We report the development of a versatile system based on the oscillating‐flow methodology in a thermal gradient system for nucleic acid analysis. Analysis of DNA and RNA samples were performed in the ...device, without additional temperature control and complexity. The technique reported in this study eliminates the need for predetermined fluidic channels for thermocycles, and complexity involved with additional incubation steps required for RNA amplification. A microfluidic device was fabricated using rapid prototyping by simply sandwiching dual side adhesive Kapton tape and a polydimethylsiloxane spacer between glass microscope slides. Amplification of the 181‐bp segment of a viral phage DNA (ΦX174) and B2M gene in human RNA samples was demonstrated using the system. The developed system enables simultaneous acquisition of amplification and melt curves, eliminating the need for postprocessing. A direct comparison between the oscillating‐flow system and a commercial real‐time polymerase chain reaction (PCR) instrument showed complete agreement in PCR data and improved sample‐to‐result time by eliminating an additional 30 min melt curve step required in commercial PCR systems.
An oscillating‐flow thermal gradient system for nucleic acid analysis is developed in this study. The authors reported the simultaneous acquisition of PCR data including amplification and melt information, eliminating the need for post‐processing time. A combination of a simple microfluidic channel, thermal gradient, and flow control resulted in a versatile PCR system that eliminates the need for predetermined amplification channels and additional temperature control for incubation steps. The oscillating‐flow PCR system was compared with a commercial real‐time PCR instrument.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
An accurate genotyping analysis is one of the critical prerequisites for lung cancer targeted therapy. Here, a quantitative polymerase chain reaction (qPCR)‐based mutation detection system, ...mutation‐selected amplification‐specific system PCR (MASS‐PCR), is developed. The specific primers and probes used in MASS‐PCR exactly match with the mutant sequence that only allows mutant gene to emit the fluorescence peak. To determine the sensitivity of MASS‐PCR, 717 lung cancer specimens, 61 formalin‐fixed paraffin‐embedded (FFPE) tissues, and 656 fresh reaction tissues are collected and undergo mutation detection of lung cancer driver genes (EGFR, KRAS, BRAF, HER2, MET, ALK, and ROS1). These samples are divided into two groups. Mutations in Group I, which has 631 fresh reaction tissues, are analyzed by MASS‐PCR and the amplification refractory mutation system PCR (ARMS‐PCR). While group II samples, 25 fresh reaction tissues and 61 FFPE tissues, are screened through MASS‐PCR and next‐generation sequencing (NGS). All results are verified by direct sequencing. MASS‐PCR shows high consistency with ARMS‐PCR (kappa value > 0.733) and NGS (kappa value = 0.79) (P < 0.001). For the samples with inconsistent MASS‐PCR and ARMS‐PCR results, DS results more likely support the MASS‐PCR results. These data suggest that MASS‐PCR is a convenient, accurate, and economical method for the detection of lung cancer driver gene mutations in clinical practice.
A quantitative polymerase chain reaction (qPCR)‐based mutation detection system, mutation‐selected amplification‐specific system PCR, is developed. The exact matched primers and probes with mutation loci contribute to improving the detection accuracy. It has good performance in detecting mutations in lung cancer driver genes.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Mycoplasma testing is a required part of release testing to ensure a cell product is sterile and safe to infuse into patients. To meet this safety standard with an increasing product volume, our ...group aimed to enable batching of DNA isolation and Mycoplasma PCR testing of cultured cell products to conserve reagents and decrease technologist time. Towards this aim, we evaluated the stability of Mycoplasma DNA in snap-frozen cells in media stored at -80°C compared to the supplier-recommended isolation from freshly harvested cells.
DNA from three cultured cell products - CAR T-cells, TGFβ-imprinted NK cells, and NK cells - was isolated both fresh on the day of harvest and after storage at -80°C following snap-freezing. Cultured cells in media were spiked with three different Mycoplasma strains at 10CFU/mL prior to DNA isolation: M. fermentans, M. orale, & M. pneumoniae. These samples were run in tandem with DNA isolated from unspiked cells in media. Mycoplasma PCR testing was performed using the MycoTOOL Mycoplasma Real-Time PCR Kit. All extraction, inhibition, and PCR controls passed acceptance criteria. All spiked cells in media frozen at -80°C for 14 (n=2) or 15 days (n=1) tested positive for the Mycoplasma species in 8 of 8 PCR reaction wells. All unspiked cells in media samples tested negative.
