Quantification Of Nucleic Acids Research Articles

Mesophilic Argonaute-Mediated Polydisperse Droplet Biosensor for Amplification-Free, One-Pot, and Multiplexed Nucleic Acid Detection Using Deep Learning.

Detection of nucleic acids from a single multiplexed and amplification-free test is critical for ensuring food safety, clinical diagnostics, and environmental monitoring. In this study, we introduced a mesophilic Argonaute protein from Clostridium butyricum (CbAgo), which exhibits nucleic acid endonuclease activity, to achieve a programmable, amplification-free system (PASS) for rapid nucleic acid quantification at ambient temperatures in one pot. By using CbAgo-mediated binding with specific guide DNA (gDNA) and subsequent targeted cleavage of wild-type target DNAs complementary to gDNA, PASS can detect multiple foodborne pathogen DNA (<102 CFU/mL) simultaneously. The fluorescence signals were then transferred to polydisperse emulsions and analyzed by using deep learning. This simplifies the process and increases the suitability of polydisperse emulsions compared to traditional digital PCR, which requires homogeneous droplets for accurate detection. We believe that PASS has the potential to become a next-generation point-of-care digital nucleic acid detection method.

StratoLAMP: Label-free, multiplex digital loop-mediated isothermal amplification based on visual stratification of precipitate.

Multiplex, digital nucleic acid detections have important biomedical applications, but the multiplexity of existing methods is predominantly achieved using fluorescent dyes or probes, making the detection complicated and costly. Here, we present the StratoLAMP for label-free, multiplex digital loop-mediated isothermal amplification based on visual stratification of the precipitate byproduct. The StratoLAMP designates two sets of primers with different concentrations to achieve different precipitate yields when amplifying different nucleic acid targets. In the detection, deep learning image analysis is used to stratify the precipitate within each droplet and determine the encapsulated targets for nucleic acid quantification. We investigated the effect of the amplification reagents and process on the precipitate generation and optimized the assay conditions. We then implemented a deep-learning image analysis pipeline for droplet detection, achieving an overall accuracy of 94.3%. In the application, the StratoLAMP successfully achieved the simultaneous quantification of two nucleic acid targets with high accuracy. By eliminating the need for fluorescence, StratoLAMP represents a unique concept toward label-free, multiplex nucleic acid assays and an analytical tool with great cost-effectiveness.

Analytical validation of the DropXpert S6 system for diagnosis of chronic myelocytic leukemia.

Digital PCR is a powerful method for absolute nucleic acid quantification and is widely used in the absolute quantification of viral copy numbers, tumor marker detection, and prenatal diagnosis. However, for most of the existing droplet-based dPCR systems, the droplet generation, PCR reaction, and droplet detection are performed separately using different instruments. Making digital PCR both easy to use and practical by integrating the qPCR workflow into a superior all-in-one walkaway solution is one of the core ideas. A new innovative and integrated digital droplet PCR platform was developed that utilizes cutting-edge microfluidics to integrate dPCR workflows onto a single consumable chip. This makes previously complex workflows fast and simple; the whole process of droplet generation, PCR amplification, and droplet detection is completed on one chip, which meets the clinical requirement of "sample in, result out". It provides high multiplexing capabilities and strong sensitivity while all measurements were within the 95% confidence interval. This study is the first validation of the DropXpert S6 system and focuses primarily on verifying its reliability, repeatability, and consistency. In addition, the accuracy, detection limit, linearity, and precision of the system were evaluated after sample collection. Among them, the accuracy assessment by calculating the absolute bias of each target gene yielded a range from -0.1 to 0.08, all within ±0.5 logarithmic orders of magnitude; the LOB for the assay was set at 0, and the LoD value calculated using probit curves is MR4.7 (0.002%); the linearity evaluation showed that the R2 value of the BCR-ABL was 0.9996, and the R2 value of the ABL metrics calculated using the ERM standard was 0.9999; and the precision evaluation showed that all samples had a CV of less than 4% for intra-day, inter-day, and inter-instrument variation. The CV of inter-batch variation was less than 7%. The total CV was less than 5%. The results of the study demonstrate that dd-PCR can be applied to molecular detection and the clinical evaluation of CML patients and provide more precise personal treatment guidance, and its reproducibility predicts the future development of a wide range of clinical applications.

