Application Of qPCR Research Articles

Verification and application of qPCR and viability-qPCR for Legionella monitoring in evaporative cooling systems complementing the conventional culture method

To date, in many countries the only legally valid method for evaporative cooling system (ECS) monitoring is the culture method. However, a duration of up to 14 days and a risk of underestimation of Legionella concentrations are seen as limitations of cultivation methods. Rapid cultivation-independent methods are an important step towards a more practicable monitoring of ECS to quickly control interventions if elevated concentrations of Legionella are found.Two commercial kits for quantitative polymerase chain reaction (qPCR) and viability-qPCR (v-qPCR) were studied, comprising sample filtration and DNA extraction. Cryopreserved Legionella pneumophila were established as calibration standard with intact (ILC) and total Legionella count (TLC) determined by flow cytometry before conducting spiking experiments in commercial mineral water and artificial process water. Final assessment was carried out using real ECS samples.Recovery and robustness ranged from 86 to 108 % for qPCR with a drop to 40–60 % for v-qPCR when compared to direct extraction, possibly attributable to cell damage during sample concentration. All methods including culture did perform well regarding linearity with R2 ≥ 0.95 for most trials. Detected concentrations in comparison to spiked Legionella counts differed with culture averaging 25 ± 7 % of spiked ILC and v-qPCR being closest to spiked concentrations with 65–144 %. In comparison, qPCR was several fold above spiked TLC concentrations. For real ECS samples Legionella spp. were detected in concentrations above 103 GU/100 mL by v-qPCR in 70–92 % of samples, depending on the kit used. Most of these samples were either culture-negative or not evaluable on agar plates.This study showed that a cryopreserved bacterial standard based examination is applicable and can be used for future v-qPCR verification. For assessment of differences in results between culture and v-qPCR/qPCR in ECS samples expert knowledge about the operating mode and used analytical methods is required. Guidelines addressing this issue could be a solution.

QPCR-based phytoplankton abundance and chlorophyll a: A multi-year study in twelve large freshwater rivers across the United States

Phytoplankton overgrowth, which characterizes the eutrophication or trophic status of surface water bodies, threatens ecosystems and public health. Quantitative polymerase chain reaction (qPCR) is promising for assessing the abundance and community composition of phytoplankton. However, applications of qPCR to indicate eutrophication and trophic status, especially in lotic systems, have yet to be comprehensively evaluated. For the first time, this study correlates qPCR-based phytoplankton abundance with chlorophyll a (the most widely used indicator of eutrophication and trophic status) in multiple freshwater rivers. From early summer to late fall in 2017, 2018, and 2019, we evaluated phytoplankton, chlorophyll a, pheophytin a, and the Trophic Level Index (TLI) in twelve large freshwater rivers in three regions (western, midcontinent, and eastern) in the United States. qPCR-based phytoplankton abundance had positive allometric correlations with chlorophyll a concentration (adjusted R2 = 0.5437, p-value < 0.001), pheophytin a concentration (adjusted R2 = 0.3378, p-value <0.001), and TLI (adjusted R2 = 0.4789, p-value < 0.001). Thus, a greater phytoplankton abundance suggests a higher trophic status. This work also presents the numerical values of qPCR-based phytoplankton abundance defining the boundaries among trophic statuses (e.g., oligotrophic, mesotrophic, and eutrophic) of freshwater rivers. The sampling sites in the midcontinent rivers were more eutrophic because they had significantly higher chlorophyll a concentrations, pheophytin a concentrations, and TLI values than the sites in the western and eastern rivers. The higher phytoplankton abundance at the midcontinent sites confirmed their higher trophic status. By linking qPCR-based phytoplankton abundance to chlorophyll a, this study demonstrates that qPCR is a promising avenue to investigate the population dynamics of phytoplankton and the trophic status (or eutrophication) of freshwater rivers.

Characterization of Taxonomic and Functional Dynamics Associated with Harmful Algal Bloom Formation in Recreational Water Ecosystems.