In summary, Mycoplasma DNA is stable in snap-frozen cultured cells in media stored at -80°C up to 15 days. This validation allows sample batching for PCR testing to reduce technologist time and maximize reagent utilization. Our group plans to perform additional testing of spiked, snap-frozen cells in media stored for > 15 days at -80°C, the stability of spiked DNA stored for > 3 days at -20°C, and to validate increased Proteinase K volume for DNA isolation to overcome varying protein content in distinct culture media.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Enteromyxum leei and Enteromyxum fugu, which are myxosporean parasites, were first found in cultured tiger puffer Takifugu rubripes in Korea. We collected four tiger puffers that showed severe ...emaciation signs for our experiments. DNA sequencing was confirmed that the tiger puffers were coinfected with E. leei and E. fugu. Furthermore, similar amounts of E. leei and E. fugu were confirmed using real‐time PCR in the intestine. To the best of our knowledge, there have been no reports of E. fugu infection in the olive flounder Paralichthys olivaceus. However, the diagnosis of inflowing water, discharged water and olive flounder samples using highly sensitive diagnostic methods confirmed the presence of E. fugu in water and fish samples from olive flounder farms near the tiger puffer farm. Therefore, the present study aimed to develop highly sensitive diagnostic methods such as real‐time and two‐step PCR for early diagnosis and follow‐up of the emaciation disease and multiplex PCR for rapid diagnosis. The multiplex PCR method exhibited the same sensitivity as the one‐step PCR method developed in this study, demonstrating its efficacy for rapid diagnosis. Therefore, the suggested methods can be utilized for the early diagnosis and rapid diagnosis of emaciation diseases and reduction of economic losses through rapid disease control.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
6.
dPCR: A Technology Review Quan, Phenix-Lan; Sauzade, Martin; Brouzes, Eric
Sensors (Basel, Switzerland),
04/2018, Volume:
18, Issue:
4
Journal Article
Peer reviewed
Open access
Digital Polymerase Chain Reaction (dPCR) is a novel method for the absolute quantification of target nucleic acids. Quantification by dPCR hinges on the fact that the random distribution of molecules ...in many partitions follows a Poisson distribution. Each partition acts as an individual PCR microreactor and partitions containing amplified target sequences are detected by fluorescence. The proportion of PCR-positive partitions suffices to determine the concentration of the target sequence without a need for calibration. Advances in microfluidics enabled the current revolution of digital quantification by providing efficient partitioning methods. In this review, we compare the fundamental concepts behind the quantification of nucleic acids by dPCR and quantitative real-time PCR (qPCR). We detail the underlying statistics of dPCR and explain how it defines its precision and performance metrics. We review the different microfluidic digital PCR formats, present their underlying physical principles, and analyze the technological evolution of dPCR platforms. We present the novel multiplexing strategies enabled by dPCR and examine how isothermal amplification could be an alternative to PCR in digital assays. Finally, we determine whether the theoretical advantages of dPCR over qPCR hold true by perusing studies that directly compare assays implemented with both methods.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Three molecular assays were used to detect and quantify white spot syndrome virus (WSSV) in DNA extracted from seston size-fractioned (0.02, 0.2, 1.2, and 20 μm) samples collected from a coastal ...lagoon and an adjacent shrimp farm. From 107 DNA extracts, only two from one sample tested positive for WSSV with nested PCR in the 1.2 and 20 μm fractions. These results were confirmed by a semi-quantitative (IQ2000TM WSSV Detection and Prevention System) and a quantitative (IQREALTM WSSV Quantitative System) detection system based, based, respectively, on nested PCR and real-time PCR. A first viral load reference value (6.54 × 104 WSSV copies/mL) was established in a seston size fraction (1.2−20 μm). The results suggest that WSSV could be associated with both resuspension of fine clays and silts, and nanoplankton and organic colloids during infectious events.
Fungal PCR has undergone considerable standardization and, together with the availability of commercial assays, external quality assessment schemes, and extensive performance validation data, is ...ready for widespread use for the screening and diagnosis of invasive fungal disease (IFD).