Paper-based analytical device for point-of-care nucleic acid quantification combining CRISPR/Cas12a and personal glucose meter

Paper-based analytical device for point-of-care nucleic acid quantification combining CRISPR/Cas12a and personal glucose meter

A ddPCR platform based on a microfluidic chip with a dual-function flow-focusing structure for sample-to-result DNA quantification analysis.

Droplet digital PCR (ddPCR) is a powerful method for absolute nucleic acid quantification with high precision and accuracy. However, complicated operational steps have hampered the use and diffusion of ddPCR. Therefore, an automated, easy-to-use, low-sample-consumption, and portable ddPCR platform is urgently needed. This paper proposes a microfluidic ddPCR platform based on a microfluidic chip that can realize the sample-to-result function by switching the rotary valve, achieving the dual function of the flow-focusing structure for droplet generation and readout. Sample, generation oil, and analysis oil were pre-added to the reservoirs. Droplets were generated due to focusing flow, and after passing through the integrated temporary storage bin in the rotary valve, the droplets and oil subsequently entered the collecting tube, improving the droplet-to-oil volume ratio for enhanced thermal cycle performance. Droplets with an average diameter of 107.44 μm and a CV of 2.38% were generated using our chip under the optimal pressures. High-performance thermal cycling was achieved through improvements of the droplet-to-oil volume ratio of the sample, the integrated heating lid, the pure copper heating base, and the temperature-controlling algorithm. Gradient quantification experiments were conducted for the HER2 and CEP17 genes extracted from breast cancer cells, yielding strong linear correlations with R2 values of 0.9996 for FAM and 0.9989 for CY5. Moreover, pronounced linearity was obtained between the detected concentrations of HER2 and CEP17, indicated by a slope of 1.0091 and an R2 of 0.9997, signifying consistent HER2 : CEP17 ratios across various sample dilutions. The outcomes of the quantitative analysis, encompassing the dynamic range and the consistency of the HER2 : CEP17 ratio using our ddPCR platform, meet the standards required for breast cancer assessment and therapy. Our ddPCR platform is automated, portable, and capable of stable droplet generation, high-efficiency amplification, realization of the sample-to-result function based on dual-function flow-focusing structure, and accuracy absolute quantification, underscoring its significant potential for ddPCR analysis in clinical diagnostics.

Gen Mutasyonunun Belirlenmesinde Rekombinaz Polimeraz Çoğaltım Tekniği Optimizasyonu Çalışmaları ve Sonuçları

Single nucleotide polymorphisms (SNPs) in human genes are very significant genetic changes and PCR (polymerase chain reaction) or NGS (next-generation sequencing) are extensively employed in SNP analysis. Thanks to the studies on the progress of new technologies, interest in the isothermal nucleic acid amplification approach has increased. As one of these methods, recombinase polymerase amplification (RPA) represents an attractive option for point-of-care nucleic acid quantification. The target SNPs selected within the scope of the study are mutations identified in the PIK3CA gene region (E542K, E545K), and DNA samples which were evaluated about PIK3CA mutations were isolated from the cancer cells MCF7, BT474, and also SKBr3. The optimization studies for the RPA reaction conditions were carried out for parameters such as assay time, temperature, primer, and also magnesium acetate concentration. According to the results of the reaction optimization studies, in which the RPA products can be obtained in the most efficient way, the assay time was determined as 20 min; the temperature as 40°C; the primer concentration as 10 µM and the MgOAc concentration as 140 mM.

An Integrated ddPCR Lab-on-a-Disc Device for Rapid Screening of Infectious Diseases.

