics MeSH
- DNA, Complementary * genetics MeSH
- Real-Time Polymerase Chain Reaction methods MeSH
- Leukocytes, Mononuclear cytology metabolism MeSH
- Humans MeSH
- RNA, Messenger genetics metabolism MeSH
- Cell Line, Tumor MeSH
- Gene Expression Profiling methods economics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
In 2020, there were numerous cases in Kazakhstan with clinical symptoms of COVID-19 but negative PCR results in nasopharyngeal and oropharyngeal swabs. The diagnosis was confirmed clinically and by CT scans (computed tomography). The problem with such negative PCR results for SARS-CoV-2 infection confirmation still exists and indicates the need to confirm the diagnosis in the bronchoalveolar lavage in such cases. There is also a lack of information about confirmation of SARS-CoV-2 infection in deceased patients. In this study, various tissue materials, including lungs, bronchi, and trachea, were examined from eight patients who died, presumably from SARS-CoV-2 infection, between 2020 and 2022. Naso/oropharyngeal swabs taken from these patients in hospitals tested PCR negative for SARS-CoV-2. This study presents a modified RNA isolation method based on a comparison of the most used methods for RNA isolation in laboratories: QIAamp Viral RNA Mini Kit and TRIzol-based method. This modified nucleic acid extraction protocol can be used to confirm SARS-CoV-2 infection by RT-qPCR in the tissues of deceased patients in disputed cases. RT-qPCR with RNA of SARS-CoV-2 re-extracted with such method from post-mortem tissues that were stored at -80 °C for more than 32 months still demonstrated high-yielding positive results.
- MeSH
- COVID-19 * virology diagnosis genetics MeSH
- Adult MeSH
- Real-Time Polymerase Chain Reaction methods MeSH
- Middle Aged MeSH
- Humans MeSH
- Nasopharynx virology MeSH
- Autopsy * methods MeSH
- Lung virology pathology diagnostic imaging MeSH
- RNA, Viral * genetics analysis MeSH
- SARS-CoV-2 * genetics MeSH
- Aged MeSH
- COVID-19 Nucleic Acid Testing methods MeSH
- Trachea virology pathology diagnostic imaging MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
Minimal/measurable residual disease (MRD) diagnostics using real-time quantitative PCR analysis of rearranged immunoglobulin and T-cell receptor gene rearrangements are nowadays implemented in most treatment protocols for patients with acute lymphoblastic leukemia (ALL). Within the EuroMRD Consortium, we aim to provide comparable, high-quality MRD diagnostics, allowing appropriate risk-group classification for patients and inter-protocol comparisons. To this end, we set up a quality assessment scheme, that was gradually optimized and updated over the last 20 years, and that now includes participants from around 70 laboratories worldwide. We here describe the design and analysis of our quality assessment scheme. In addition, we here report revised data interpretation guidelines, based on our newly generated data and extensive discussions between experts. The main novelty is the partial re-definition of the "positive below quantitative range" category by two new categories, "MRD low positive, below quantitative range" and "MRD of uncertain significance". The quality assessment program and revised guidelines will ensure reproducible and accurate MRD data for ALL patients. Within the Consortium, similar programs and guidelines have been introduced for other lymphoid diseases (e.g., B-cell lymphoma), for new technological platforms (e.g., digital droplet PCR or Next-Generation Sequencing), and for other patient-specific MRD PCR-based targets (e.g., fusion genes).