Harmful algal bloom (HAB) formation leads to the eutrophication of water ecosystems and may render recreational lakes unsuitable for human use. We evaluated the applicability and comparison of metabarcoding, metagenomics, qPCR, and ELISA-based methods for cyanobacteria/cyanotoxin detection in bloom and non-bloom sites for the Great Lakes region. DNA sequencing-based methods robustly identified differences between bloom and non-bloom samples (e.g., the relative prominence of Anabaena and Planktothrix). Shotgun sequencing strategies also identified the enrichment of metabolic genes typical of cyanobacteria in bloom samples, though toxin genes were not detected, suggesting deeper sequencing or PCR methods may be needed to detect low-abundance toxin genes. PCR and ELISA indicated microcystin levels and microcystin gene copies were significantly more abundant in bloom sites. However, not all bloom samples were positive for microcystin, possibly due to bloom development by non-toxin-producing species. Additionally, microcystin levels were significantly correlated (positively) with microcystin gene copy number but not with total cyanobacterial 16S gene copies. In summary, next-generation sequencing-based methods can identify specific taxonomic and functional targets, which can be used for absolute quantification methods (qPCR and ELISA) to augment conventional water monitoring strategies.

Relative gene expression analysis of catalase in environmental RNA from Japanese medaka exposed to toxic chemicals

AbstractEnvironmental RNA (eRNA) has the potential as a non‐invasive tool for assessing the physiological status of macro‐organisms, yet the quantitative method for relative gene expression analysis is still underexplored. To bridge this gap, this study introduces a relative quantification method for eRNA, employing quantitative PCR (qPCR) to evaluate the expression of the antioxidant gene, catalase (cat), in the Japanese medaka Oryzias latipes exposed to two toxic chemicals for 96 h: chlorpyrifos (CPS) and carbamazepine. Our results showed that, in eRNA, genes frequently used as a reference for tissue or cells in conventional expression analysis correlated with each other. Also, one of them (elfa) exhibited less variability, showing its potential suitability as a reference gene in eRNA analyses. Additionally, cat expression levels in eRNA increased with increasing CPS concentrations, in a concentration‐response manner. These results suggest the promising potential of qPCR applications for eRNA in the monitoring of an organism's health and response to environmental changes. However, we observed disparities in gene expression levels of cat and beta‐actin (actb) between eRNA and whole fish body, indicating eRNA might have a biased origin. Further research is needed to uncover the origin of eRNA and determine the limitations and applicability domain of eRNA analysis.

Establishment of a Rapid LAMP Assay for Aeromonas hydrophila and Comparison with the Application of qPCR.

The development of an exceptionally sensitive diagnostic technique for early identification of aquaculture diseases, specifically Aeromonas hydrophila, is essential for efficient management of disease outbreaks at aquaculture locations. In this research, a swift and sensitive diagnostic assay employing Loop-mediated isothermal amplification (LAMP) of Aeromonas hydrophila was devised and compared to the conventional qPCR method documented by Rong Wang. Validation of the diagnostic assay was carried out using actual samples obtained from aquaculture fish. The findings revealed that based on the rapid detection of crude bacterial genomic DNA, the fluorescent LAMP assay possessed a lower limit of detection (LOD) of 0.559 ng/μL (0.315-1.693, 95% CI), while the LOD for qPCR stood at 4.301 ng/μL (2.084-8.876, 95% CI). Both techniques demonstrated outstanding specificity, exhibiting no cross-reactivity with bacteria from the same or closely related genera. A total of 74 fish samples suspected to be infected with the fish disease were gathered, with 26 and 23 samples testing positive for Aeromonas hydrophila via LAMP and qPCR, respectively. The concordance analysis for LAMP and qPCR methods generated a Kappa value of 0.909 (0.778-1.000, 95% CI), signifying a high degree of diagnostic consensus. This study highlights that the LAMP assay eliminates the thermal cycle temperature change process of qPCR, uses lysate to crudely extract bacterial genomic DNA, and can complete the detection within 40 min, rendering it a practical and efficient alternative for monitoring disease outbreaks at aquaculture sites.

Genomic copy number variability at the genus, species and population levels impacts in situ ecological analyses of dinoflagellates and harmful algal blooms