Digital droplet PCR (ddPCR) is a powerful amplification technique for absolute quantification of viral nucleic acids. Although commercial ddPCR devices are effective in the lab bench tests, they cannot meet current urgent requirements for on-site and rapid screening for patients. Here, we have developed a portable and fully integrated lab-on-a-disc (LOAD) device for quantitively screening infectious disease agents. Our designed LOAD device has integrated (i) microfluidics chips, (ii) a transparent circulating oil-based heat exchanger, and (iii) an on-disc transmitted-light fluorescent imaging system into one compact and portable box. Thus, droplet generation, PCR thermocycling, and analysis can be achieved in a single LOAD device. This feature is a significant attribute for the current clinical application of disease screening. For this custom-built ddPCR setup, we have first demonstrated the loading and ddPCR amplification ability by using influenza A virus-specific DNA fragments with different concentrations (diluted from the original concentration to 107 times), followed by analyzing the droplets with an external fluorescence microscope as a standard calibration test. The measured DNA concentration is linearly related to the gradient-dilution factor, which validated the precise quantification for the samples. In addition to the calibration tests using DNA fragments, we also employed this ddPCR-LOAD device for clinical samples with different viruses. Infectious samples containing five different viruses, including influenza A virus (IAV), respiratory syncytial virus (RSV), varicella zoster virus (VZV), Zika virus (ZIKV), and adenovirus (ADV), were injected into the device, followed by analyzing the droplets with an external fluorescence microscope with the lowest detected concentration of 20.24 copies/µL. Finally, we demonstrated the proof-of-concept detection of clinical samples of IAV using the on-disc fluorescence imaging system in our fully integrated device, which proves the capability of this device in clinical sample detection. We anticipate that this integrated ddPCR-LOAD device will become a flexible tool for on-site disease detection.

Constant Pressure-Regulated Microdroplet Polymerase Chain Reaction in Microfluid Chips: A Methodological Study.

Digital polymerase chain reaction (PCR) technology in microfluidic systems often results in bubble formation post-amplification, leading to microdroplet fragmentation and compromised detection accuracy. To solve this issue, this study introduces a method based on the constant pressure regulation of microdroplets during PCR within microfluidic chips. An ideal pressure reference value for continuous pressure control was produced by examining air solubility in water at various pressures and temperatures as well as modeling air saturation solubility against pressure for various temperature scenarios. Employing a high-efficiency constant pressure device facilitates precise modulation of the microfluidic chip's inlet and outlet pressure. This ensures that air solubility remains unsaturated during PCR amplification, preventing bubble precipitation and maintaining microdroplet integrity. The device and chip were subsequently utilized for quantitative analysis of the human epidermal growth factor receptor (EGFR) exon 18 gene, with results indicating a strong linear relationship between detection signal and DNA concentration within a range of 101-105 copies/μL (R2 = 0.999). By thwarting bubble generation during PCR process, the constant pressure methodology enhances microdroplet stability and PCR efficiency, underscoring its significant potential for nucleic acid quantification and trace detection.

Absolute quantification of nucleic acid on digital microfluidics platform based on superhydrophobic–superhydrophilic micropatterning

Digital microfluidics (DMF) shows great potential in the manipulation of individual droplets. However, the limited number of electrode control wirings for droplet control intrinsically restrains the massive generation of pL/nL individual droplets, fundamentally hindering the realization of high throughput applications on DMF such as digital nucleic acid amplification and analysis. This work demonstrates on-chip high throughput digital loop-mediated isothermal amplification (dLAMP) on a DMF platform based on a functional substrate with super-wettability contrast. Exploiting the passive dispensing technique as empowered by electrowetting on dielectric (EWOD) effect, a total of 1818 individual droplets with high uniformity are generated on the super-hydrophilic microarray within 1 min. Separation within the oil phase without tubes and valves can avoid evaporation, ensuring a precise total reaction volume. Absolute quantification of λDNA with a dynamic range of 1–1000 copies/μL is achieved by counting the amount of positive microdroplets after on-chip incubation, demonstrating the strong potential for rapid, low-cost, reliable, and quantitative nucleic acid analysis with high accuracy.

Quality and Quantity of Nucleic Acids Extracted from Formalin-Fixed Paraffin-Embedded Lymphoma Biopsies from Nigerian Archived Biopsy.