Drawing on the experience and knowledge of the leads of the various working parties of the Fungal PCR initiative, this review will address general considerations concerning the use of molecular tests for the diagnosis of IFD, before focusing specifically on the technical and clinical aspects of molecular testing for the main causes of IFD and recent technological developments.
For infections caused by Aspergillus, Candida, and Pneumocystis jirovecii, PCR testing is recommended, and combination with serological testing will likely enhance the diagnosis. For other IFD (e.g. mucormycosis), molecular diagnostics represent the only non-classical mycological approach toward diagnoses, and continued performance validation and standardization have improved confidence in such testing. The emergence of antifungal resistance can be diagnosed, in part, through molecular testing. Next-generation sequencing has the potential to significantly improve our understanding of fungal phylogeny, epidemiology, pathogenesis, mycobiome/microbiome, and interactions with the host, while identifying novel and existing mechanisms of antifungal resistance and novel diagnostic/therapeutic targets.
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•Simple and accurate spectrophotometric method for the determination of celecoxib and tramadol.•Principle component regression chemometric model was applied.•The model optimized and ...validated.
Celecoxib and tramadol have been combined in a novel FDA-approved medication to address acute pain disorders requiring opioid treatment when other analgesics proved either intolerable or ineffective. The absorbance spectra of celecoxib and tramadol exhibit significant overlap, posing challenges for their individual quantification. This study introduces a spectrophotometric quantification approach for celecoxib and tramadol using a principle component regression assistive model to assist resolving the overlapped spectra and quantifying both drugs in their binary mixture. The model was constructed by establishing calibration and validation sets for the celecoxib and tramadol mixture, employing a five-level, two-factor experimental design, resulting in 25 samples. Spectral data from these mixtures were measured and preprocessed to eliminate noise in the 200–210 nm range and zero absorbance values in the 290–400 nm range. Consequently, the dataset was streamlined to 81 variables. The predicted concentrations were compared with the known concentrations of celecoxib and tramadol, and the errors in the predictions were evidenced calculating root mean square error of cross-validation and root mean square error of prediction. Validation results demonstrate the efficacy of the models in predicting outcomes; recovery rates approaching 100 % are demonstrated with relative root mean square error of prediction (RRMSEP) values of 0.052 and 0.164 for tramadol and celecoxib, respectively. The selectivity was further evaluated by quantifying celecoxib and tramadol in the presence of potentially interfering drugs. The model demonstrated success in quantifying celecoxib and tramadol in laboratory-prepared tablets, producing metrics consistent with those reported in previously established spectrophotometric methods.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Blood samples are widely used for PCR-based DNA analysis in fields such as diagnosis of infectious diseases, cancer diagnostics, and forensic genetics. In this study, the mechanisms behind ...blood-induced PCR inhibition were evaluated by use of whole blood as well as known PCR-inhibitory molecules in both digital PCR and real-time PCR. Also, electrophoretic mobility shift assay was applied to investigate interactions between inhibitory proteins and DNA, and isothermal titration calorimetry was used to directly measure effects on DNA polymerase activity. Whole blood caused a decrease in the number of positive digital PCR reactions, lowered amplification efficiency, and caused severe quenching of the fluorescence of the passive reference dye 6-carboxy-X-rhodamine as well as the double-stranded DNA binding dye EvaGreen. Immunoglobulin G was found to bind to single-stranded genomic DNA, leading to increased quantification cycle values. Hemoglobin affected the DNA polymerase activity and thus lowered the amplification efficiency. Hemoglobin and hematin were shown to be the molecules in blood responsible for the fluorescence quenching. In conclusion, hemoglobin and immunoglobulin G are the two major PCR inhibitors in blood, where the first affects amplification through a direct effect on the DNA polymerase activity and quenches the fluorescence of free dye molecules, and the latter binds to single-stranded genomic DNA, hindering DNA polymerization in the first few PCR cycles.
Graphical abstract
PCR inhibition mechanisms of hemoglobin and immunoglobulin G (IgG). Cq quantification cycle, dsDNA double-stranded DNA, ssDNA single-stranded DNA
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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