The application of meta-barcoding, qPCR, and metagenomics to aquatic eukaryotic microbial communities requires knowledge of genomic copy number variability (CNV). CNV may be particularly relevant to functional genes, impacting dosage and expression, yet little is known of the scale and role of CNV in microbial eukaryotes. Here, we quantify CNV of rRNA and a gene involved in Paralytic Shellfish Toxin (PST) synthesis (sxtA4), in 51 strains of 4 Alexandrium (Dinophyceae) species. Genomes varied up to threefold within species and ~7-fold amongst species, with the largest (A. pacificum, 130 ± 1.3 pg cell−1 /~127 Gbp) in the largest size category of any eukaryote. Genomic copy numbers (GCN) of rRNA varied by 6 orders of magnitude amongst Alexandrium (102– 108 copies cell−1) and were significantly related to genome size. Within the population CNV of rRNA was 2 orders of magnitude (105 – 107 cell−1) in 15 isolates from one population, demonstrating that quantitative data based on rRNA genes needs considerable caution in interpretation, even if validated against locally isolated strains. Despite up to 30 years in laboratory culture, rRNA CNV and genome size variability were not correlated with time in culture. Cell volume was only weakly associated with rRNA GCN (20–22% variance explained across dinoflagellates, 4% in Gonyaulacales). GCN of sxtA4 varied from 0–102 copies cell−1, was significantly related to PSTs (ng cell−1), displaying a gene dosage effect modulating PST production. Our data indicate that in dinoflagellates, a major marine eukaryotic group, low-copy functional genes are more reliable and informative targets for quantification of ecological processes than unstable rRNA genes.

Identification and validation of immune-associated NETosis subtypes and biomarkers in anti-neutrophil cytoplasmic antibody associated glomerulonephritis.

NETosis is a new form of cell death, marked by DNA chromatin release from dead neutrophils. While it aids in microbe defense, it may worsen inflammation in autoimmune diseases, causing tissue harm. The impact of NETosis on Anti-neutrophil Cytoplasmic Antibody-associated Glomerulonephritis (ANCA-GN) remains unexplored and requires investigation. First, a weighted gene co-expression network analysis (WGCNA) was conducted to uncover differential expression of neutrophil extranuclear trap-associated genes (DE-NETs) in ANCA-GN. The NETosisScore model was established through the single sample gene set enrichment analysis (ssGSEA), which categorized all patients into high-risk and low-risk groups. The accuracy of model was assessed by ROC curve. The biological function of various subgroups was explored through Gene Set Variation Analysis (GSVA), while the abundance of immune cell infiltration was measured with CIBERSORT. Furthermore, the key NETosis-related genes (NRGs) were identified using threemachine learning algorithms, and their relationship with renal function was analyzed through the NephroseqV5 database. Through the application of qPCR and immunohistochemical staining techniques, the mRNA and protein expression levels of NRGs were determined in patients with ANCA-GN andcontrol. A NETosisScore model was developed from 18 DE-NETs using the ssGSEA algorithm. The model's ability to predict ANCA-GN patients with a ROC AUC of 0.921. The high-risk group in ANCA-GN showed enrichment of immune-related pathways and greater infiltration of immune cells, as revealed by KEGG enrichment analysis and CIBERSORT. Using three machine learning algorithms, we identified six NRGs. Significant positive correlations were found between NRGs and CCR, macrophages, T-cell co-inhibition, and TIL. Further KEGG analysis revealed that the functions of NRGs may be closely related to the toll-like receptor signaling pathway. The levels of NRGs increased as kidney function declined and were positively correlated with Scr (serum creatinine) and negatively correlated with GFR (glomerular filtration rate), qPCR analysis showed increased expression of most NRGs in ANCA-GN patients. Furthermore, immunohistochemical staining confirmed higher expression of all NRGs in ANCA-GN patients. NETosisScore model accurately predicts high-risk patients in ANCA-GN with enriched immune pathways, 6 NRGs identified as potential biomarkers.

Microbial water quality of pond Alleben (Gaziantep, Turkey) in winter and climatic changes in the region

Abstract This study assessed the microbial quality of water sources in winter by microbiological methods and molecular-based quantitative polymerase chain reaction (qPCR) and potential future bacterial loads in the Alleben pond based on the increase in water temperature. Total bacteria, total coliform, and Escherichia coli levels using the qPCR were significantly higher in the water samples than those enumerated by culture-dependent microbiological methods. These results showed that our qPCR approach is suitable to estimate the relative abundances of uncultured and cultured indicator microorganisms affected by the water characteristics. Based on our climate data, the daily average rate of 1.8 °C/increase associated with air temperature for the pond was recorded. As a result of the warming of the water surface, temperature thresholds in winter may breach and, thus, the physicochemical and biological properties of the pond may change by increasing bacterial growth rates. This study is the first analysis to apply combined climate change with molecular approaches to better understand how bacterial concentrations in aquatic environments may change in the future based on air temperature variations for the Gaziantep region. This analysis indicates developing surface water treatment measures and climate adaptation strategies that may reduce bacterial contamination in the Alleben pond.