Integrity of nucleic acids derived from archived formalin-fixed paraffin-embedded (FFPE) cancer specimens affects diagnosis, prognosis, and therapy. Several factors affect the quality and quantity of extracted nucleic acids and one of such factors is storage period. We investigated the impact of storage duration on the quality and quantity of nucleic acids extracted from archived FFPE lymphoma biopsies in Nigeria. A total of 53 FFPE biopsies diagnosed as lymphoma stored over several years (2008-2019) were analyzed. They were 22 chronic lymphocytic leukemia (CLL) cases, 17 Hodgkin lymphoma (HL) cases, and 14 diffuse large B-cell lymphoma, not otherwise specified (DLBCL, NOS). DNA was extracted from all the lymphoma samples which were analyzed for integrity and amplifiability using the four pairs of control genes polymerase chain reaction (PCR) primers of BIOMED-2 protocol, whereas RNA extraction was from 6 CLL cases used for qPCR analysis of RNU43. For CLL, the mean DNA yield was 193.6 ng/µl (range: 3.0-533.0 ng/µl), whereas the mean A260/A280 ratio was 1.7 (1.2-1.9). For DLBCL, NOS, and HL, 255.5 ng/µl (range: 32.9-605.4 ng/µl), 1.8 (1.5-2.0) and 242.7 ng/µl (range: 1.3-886.0 ng/µl), and 1.7 (0.9-1.8), respectively. The extracted DNA gave amplifiable products of at least 200bp, whereas the RNA analysis showed CT values of <38 in all the samples. The mean RNA yield was 462.2 ng/µl (range: 74.7-1082.1), whereas the mean A260/A280 was 1.7 (1.5-1.8). Quantity and quality of nucleic acids from FFPE tissues stored for different time periods showed no significant difference in yield and quality.

The FLT3-like Gene Expression Signature Predicts Response to Quizartinib in Wild-Type FLT3 Acute Myeloid Leukemia: An Analysis of the Pethema Quiwi Trial

Background The identification of predictive biomarkers is crucial for guiding treatment decisions in acute myeloid leukemia (AML). Previously, we identified a FLT3-like gene expression signature in FLT3 wild-type AML patients, which clustered a variable proportion of FLT3 wild-type patients with FLT3-ITD and TKD mutated patients. The QUIWI trial was a randomized, placebo-controlled, phase II study preliminary showing a significant increase in overall survival (OS) in wild-type FLT3 AML patients treated with the FLT3 inhibitor quizartinib (Quiza) plus standard chemotherapy. This preplanned correlative study was designed to assess the value of the FLT3-like signature to predict responses to Quiza. Methods We performed RNA sequencing (RNAseq) analysis on a subset of patients from the clinical trial. RNA was extracted using standard methods, followed by assessment of nucleic acid integrity (TapeStation) and quantification (Qubit). Total mRNA sequencing was performed using polyA RNAseq with TruSeq technology. A total of 206 adequate samples from bone marrow and peripheral blood were sequenced (161 from FLT3-ITD negative enrolled in QUIWI; and 55 from FLT3-ITD positive patients who were screen failure of the QUIWI trial by this reason). The sequences were aligned to the GRCh37 reference genome using the Hisat algorithm. Gene expression quantification was performed using the Bioconductor workflow, and gene expression estimates (FPKM) were obtained. The gene expression estimates were log2 normalized. Finally, those genes mapping to the original FLT3-like signature (595 genes) were selected for downstream analysis. Clustering was based on the hierarchical method, using standard euclidean distance metrics. OS was defined as time from start of screening to death. Event-free survival (EFS) was defined as time from randomization to failure to achieve CR/CRi after 1 or 2 cycles, death in CR/CRi, or relapse (whichever occurred the first). Relapse-free survival was defined as time from randomization to disease relapse or death by any cause. Results Among the total 206 patients, a cluster of 54.37% (N=112) was enriched in FLT3-mutant cases (71.11% of cases, Figure 1A). This subgroup comprised 49.67% of wild-type FLT3 cases (N=80), hereafter FLT3-like patients. In the group of not FLT3-like patients, no differences were identified between the placebo and Quiza group in the total number of deaths (Fisher's p-value, 0.63), EFS (cox p-value, 0.83; HR 1.07 [0.56-2.06]), RFS (cox p-value 0.76; HR 0.88 [0.38-2.01]) and OS (cox p-value, 0.62; HR 1.22 [0.55-2.67]). Among FLT3-like patients, significant differences were identified in the total number of deaths (Fisher's p-value, 0.004), EFS (cox p-value, 0.009; HR 0.45 [0.25-0.82], RFS (cox p-value 0.01, HR 0.37 [0.18-0.79]) and OS (cox p-value 0.01, HR, 0.41 [0.20-0.84]) ( Figure 1B). No statistically significant association was observed between the FLT3-like pattern and the ELN-17 classification: 30.4% were low risk, 40.5% intermediate risk, 29.1% high risk. Conclusion The FLT3-like gene expression signature successfully identified a subset of patients who derived the most benefit from Quiz, while patients without the FLT3-like signature did not demonstrate a benefit compared with placebo. These findings support the use of the FLT3-like signature as a potential biomarker to identify those wild-type FLT3 AML patients who may benefit from Quiz, providing a valuable insight for personalized treatment in AML.