- MeSH
- Precursor Cell Lymphoblastic Leukemia-Lymphoma genetics diagnosis MeSH
- Gene Rearrangement MeSH
- Genes, Immunoglobulin MeSH
- Real-Time Polymerase Chain Reaction methods standards MeSH
- Humans MeSH
- Neoplasm, Residual * genetics diagnosis MeSH
- Practice Guidelines as Topic standards MeSH
- Quality Assurance, Health Care MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Východiska: Dlaždicobuněčný karcinom ústní dutiny (oral squamous cell carcinoma – OSCC) je jedným z nejběžnějších nádorů ze skupin dlaždicobuněčných karcinomů hlavy a krku. Zvyšující se výskyt karcinomů ústní dutiny a jejich zjištění v pokročilých stadiích je celosvětovým zdravotním problémem. Stále více údajů svědčí o tom, že při růstu a progresi zhoubných nádorů hrají důležitou roli microRNA (miRNAs), zatímco o významu miR-7113-3p and miR-6721-5p v OSCC nejsou k dispozici žádné informace. Tento článek pojednává o zkoumání exprese MAP2K1, miR-7113-3p a miR-6721-5p pro možné biologické funkce při rozvoji dlaždicobuněčného karcinomu ústní dutiny. Materiál a metody: Pomocí kvantitativní polymerázové řetězové reakce v reálném čase jsme stanovili expresi mRNA u MAP2K1, miR-7113-3p a miR-6721-5p v čerstvě zmražených tkáních OSCC a v čerstvě zmražených přilehlých normálních tkáních 30 pacientů a zkoumali jsme jejich vztah ke klinickým parametrům. Výsledky: Exprese MAP2K1 v nádorové tkáni byla oproti normálním tkáním významně vyšší, zatímco exprese miR-7113-3p a miR-6721-5p byla významně nižší. Také byla pozorována statistická korelace p = 0,04 mezi zvýšenou expresí MAP2K1 a perineurální invazí. Navíc jsme zaznamenali, že mezi down-regulací miR-7113-3p a zvýšenou expresí MAP2K1 je pozitivní korelace (p = 0,0218) a mezi down-regulací miR-6721-5p a zvýšenou expresí MAP2K1 je negativní korelace (p = 0,7771). Závěr: Z těchto nálezů vyplývá, že u pacientů s OSCC mohou miR-7113-3p a miR-6721-5p sloužit jako prospektivní biomarkery, které by v budoucnu mohly být využívány k detekci OSCC v časném stadiu. Zvýšená exprese MAP2K1 je spojena s rozvojem OSCC a perineurální invazí.
Background: Oral squamous cell carcinoma (OSCC) is one of the most common cancers in the head and neck squamous cell cancer group. The increasing frequency of oral carcinomas and their late-stage appearance is a major worldwide health concern. MicroRNAs (miRNAs) appear to play an important role in cancer growth and progression, according to growing data, whereas no information is available regarding miR-7113-3p and miR-6721-5p involvement in OSCC. In this article, the expression of MAP2K1, miR-7113-3p, and miR-6721-5p was examined for possible biological functions in the advancement of oral squamous cell carcinoma. Material and methods: We used quantitative real-time PCR (to examine the mRNA expression of MAP2K1, miR-7113-3p, and miR-6721-5p in fresh frozen OSCC tissues and adjacent normal fresh frozen tissues from 30 patients, and we investigated their relationship with clinical parameters. Results: MAP2K1 expression was found to be dramatically increased in tumor tissues than in normal tissues, whereas miR7113-3p and miR-6721-5p expression was significantly decreased. Furthermore, a statistical correlation of P = 0.04 was also observed between increased MAP2K1 expression and perineural invasion. Additionally, we noted that the downregulation of miR-7113-3p appears to correlate positively with overexpression of MAP2K1 (P = 0.0218), and a negative correlation was observed between downregulation of miR-6721-5p and overexpression of MAP2K1 (P = 0.7771). Conclusion: Based on these findings, miR-7113-3p and miR-6721-5p might be prospective biomarkers for OSCC patients, and could be utilized to detect OSCC at an early stage for future diagnosis. MAP2K1 overexpression has been linked to the development of OSCC and perineural invasion.