Mining Lepeophtheirus salmonis RNA-Seq data for qPCR reference genes and their application in Caligus elongatus

Lepeophtheirus salmonis and Caligus elongatus are two parasitic copepod species posing a significant threat to salmonid aquaculture. Consequently, several gene expression studies are executed each year to gain new knowledge and treatment strategies. Though, to enable accurate gene expression measurements by quantitative real time PCR, stable reference genes are needed. Previous studies have mainly focused on a few genes selected based on their function as housekeeping genes, as these are often stably expressed in various cells and tissues. In the present study, however, RNA-sequencing data from 127 L. salmonis samples from different life stages and diverse environmental conditions were used to identify new candidate reference genes displaying low variation. From this, six genes were selected, and the stability validated by qPCR on samples from different life stages. Since neither a genome nor comprehensive RNA sequencing data are available for C. elongatus, homologous genes to those identified for L. salmonis were identified within a C. elongatus transcriptome assembly and validated by qPCR in different life stages. Overall, the genes eukaryotic translation initiation factor 1A (EIF1A) and serine/threonine-protein phosphatase 1 (PP1) displayed the highest stability in L. salmonis, while the combination of PP1 and ribosomal protein S13 (RPS13) was found to have the highest stability in C. elongatus. These genes are well-suited reference genes for qPCR applications which allow for accurate normalization of target genes.

Quantitative PCR Assay as a Tool for the Detection of Lactobacilli in Sicilian Table Olives Produced at an Industrial Scale

Table olives are an important fermented product of the Mediterranean area consumed all over the world. In our era, the food industry requires a safe and stable final product with desirable characteristics for the consumer. In the present study, two different experimental fermentations (L, with Lactiplantibacillus plantarum strains, and LY, with L. plantarum strains and Wickerhamomyces anomalus strain) were conducted and monitored up to 180 days and compared with a spontaneous fermentation, used as control (C). The safety and stability of table olives were determined by applying a plate count and quantitative real-time PCR (qPCR) approach. Compared with the control sample (C), experimental fermentations showed a faster acidification and a good inhibition rate of spoilage bacteria, indicating the safety of the process. Quantitative PCR data confirmed the abundance of the Lactobacillus group in both experimental table olives, confirming the importance of the starter cultures for the stability of the final product. In conclusion, the use of starter cultures ensures the safety of industrially produced table olives, and the application of qPCR seems to be a promising tool to detect and quantify lactobacilli as a positive biomarker of table olive fermentation.

Globular adiponectin-mediated vascular remodeling by affecting the secretion of adventitial-derived tumor necrosis factor-α induced by urotensin II.

In this study, we explored how adiponectin mediated urotensin II (UII)‍-induced tumor necrosis factor-‍α (TNF-‍α) and α‍-smooth muscle actin (α‍-SMA) expression and ensuing intracellular signaling pathways in adventitial fibroblasts (AFs). Growth-arrested AFs and rat tunica adventitia of vessels were incubated with UII and inhibitors of signal transduction pathways for 1‍‒‍24 h. The cells were then harvested for TNF-α receptor (TNF-‍α-R) messenger RNA (mRNA) and TNF-‍α protein expression determination by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Adiponectin and adiponectin receptor (adipoR) expression was measured by RT-PCR, quantitative real-time PCR (qPCR), immunohistochemical analysis, and cell counting kit-8 (CCK-8) cell proliferation experiments. We then quantified TNF-α and α-SMA mRNA and protein expression levels by qPCR and immunofluorescence (IF) staining. RNA interference (RNAi) was used to explore the function of the adipoR genes. To investigate the signaling pathway, we applied western blotting (WB) to examine phosphorylation of adenosine 5'-monophosphate (AMP)‍-activated protein kinase (AMPK). In vivo, an adiponectin (APN)‍-knockout (APN-KO) mouse model mimicking adventitial inflammation was generated to measure TNF-α and α‍-SMA expression by application of qPCR and IF, with the goal of gaining a comprehensive atlas of adiponectin in vascular remodeling. In both cells and tissues, UII promoted TNF-α protein and TNF-α-R secretion in a dose- and time-dependent manner via Rho/protein kinase C (PKC) pathway. We detected marked expression of adipoR1, T-cadherin, and calreticulin as well as a moderate presence of adipoR2 in AFs, while no adiponectin was observed. Globular adiponectin (gAd) fostered the growth of AFs, and acted in concert with UII to induce α-SMA and TNF-α through the adipoR1/T-cadherin/calreticulin/AMPK pathway. In AFs, gAd and UII synergistically induced AMPK phosphorylation. In the adventitial inflammation model, APN deficiency up-regulated the expression of α-SMA, UII receptor (UT), and UII while inhibiting TNF-‍α expression. From the results of our study, we can speculate that UII induces TNF‍-‍α protein and TNF-‍α‍-R secretion in AFs and rat tunica adventitia of vessels via the Rho and PKC signal transduction pathways. Thus, it is plausible that adiponectin is a major player in adventitial progression and could serve as a novel therapeutic target for cardiovascular disease administration.