DNA-Aptamer-Based qPCR Using Light-Up Dyes for the Detection of Nucleic Acids.

Quantitative polymerase chain reaction (qPCR) is widely used in detection of nucleic acids, but existing methods either lack sequence-specific detection or are costly because they use chemically modified DNA probes. In this work, we apply a DNA aptamer and light-up dye-based chemistry for qPCR for nucleic acid quantification. In contrast to the conventional qPCR, in our method, we observe an exponential decrease in fluorescence upon DNA amplification. The qPCR method we developed produced consistent Ct vs log10 (DNA amount) standard curves, which have a linearfit with R2 value > 0.99. This qPCR technique was validated by quantifying gene targets from Streptococcus zooepidemicus (SzhasB) and Mycobacterium tuberculosis (MtrpoB). We show that our strategy is able to successfully detect DNA at as low as 800 copies/μL. To the best of our knowledge, this is the first study demonstrating the application of light-up dyes and DNA aptamers in qPCR.

Light-Driven Photocatalytic-Photothermal Synergetic System for Portable and Sensitive Nucleic Acid Quantification.

Photosensitizers and photothermal agents have attracted increasing attention for in vitro diagnosis, but the combination remains challenging. Herein, a light-driven photocatalytic-photothermal synergetic system integrated microfluidic distance-based analytical device (PCPT-μDAD) for visual, portable, sensitive, and quantitative detection of targets was developed. Target DNA was recognized and initiated the hybridization chain reaction to form a double-stranded DNA/SYBR Green I (dsDNA/SG-I) complex. By applying the photosensitization of the dsDNA/SG-I complex and the photothermal effect of oxidized 3,3',5,5'-tetramethylbenzidine, the target concentration can effectively translate into a visual distance signal readout. Importantly, the light-driven PCPT-μDAD greatly improves the controllability of catalytic reactions and signal amplification efficiency. The light-driven PCPT-μDAD shows a low limit of detection (fM level), good stability, and high reproducibility for nucleic acid detection.

An integrated and multi-functional droplet-based microfluidic platform for digital DNA amplification

Digital DNA amplification is a powerful method for detecting and quantifying rare nucleic acids. In this study, we developed a multi-functional droplet-based platform that integrates the traditional digital DNA amplification workflow into a one-step device. This platform enables efficient droplet generation, transition, and signal detection within a 5-min timeframe, distributing the sample into a uniform array of 4 × 104 droplets (variation <2%) within a chamber. Subsequent in-situ DNA amplification, fluorescence detection, and signal analysis were carried out. To assess the platform's performance, we quantitatively detected the human epidermal growth factor receptor (EGFR) mutation and human papillomavirus (HPV) mutation using digital polymerase chain reaction (dPCR) and digital loop-mediated isothermal amplification (dLAMP), respectively. The fluorescence results exhibited a positive, linear, and statistically significant correlation with target DNA concentrations ranging from 101 to 105 copies/μL, demonstrating the capability and feasibility of the integrated device for dPCR and dLAMP. This platform offers high-throughput droplet generation, eliminates droplet fusion and transition, is user-friendly, reduces costs compared to current methods, and holds potential for thermocycling and isothermal nucleic acid quantification with high sensitivity and accuracy.