- Keywords
- miR-7113-3p, miR-6721-5p,
- MeSH
- Squamous Cell Carcinoma of Head and Neck * diagnostic imaging genetics MeSH
- Real-Time Polymerase Chain Reaction methods MeSH
- Humans MeSH
- MAP Kinase Kinase 1 genetics MeSH
- Biomarkers, Tumor analysis MeSH
- Gene Expression Profiling classification methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Clinical Study MeSH
- MeSH
- Chorionic Gonadotropin * blood MeSH
- Adult MeSH
- Real-Time Polymerase Chain Reaction methods MeSH
- Middle Aged MeSH
- Humans MeSH
- Microsatellite Repeats MeSH
- Young Adult MeSH
- Hydatidiform Mole classification blood physiopathology MeSH
- Nomograms * MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Young Adult MeSH
- Female MeSH
- Publication type
- Clinical Study MeSH
Kontext a cíl výzkumu: Nealkoholické ztukovatění jater (nonalcoholic fatty liver disease, NAFLD) má závažné ekonomické dopady na zdravotnictví celosvětově a na Ukrajině obzvláště. Hlavní příčinou mortality pacientů s NAFLD jsou kardiovaskulární onemocnění (KVO). Za potenciální mechanismus rozvoje ischemické choroby srdeční (ICHS) u pacientů s NAFLD lze považovat změny ve složení střevní mikrobioty. Cílem našeho výzkumu bylo zjistit změny koncentrací hlavních fylotypů střevní mikrobioty, kmenů Bacteroidetes, Firmicutes a Actinobacteria, a kvantifikovat koncentrace kmenů Firmicutes/Bacteroidetes u pacientů s NAFLD a současně s ICHS. Materiál a metody: Do studie bylo zařazeno 109 jedinců s NAFLD (25 současně s arteriální hypertenzí [AH] a 24 současně s ICHS). Složení střevní mikrobioty bylo hodnoceno metodou qPCR. Výsledky a závěry: U obou podskupin, s ICHS a s AH jako komorbiditami, byl pozorován výrazný trend ke zvyšování koncentrací Bacteroidetes (o 37,11 %, resp. 21,30 %) a snižování koncentrací kmene Firmicutes (o 7,38 %, resp. 7,77 %), přičemž nalezené změny nedosahovaly statistické významnosti. Ve srovnání s pacienty pouze s NAFLD bylo u nemocných s NAFLD plus ICHS zaznamenáno statisticky významné snížení koncentrací kmene Actinobacteria o 41,37 % (p ˂ 0,05). U pacientů s NAFLD plus AH byly koncentrace kmene Actinobacteria nižší o 12,35 % (statisticky nevýznamný rozdíl). Byly nalezeny změny ve složení střevní mikrobioty, konkrétně nižší koncentrace kmene Actinobacteria u pacientů s ICHS; toto zjištění si vyžádá další výzkum.
Background and the research aim: Nonalcoholic fatty liver disease (NAFLD) bears serious economic consequences for the health care system worldwide and Ukraine, in particular. Cardiovascular diseases (CVD) are the main cause of mortality in NAFLD patients. Changes in the gut microbiota composition can be regarded as a potential mechanism of CVD in NAFLD patients. The research aim was the investigation changes in major gut microbiota phylotypes, Bacteroidetes, Firmicutes, and Actinobacteria with quantification of Firmicutes/Bacteroidetes in NAFLD patients with concomitant CVD. Materials and methods: There were 109 NAFLD subjects (25 with concomitant arterial hypertension [AH] and 24 with coronary artery disease [CAD]) enrolled. The gut microbiota composition was assessed by qPCR. Results and conclusions: There was a marked tendency towards an increase in the concentration of Bacte- roidetes (by 37.11% and 21.30%, respectively) with a decrease in Firmicutes (by 7.38% and 7.77%, respectively) found in both groups with comorbid CAD and AH with the identified changes not reaching a statistical significance. A statistically significant decrease in the concentration of Actinobacteria was revealed in pa- tients with NAFLD with concomitant CAD at 41.37% (p <0.05) as compared with those with an isolated NAFLD. In patients with concomitant AH, the content of Actinobacteria dropped by 12.35%, which was statistically insignificant. There were changes found in the intestinal microbiota composition, namely decrease in Actinobacteria in patients with CAD, which requires further research.
- MeSH
- Adult MeSH
- Endotoxins analysis MeSH
- Myocardial Ischemia etiology complications MeSH
- Cardiovascular Diseases * etiology complications MeSH
- Comorbidity MeSH
- Real-Time Polymerase Chain Reaction methods MeSH
- Middle Aged MeSH
- Humans MeSH
- Non-alcoholic Fatty Liver Disease complications MeSH
- Aged MeSH
- Statistics as Topic MeSH
- Gastrointestinal Microbiome * MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Aged MeSH
- Publication type
- Clinical Study MeSH
- Geographicals
- Ukraine MeSH
Accuracy and transparency of scientific data are becoming more and more relevant with the increasing concern regarding the evaluation of data reproducibility in many research areas. This concern is also true for quantifying coding and noncoding RNAs, with the remarkable increase in publications reporting RNA profiling and sequencing studies. To address the problem, we propose the following recommendations: (a) accurate documentation of experimental procedures in Materials and methods (and not only in the supplementary information, as many journals have a strict mandate for making Materials and methods as visible as possible in the main text); (b) submission of RT-qPCR raw data for all experiments reported; and (c) adoption of a unified, simple format for submitted RT-qPCR raw data. The Real-time PCR Data Essential Spreadsheet Format (RDES) was created for this purpose.