Limitations of qPCR to estimate DNA quantity: An RFU method to facilitate inter-laboratory comparisons for activity level, and general applicability

The application of qPCR to estimate the quantity of DNA present is usually based upon a short amplicon (typically c.80bp) and a longer amplicon (typically c.200–300bp) where the latter is used to determine the amount of degradation present in a sample. The data are used to make decisions about a) whether there is sufficient template to amplify? b) how much of the elution volume to forward to PCR? A typical multiplex amplifies template in the region of 100–500bp. Consequently, the results from an 80bp amplicon will tend to overestimate the actual amplifiable quantity that is present in a degraded sample. To compensate, a method is presented that relates the quantity of amplifiable DNA to the average RFU of the amplified fragments. This provides greatly improved accuracy of the estimated quantity of DNA present, which may differ by more than an order of magnitude compared to qPCR. The relative DNA quantities can be apportioned per contributor once mixture proportions are ascertained with probabilistic genotyping software (EuroForMix). The motivation for this work was to provide an improved method to generate data to prepare distributions that are used to inform activity level propositions. However, other applications will benefit, particularly those where extraction and quantification are bypassed: For example direct PCR and Rapid DNA technology. The overall aim of this work was to provide a method of quantification that is standardised and can be used to compare results between different laboratories that use different multiplexes. A software solution ”ShinyRFU” is provided to aid calculations.

Pneumococcal carriage in unvaccinated children at the time of vaccine implementation into the national immunization program in Poland

We investigated pneumococcal carriage among unvaccinated children under five years of age at a time when the conjugate polysaccharide vaccine (PCV) was introduced in Poland into the national immunization program (NIP). Paired nasopharyngeal swab (NPS) and saliva samples collected between 2016 and 2020 from n = 394 children were tested with conventional culture and using qPCR. The carriage rate detected by culture was 25.4% (97 of 394), by qPCR 39.1% (155 of 394), and 40.1% (158 of 394) overall. The risk of carriage was significantly elevated among day care center attendees, and during autumn/winter months. Among isolates cultured, the most common serotypes were: 23A, 6B, 15BC, 10A, 11A. The coverage of PCV10 and PCV13 was 23.2% (23 of 99) and 26.3% (26 of 99), respectively. Application of qPCR lead to detection of 168 serotype carriage events, with serogroups 15, 6, 9 and serotype 23A most commonly detected. Although the highest number of carriers was identified by testing NPS with qPCR, saliva significantly contributed to the overall number of detected carriers. Co-carriage of multiple serotypes was detected in 25.3% (40 of 158) of carriers. The results of this study represent a baseline for the future surveillance of effects of pneumococcal vaccines in NIP in Poland.

Improvement and Application of qPCR (Real-Time Quantitative Polymerase Chain Reaction) Data Processing Method for Home-Made Integrated Nucleic Acid Detection System.

Because it has many advantages such as rapidity and accuracy, nucleic acid detection is applied to infectious disease diagnosis more and more. An automatic integrated nucleic acid detection system based on real-time PCR is developed by our research group to conduct point-of-care testing of infectious pathogens. The home-made detection system collects fluorescence data in each PCR cycle through an integrated dual-channel fluorescence detection module and then real-time fluorescence curves are drawn by the software, which can tell the results of the diagnostics after some processing and analysis. However, owing to the disturbance of the environment or the imperfect of nucleic acid extraction before PCR, the fluorescence curves sometimes may contain several abnormal points. For the purpose of enhancing its ability to deal with these iffy curves and improve the accuracy of the testing results, in this study, the SDM-based qPCR data processing algorithm was studied and 11 groups of qPCR data that have different flaws from the clinical samples detected by this system were chosen to prove the practicability of the method. In comparison with the conventional threshold-based method, the Cq values calculated by the SDM-based method were more close to the actual values, meaning it can overcome the shortcomings of the conventional methods such as being unable to accommodate noise and being unable to avoiding abnormal data. With the improvement of this data processing algorithm, the stability of our system and the reliability and accuracy of the results are greatly improved.