In Vitro Cytocompatibility Assessment of Novel 3D Chitin/Glucan- and Cellulose-Based Decellularized Scaffolds for Skin Tissue Engineering

Background: Naturally derived sustainable biomaterials with high flexibility, mechanical properties, biocompatibility, and the ability to manipulate surface chemistry, providing a natural cellular environment, can be used for tissue engineering applications. However, only a few researchers have demonstrated the exploitation of natural architectures for constructing three-dimensional scaffolds. The chemical decellularization technique for fabricating natural scaffolds and their cytocompatibility assessment for tissue engineering applications need to be thoroughly explored and evaluated. Methods: Decellularization of natural scaffolds has been performed via a chemical method using anionic detergent sodium dodecyl sulfate (SDS) which was used for the in vitro culturing of murine embryonic NIH/3T3 fibroblasts. Techniques such as field-emission scanning electron microscopy (FE-SEM), compressive testing and swelling ratio, and biodegradation were performed to characterize the properties of fabricated decellularized natural scaffolds. Nucleic acid quantification, DAPI, and H&amp;E staining were performed to confirm the removal of nuclear components. In vitro cytocompatibility and live/dead staining assays were performed to evaluate cultured fibroblasts’ metabolic activity and qualitative visualization. Results: 3D chitin/glucan- and cellulose-based scaffolds from edible mushroom (stem) (DMS) and unripe jujube fruit tissue (DUJF) were fabricated using the chemical decellularization technique. FE-SEM shows anisotropic microchannels of highly microporous structures for DMS and isotropic and uniformly arranged microporous structures with shallow cell cavities for DUJF. Both scaffolds exhibited good mechanical properties for skin tissue engineering and DUJF showed a higher compressive strength (200 kPa) than DMS (88.3 kPa). It was shown that the DUJF scaffold had a greater swelling capacity than the DMS scaffold under physiological conditions. At 28 days of incubation, DUJF and DMS displayed approximately 14.97 and 15.06% biodegradation, respectively. In addition, DUJF had greater compressive strength than DMS. Compared to DMS scaffolds, which had a compressive stress of 0.088 MPa at a 74.2% strain, the DUJF scaffolds had a greater compressive strength of 0.203 MPa at a 73.6% strain. The removal of nuclear DNA in the decellularized scaffolds was confirmed via nucleic acid quantification, DAPI, and H&amp;E staining. Furthermore, both of these scaffolds showed good adherence, proliferation, and migration of fibroblasts. DMS showed better biocompatibility and high viability of cells than DUJF. Conclusions: This sustainable scaffold fabrication strategy is an alternative to conventional synthetic approaches for the in vitro 3D culture of mammalian cells for various tissue engineering and cultured meat applications.

Exploring Potential Molecular Mechanisms of Drug Response in FLT3-ITD Negative AML Patients Treated with Quizartinib Vs Placebo Plus Standard Chemotherapy in the Quiwi Trial

