ddPCR

BACKGROUND: The prevalence of multidrug-resistant tuberculosis (MDR-TB) among Korean tuberculosis patients is about 4.1%, which is higher than the OECD average of 2.6%. Inadequate drug use and poor patient compliance increase MDR-TB prevalence through selective pressure. Therefore, prompt detection of drug resistance in tuberculosis patients at the time of diagnosis and quantitative monitoring of these resistant strains during treatment are crucial. METHODS: A multiplex droplet digital PCR (ddPCR) assay was developed and assessed using DNA material of nine Mycobacterium tuberculosis strains with known mutation status that were purchased from the Korean National Tuberculosis Association...

INTRODUCTION: Chronic intestinal failure patients (CIF) require a central venous access device (CVAD) to administer parenteral nutrition. Most serious complication related to a CVAD is a central line-associated bloodstream infection (CLABSI). The golden standard to diagnose a CLABSI are blood cultures, however, they may require 1-5 days before getting a result. Droplet digital polymerase chain reaction (ddPCR) for the detection of pathogen 16S/28S rRNA is a novel culture-independent molecular technique that has been developed to enhance and expedite infection diagnostics within two and a half hours...

BACKGROUND: Circulating tumor DNA has emerging clinical applications in several cancers; however, previous studies have shown low sensitivity in glioma. We investigated if 3 key glioma gene mutations IDH1 , TERTp , and EGFRvIII could be reliably detected in plasma by droplet digital polymerase chain reaction (ddPCR) thereby demonstrating the potential of this technique for glioma liquid biopsy. METHODS: We analyzed 110 glioma patients from our biobank with a total of 359 plasma samples (median 4 samples per patient)...

Isogenic H/N/KRAS-less mouse embryonic fibroblast (MEF) cell lines have been developed to assist in cell-based assays of RAS inhibitors. The quality control assessment of a panel of these isogenic MEFs is described here, with a focus on ensuring the proper insertion of the desired mutant RAS transgene, a determination of gene copy number, and an investigation of potential off-target mutations which could lead to phenotypes which are undesired in downstream experiments. Using this suite of quality control tools, a MEF cell line can be readily validated, and researchers can be assured of the rationale for an observed phenotype...

BACKGROUND: Primary central nervous system lymphoma (PCNSL) are rare mature B-cell lymphoproliferative diseases characterized by a high incidence of MYD88 L265P and CD79B Y196 hotspot mutations. Diagnosis of PCNSL can be challenging. The aim of the study was to analyze the detection rate of the MYD88 L265P and CD79B Y196 mutation in cell free DNA (cfDNA) in plasma of patients with PCNSL. METHODS: We analyzed by digital droplet PCR (ddPCR) to determine presence of the MYD88 L265P and CD79B Y196 hotspot mutations in cfDNA isolated from plasma of 24 PCNSL patients with active disease...

Pulmonary invasive fungal infection in immunocompromised hosts is difficult to diagnose, and current tools for diagnosis or monitoring of response to antifungal treatments have inherent limitations. Droplet digital PCR (ddPCR) has emerged as a promising tool for pulmonary pathogen detection with high sensitivity. This study presents a novel ddPCR panel for rapid and sensitive identification of pulmonary fungal pathogens. First, a ddPCR method for detecting three fungal genera, including Pneumocystis , Aspergillus , and Cryptococcus , was established and evaluated...

Heat stress and coral diseases are the predominant factors causing the degradation of coral reef ecosystems. Over recent years, Vibrio coralliilyticus was identified as a temperature-dependent pathogen causing tissue lysis in Pocillopora damicornis and one of the primary pathogens causing bleaching and mortality in other corals. Yet current detection techniques for V. coralliilyticus rely primarily on qPCR and ddPCR, which cannot meet the requirements for non-invasive and real-time detection. Herein, we developed an effective electrochemical biosensor modified by an Au-MoS2 /rGO (AMG) nanocomposites and a specific capture probe to dynamically detect V...

BACKGROUND AIMS: Gene therapy using lentiviral vectors (LVs) that harbor a functional β-globin gene provides a curative treatment for hemoglobinopathies including beta-thalassemia and sickle cell disease. Accurate quantification of the vector copy number (VCN) and/or the proportion of transduced cells is critical to evaluate the efficacy of transduction and stability of the transgene during treatment. Moreover, commonly used techniques for LV quantification, including real-time quantitative polymerase chain reaction (PCR) or fluorescence-activated cell sorting, require either a standard curve or expression of a reporter protein for the detection of transduced cells...

Hepatitis E virus (HEV) represents an emerging risk in industrialized countries where the consumption of contaminated food plays a pivotal role. Quantitative real-time RT-PCR (RT-qPCR) is one of the most suitable methods for the detection and quantification of viruses in food. Nevertheless, quantification using RT-qPCR has limitations. Droplet digital PCR (ddPCR) provides the precise quantification of nucleic acids without the need for a standard curve and a reduction in the effect on virus quantification due to the presence of inhibitors...