Development and Testing of Species-specific Quantitative PCR Assays for Environmental DNA Applications.

New, non-invasive methods for detecting and monitoring species presence are being developed to aid in fisheries and wildlife conservation management. The use of environmental DNA (eDNA) samples for detecting macrobiota is one such group of methods that is rapidly becoming popular and being implemented in national management programs. Here we focus on the development of species-specific targeted assays for probe-based quantitative PCR (qPCR) applications. Using probe-based qPCR offers greater specificity than is possible with primers alone. Furthermore, the ability to quantify the amount of DNA in a sample can be useful in our understanding of the ecology of eDNA and the interpretation of eDNA detection patterns in the field. Careful consideration is needed in the development and testing of these assays to ensure the sensitivity and specificity of detecting the target species from an environmental sample. In this protocol we will delineate the steps needed to design and test probe-based assays for the detection of a target species; including creation of sequence databases, assay design, assay selection and optimization, testing assay performance, and field validation. Following these steps will help achieve an efficient, sensitive, and specific assay that can be used with confidence. We demonstrate this process with our assay designed for populations of the mucket (Actinonaias ligamentina), a freshwater mussel species found in the Clinch River, USA.

Molecular Detection Mapping and Analysis Platform for R (MDMAPR) facilitating the standardization, analysis, visualization, and sharing of qPCR data and metadata.

Quantitative polymerase chain reaction (qPCR) has been used as a standard molecular detection tool in many scientific fields. Unfortunately, there is no standard method for managing published qPCR data, and those currently used generally focus on only managing raw fluorescence data. However, associated with qPCR experiments are extensive sample and assay metadata, often under-examined and under-reported. Here, we present the Molecular Detection Mapping and Analysis Platform for R (MDMAPR), an open-source and fully scalable informatics tool for researchers to merge raw qPCR fluorescence data with associated metadata into a standard format, while geospatially visualizing the distribution of the data and relative intensity of the qPCR results. The advance of this approach is in the ability to use MDMAPR to store varied qPCR data. This includes pathogen and environmental qPCR species detection studies ideally suited to geographical visualization. However, it also goes beyond these and can be utilized with other qPCR data including gene expression studies, quantification studies used in identifying health dangers associated with food and water bacteria, and the identification of unknown samples. In addition, MDMAPR’s novel centralized management and geospatial visualization of qPCR data can further enable cross-discipline large-scale qPCR data standardization and accessibility to support research spanning multiple fields of science and qPCR applications.

Principles of computer-controlled linear motion applied to an open-source affordable liquid handler for automated micropipetting

OTTO is an open-source automated liquid handler that can be fabricated at a cost of $1,500 using off-the-shelf and 3D-printable parts as an alternative to commercial devices. Open-source approaches have been applied to build syringe pumps, centrifuges, and other laboratory equipment. These devices are affordable but generally rely on a single motor to perform simple operations and thus do not fully utilize the potential of the Maker Movement. Open-source linear actuators and microcontrollers enable the fabrication of more complex laboratory instruments that rely on 3D positioning and accurate dispensing of fluids, such as automated liquid handlers. These instruments can be built rapidly and affordably, thereby providing access to highly reproducible sample preparation for common biological assays such as qPCR. We applied the design principles of speed and accuracy, unattended automation, and open-source components to build an automated liquid handler that controls micropipetting of liquids in 3D space at speeds and positional resolutions required for qPCR. In benchmarking studies, OTTO showed accuracy and sample preparation times comparable to manual qPCR. The ability to control linear motion and liquid dispensing using affordable off-the-shelf and 3D-printable parts can facilitate the adoption of open-source automated liquid handlers for qPCR, bioplotting, and other bioinstrumentation applications.

Microbiological examination of water and aerosols from four industrial evaporative cooling systems in regard to risk of Legionella emissions and methodological suggestions for surveillance.