Introduction Wild-type FLT3 AML presents significant therapeutic challenges due to limited treatment options. The QUIWI trial investigated quizartinib (Quiz) combined with standard chemotherapy, showing promising results. To understand drug response and refractoriness, we conducted a correlative analysis, exploring differential expression patterns among distinct patient groups. We reasoned that the molecular fingerprints of good responders to Quiz plus chemotherapy would be different with respect to patients who respond only to standard chemotherapy, helping to elucidate critical molecular mechanisms associated with Quiz response. Our study provides a basis for personalized therapeutic strategies in FLT3-ITD negative AML. Methods We performed RNA sequencing (RNAseq) analysis on a subset of patients from the clinical trial. RNA was extracted using standard methods, followed by assessment of nucleic acid integrity (TapeStation) and quantification (Qubit). Total mRNA sequencing was performed using polyA RNAseq with TruSeq technology. A total of 206 adequate samples from bone marrow and peripheral blood were sequenced, out of which 55 cases were FLT3-ITD and were discarded for this analysis. For this analysis, only samples with a minimum of 20% blasts were considered. The sequences were aligned to the GRCh37 reference genome using the Hisat algorithm. Differential expression was assessed with the DESeq2 algorithm, using Blast proportion to adjust for disparities among the samples. P-values were adjusted for multiple testing using the FDR method. OS was defined as time from start of screening to death. Gene ontology and pathways analysis were performed using the WebGestalt portal. Results In this study, we compared the gene expression profiles of Quiz versus placebo-treated patients categorized as good responders, defined arbitrarily as those who continue alive and have a minimum follow-up of 6 months after diagnosis. Out of 82 eligible patients, we identified 100 (q-value &amp;lt; 0.05) and 172 (q-value &amp;lt; 0.1) differentially expressed genes between the Quiz and Placebo groups. Notably, 132 genes were overexpressed in the Quiz group, while 40 genes showed overexpression in the placebo group. Moreover, we observed a highly significant overexpression of ribosomal genes in the Quiz group (17 overlaps, KEGG pathways q-value &amp;lt; 2.2 x 10 -16), and a significant overexpression of genes related to the attenuation of the heat shock protein transcriptional response, which is involved in protein folding, in the placebo group (Reactome q-value, 0.008; gene ids: HSPA1A, HSPA1B and DNAJB1). Additionally, the Quiz group exhibited a significant overexpression of transmembrane receptor protein tyrosine phosphatase-related genes (Gene Ontology Molecular Function q-value, 0.02; gene ids: PTPRB, PTPRG and PTPRU). Conclusion This correlative analysis of the Quiwi trial revealed critical molecular mechanisms associated with Quiz response. The overexpression of ribosomal and transmembrane receptor protein tyrosine phosphatase-related genes in good responders within the Quiz group, along with the overexpression of the heat shock pathway genes in the placebo group, aligns with existing knowledge about the FLT3 pathway biology and the molecular determinants to tyrosine-kinase inhibitors. These findings offer potential biomarkers for personalized therapeutic approaches to improve clinical outcomes in this challenging AML population.

Photofabricated channel-digital microfluidics (pCDMF): A promising lab-on-a-chip platform for fully integrated digital PCR

Microfluidics-based digital PCR (dPCR) is a potent technique capable of absolute quantification of nucleic acids with ultrahigh sensitivity. However, an indispensable process, nucleic acid extraction (NAE) remains hard to be integrated with dPCR into a monolithic device, because both of them involve complicated processes and exquisite fluid manipulation. An ideal solution to this issue is to integrate the two main microfluidic platforms, channel-based microfluidics (CMF) and digital microfluidics (DMF), which, however, remains very challenging. To address these challenges, we herein develop a photofabricated channel-digital microfluidic chip (pCDMF) with significant advantages including: the fabrication of pCDMF is very convenient due to the flexible material choices of photocuring, without tedious process of hydrophobic coating; a parallel-plate configuration can be easily formed, enabling all basic functions of DMF; various channels and microstructures can be freely designed, allowing for fluidic functions of CMF. It is therefore the first example of full integration of DMF and CMF as a monolithic device, with capabilities to precisely handle both discrete droplets and continuous fluids. Finally, an all-in-one device for sample-to-answer digital PCR has been successfully demonstrated. We also envisioned that the pCDMF may open an alternative route toward the fully integrated lab-on-a-chip for various biological applications.

An approach for integrating droplet generation and detection in digital polymerase chain reaction applications based on a bifunctional microfluidic cross-structure

Digital polymerase chain reaction (dPCR) is an approach for absolute nucleic acid quantification with high sensitivity. Although several successful commercial dPCR devices have been developed to date, further miniaturizing device dimensions, decreasing cross-contamination, and improving automation level are still research highlights. In this study, we developed a fully contamination-free dPCR detection chip with fluorescence flow cytometry and micro droplet approach. A bifunctional cross-structure (BFCS) was designed to realize monodisperse sample droplet generation in forward flow and droplet detection in backward flow with simple pneumatic control and fixed chip position. In order to improve droplet detection efficiency and accuracy, droplets morphology and sequence pattern during microfluidic droplet generation and backward flow droplet detection at the same cross-structure were observed and analyzed under different pneumatic pressures. In addition, during backward flow droplet detection, an optimized declination angle of the chip was applied to increase droplet reflux rates. For the validation of PCR performance, temperature changing processes during PCR cycles were achieved by heating the monodispersed droplet array with a customized PCR amplification device. The fluorescence signal of each droplet right after passing the cross-structure was excitated and detected. The absolute quantification ability of our integrated dPCR microfluidic chip utilizing flow fluorescence cytometry was tested and verified with Influenza A virus gene (from 7.5 copies/μL to 30000 copies/μL). Thus, our platform provides a novel and integrated approach for ddPCR analysis.