The early detection of cognitive decline in Parkinson's disease is important for providing drug therapy and non-pharmacological management. The circulating microRNAs present in plasma are promising biomarkers of PD with dementia (PDD) due to their critical roles in synaptic plasticity and the regulation of neurodegeneration-associated proteins. In this study, we aimed to identify plasma microRNAs that may differentiate PD with or without cognitive impairment. Global microRNA expression was obtained from a discovery set of 123 participants who were divided into four groups, namely normal controls (HC), PD with no dementia (PDND), PD with mild cognitive impairment (PD-MCI), and PDD, using next-generation sequencing...

This research investigated the factors associated with the quantitative detection of Paratrichodorus allius in soil using droplet digital PCR (ddPCR). Small-sized nematodes exhibited significantly lower DNA quantities compared to their medium and large counterparts. Soil pre-treatments (room temperature drying and 37 °C oven-drying) demonstrated no substantial impact on ddPCR detection, and soil storage (0-3 months at 4 °C) exhibited negligible alterations in DNA quantities. A commercial DNA purification kit improved the resulting quality of ddPCR, albeit at the cost of a notable reduction in DNA quantity...

Malaria remains a significant global public health concern, with a recent increase in the number of zoonotic malaria cases in Southeast Asian countries. However, limited reports on the vector for zoonotic malaria exist owing to difficulties in detecting parasite DNA in Anopheles mosquito vectors. Herein, we demonstrate for the first time that several Anopheles mosquitoes contain simian malaria parasite DNA using droplet digital PCR (ddPCR), a highly sensitive PCR method. An entomological survey was conducted to identify simian malaria vector species at Phra Phothisat Temple (PPT), central Thailand, recognized for a high prevalence of simian malaria in wild cynomolgus macaques...

Background: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular life-threatening disorder. Owing to high carrier frequency, population-wide SMA screening to quantify the copy number of SMN gene is recommended by American College of Medical Genetics and Genomics. An accurate, reliable, short runaround time and cost-effective method may be helpful in mass population screening for SMA. Methods: Multiplex ligation-dependent probe amplification (MLPA) is a gold standard to estimate the copy number variation (CNV) for SMN1 and SMN2 genes...

BACKGROUND & METHODS: In this study, a novel restriction enzyme (RE) digestion-based droplet digital polymerase chain reaction (ddPCR) assay was designed for cg005575921 within the AHRR gene body and compared with matching results obtained by bisulfite conversion (BIS) ddPCR and Illumina DNA methylation array. RESULTS: The RE ddPCR cg05575921 assay appeared concordant with BIS ddPCR (r2  = 0.94, P < 0.0001) and, when compared with the Illumina array, had significantly better smoking status classification performance for current versus never smoked (AUC 0...

OBJECTIVES: We hypothesize that digital droplet polymerase chain reaction (ddPCR) would optimize the treatment strategies in epidermal growth factor receptor (EGFR) -tyrosine kinase inhibitors (TKIs) relapsed patients. In this study, we compared the efficacy of third-generation TKIs with various T790M statuses via ddPCR and next-generation sequencing (NGS). METHODS: NGS was performed on blood samples of patients progressed from previous EGFR-TKIs for resistance mechanism...

OBJECTIVE: :We sought to determine if standard influenza and pneumococcal vaccines can be used to stimulate HIV reservoirs during antiretroviral therapy (ART). DESIGN: :Prospective, randomized, double-blinded, placebo-controlled, crossover trial of two clinically recommended vaccines (influenza and pneumococcal). METHODS: :Persons with HIV on ART (N = 54) were enrolled in the clinical trial. Blood was collected at baseline and days 2,4,7,14 and 30 postimmunizations...

This study aimed to investigate the prognostic value of a novel droplet digital polymerase chain reaction (DDPCR) assay in sepsis patients. In this prospective cohort study, univariable and multivariable Cox regressions were used to assess risk factors for 28-day mortality. We also monitored pathogen load together with clinical indicators in a subgroup of the cohort. A total of 107 sepsis patients with positive baseline DDPCR results were included. Detection of poly-microorganisms [adjusted hazard ratio (HR) = 3...

Pruritus is a common irritating sensation that provokes the desire to scratch. Environmental and genetic factors contribute to the onset of pruritus. Moreover, itch can become a major burden when it becomes chronic. Interestingly, the rare Collagen VI alpha 5 (COL6A5) gene variant p.Glu2272* has been identified in two families and an independent patient with chronic neuropathic itch. These patients showed reduced COL6A5 expression in skin and normal skin morphology. However, little progress has been made until now toward understanding the relationships between this mutation and chronic itch...

Recent studies have shown that alterations of the androgen receptor (AR) are associated with resistance to AR-directed therapy in prostate cancer. Thus, it is crucial to develop robust detection methods for AR alterations as predictive biomarkers to enable applicability in clinical practice. We designed and validated five multiplex droplet digital PCR (ddPCR) assays for reliable detection of 12 AR targets including AR amplification, AR-V7 and 10 AR hotspot mutations as well as AR and KLK3 gene expression from plasma derived cell-free (cf)DNA and cfRNA...

PURPOSE: Bloodstream infection(BSI) is linked with high mortality, underscoring the significance of prompt etiological diagnosis for timely and precise treatment. This study aims to investigate the diagnostic value of droplet digital polymerase chain reaction(ddPCR) in combination with conventional inflammatory markers [interleukin-6(IL-6) and procalcitonin(PCT)] concerning disease progression and treatment prognosis in BSI patients. Furthermore, the study aims to explore a more efficient clinical application strategy...