The hygienic risk associated with evaporative cooling systems in Germany is currently only assessed by determining concentrations of Legionella spp. in the corresponding cooling waters. Relevant for the health risk is however the load of Legionella in emitted aerosols. In this work aerosol emissions from four industrial cooling systems (A - D) were analyzed. A microbiological air bioburden factor (MABF) is suggested to be useful to assess the overall microbiological load of emitted air and to judge the efficiency of droplet separation and overall microbiological retention. Whereas the MABF by itself only serves as a technical quality assurance (QA) parameter, the hygienic relevance has to be seen in combination with the assessment of Legionella either contained in the aerosol or in the cooling water. Plate counting of colonies was an appropriate method to quantify Legionella spp. in aerosols given the short time of flight at the chosen sampling locations and resulting low risk of desiccation. qPCR data on the other hand proved more reproducible than the culture approach to quantify Legionella spp. concentrations in cooling water-. The application of qPCR also allowed to assess the relative proportion of Legionella pneumophila within the total pool of Legionella which adds epidemiological relevance to risk assessment. A traffic light system was proposed to guide interpretation of qPCR data. The four industrial systems greatly differed in all measured parameters leading to different associated risks.

Loop-mediated isothermal amplification (LAMP) reaction as viable PCR substitute for diagnostic applications: a comparative analysis study of LAMP, conventional PCR, nested PCR (nPCR) and real-time PCR (qPCR) based on Entamoeba histolytica DNA derived from faecal sample

BackgroundThis study reports the analytical sensitivity and specificity of a Loop-mediated isothermal amplification (LAMP) and compares its amplification performance with conventional PCR, nested PCR (nPCR) and real-time PCR (qPCR). All the assays demonstrated in this study were developed based on Serine-rich Entamoeba histolytica protein (SREHP) gene as study model.ResultsA set of SREHP gene specific LAMP primers were designed for the specific detection of Entamoeba histolytica. This set of primers recorded 100% specificity when it was evaluated against 3 medically important Entamoeba species and 75 other pathogenic microorganisms. These primers were later modified for conventional PCR, nPCR and qPCR applications. Besides, 3 different post-LAMP analyses including agarose gel electrophoresis, nucleic acid lateral flow immunoassay and calcein-manganese dye techniques were used to compare their limit of detection (LoD). One E. histolytica trophozoite was recorded as the LoD for all the 3 post-LAMP analysis methods when tested with E. histolytica DNA extracted from spiked stool samples. In contrast, none of the PCR method outperformed LAMP as both qPCR and nPCR recorded LoD of 100 trophozoites while the LoD of conventional PCR was 1000 trophozoites.ConclusionsThe analytical sensitivity comparison among the conventional PCR, nPCR, qPCR and LAMP reveals that the LAMP outperformed the others in terms of LoD and amplification time. Hence, LAMP is a relevant alternative DNA-based amplification platform for sensitive and specific detection of pathogens.

Application of qPCR for multicopper oxidase gene (MCO) in biogenic amines degradation by Lactobacillus casei

Degradation of undesirable biogenic amines (BAs) in foodstuffs by microorganisms is considered one of the most effective ways of eliminating their toxicity. In this study, we designed two sets of primers for the detection and quantification of the multicopper oxidase gene (MCO), which encodes an enzyme involved in BAs degradation, and endogenous (glyceraldehyde-3-phosphate dehydrogenase) gene (GAPDH) in Lactobacillus casei group by real-time PCR (qPCR). We tested 15 Lactobacillus strains in the screening assays (thus, MCO gene possessing assay (PCR) and monitoring of BAs degradation by HPLC-UV), in which Lactobacillus casei CCDM 198 exhibited the best degradation abilities. For this strain, we monitored the expression of the target gene (MCO) in time (qPCR), the effect of redox treatments (cysteine, ascorbic acid) on the expression of the gene, and the ability to degrade BAs not only in a modified MRS medium (MRS/2) but also in a real food sample (milk). Moreover, decarboxylase activity (ability to form BAs) of this strain was excluded. According to the results, CCDM 198 significantly (P < 0.05) reduced BAs (putrescine, histamine, tyramine, cadaverine), up to 25% decline in 48 h. The highest level of relative expression of MCO (5.21 ± 0.14) was achieved in MRS/2 media with cysteine.