B-388 Clinical Performance of Xpert BCR-ABL Ultra Assay for Isoform p210 Quantitation

Abstract Background Chronic myelogenous leukemia (CML) is one of the most common hematologic neoplasms. More than 95% of patients with CML have the distinctive Philadelphia chromosome (Ph1) that results from a translocation between the chromosomes 9 and 22, and consequently in the BCR::ABL1 fusion gene. This fusion gene produces a tyrosine kinase with deregulated activity that plays a key role in the development of CML. Monitoring BCR::ABL transcript levels in patients on tyrosine kinase inhibitor (TKI) therapy using real-time quantitative PCR is standard of care in the management of CML patients. The test utilizes a quantitative, real-time reverse transcription polymerase chain reaction (RT-qPCR) to measure BCR::ABL1 to ABL1 percent ratios on the International Scale (%IS), in t(9;22) positive CML patients during monitoring of treatment with TKIs. In this context, this study evaluated clinical performance of the Xpert® BCR-ABL Ultra assay for quantitation of BCR::ABL1 p210 transcripts. Methods The clinical performance of assay was evaluated by comparison to an RT-qPCR assay routinely used in our institution, which also detects and quantifies the mRNA transcripts for the p210 translocation types and uses the ABL gene as an endogenous control. In total, 30 fresh peripheral blood samples of CML patients with the level of BCR::ABL1 transcripts (international scale, IS) between &amp;gt;50% and undetectable were enrolled in this study. All steps of protocols were performed according to manufacturer ́s instructions. Results The comparison of two assay revealed consistente molecular response (MR) level. Linear regression analysis showed high correlation between the assays (R2 = 0.974, P &amp;lt; 0.0001) The slope of regression curve were not significantly different from the value of 1 (1.003; 95% CI, range 0.943–1.069). The evaluation of MR level was identical in 21/28 (75%) samples. Two samples were excluded from the study due to failure to control ABL1 (Ct &amp;gt; 18). The median of ABL1 copies per sample was 66 011 copies (range 20 010–236 171) for conventional RT-qPCR assay. Xpert BCR-ABL Ultra reports %IS (MR), but this test does not report ABL1 copy numbers. If the Xpert BCR-ABL Ultra test reports a positive or negative result and the ABL1 Ct value is below 18, a minimum of 32 000 ABL1 copy numbers is guaranteed in the reaction. The BCR::ABL1 negativity was concordant in 2/8 samples. Conclusion We showed that the results Xpert® BCR-ABL Ultra assay (MR) are in good correlation with the results of RT-qPCR conventional method.The Xpert® BCR-ABL Ultra assay simplifies the generation of results by integrating nucleic acid extraction, amplification, and quantification of the BCR::ABL1 p210 transcripts in peripheral blood specimens. The assay turn-around-time is approximately 3 h. This does not differentiate between b2a2 or b3a2 fusion transcripts.This assay is unable to detect BCR::ABL1 p190 or other rarer translocations.

Facile prepared microfluidic chip for multiplexed digital RT-qPCR test.

The chip-based digital polymerase chain reaction (PCR) is an indispensable technique for amplifying and quantifying nucleic acids, which has been widely employed in molecular diagnostics at both fundamental and clinical levels. However, the previous designs have yet to achieve widespread application due to limitations in complex chip fabrication, pretreatment procedures, special surface properties, and low throughput. This study presents a facile digital microfluidic chip driven by centrifugal force for digital PCR analysis. Interestingly, regardless of the hydrophilicity or hydrophobicity of the inner chip surface, an efficient digitization process can be achieved. PCR reagents introduced into the inlet can be allocated to 9600 microchambers and subsequently isolated by the immiscible phase (silicone oil). The centrifugal priming approach offers a facile means to achieve high-throughput analysis. The design was further employed for the quantification of nucleic acids using digital PCR. The calculated result exhibited a strong correlation with the measured value at the concentrations from 1 copy/μL to 1000 copies/μL (R2 =0.99). Additionally, the chip also allowed digital multiplexed analysis, thereby indicating its potential for multi-target detection applications.