Droplet Digital Polymerase Chain Reaction Research Articles

Droplet Digital PCR: A New Molecular Method to Detect G1105S/V Mutations in Plasmopara viticola CesA3 Gene

Plasmopara viticola is the causal agent of Grapevine Downy Mildew (GDM), which is a devastating disease of grapevines in humid temperate regions. The most employed method for protecting grapevines against GDM is the application of chemical fungicides. In Spain, Carboxylic Acid Amides (CAAs) are a fungicide group currently utilized in GDM control. In P. viticola, resistance to CAAs is conferred by G1105S and G1105V mutations in the CesA3 gene. Droplet digital polymerase chain reaction (ddPCR) is an innovative technique that combines PCR and droplet microfluidics to disperse the sample into thousands of water-in-oil droplets in which an amplification reaction is individually performed. In this study, we set up a ddPCR protocol to quantify S1105 and V1105 mutations conferring resistance to CAAs in P. viticola. The optimal PCR conditions were established, and the sensitivity and precision of the protocol were assessed. Four P. viticola populations coming from commercial vineyards in northern Spain were analyzed, and different allele frequencies were found in the analyzed samples corresponding to the different fungicide management strategies, ranging from 7.72% to 100%. Knowing the level of mutated alleles allows for designing resistance management strategies suited for each location. This suggests that similar ddPCR assays could be developed for studying mutations implicated in fungicide resistance in other fungicide groups and plant pathogens.

Methodology and clinical utility of longitudinal UBA1 tracking in VEXAS syndrome.

Vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic (VEXAS) is a haemato-inflammatory syndrome genetically defined by somatic mutations in the X-linked UBA1 gene, typically Val/Thr/Leu substitutions at the Met41 hotspot. Clinical manifestations are heterogeneous and refractory to most haemato-rheumatological treatments. To date, no guidelines exist for the management of VEXAS, and scarce is the evidence on methodology and clinical significance of longitudinal UBA1 clonal burden evaluation upon therapy. Here, we validated a method to quantify UBA1 clonal burden and explored its applicability in patients with VEXAS. Given the different treatment interactions, droplet digital polymerase chain reaction (ddPCR) may allow for informed therapeutic decisions and implementation of personalized strategies.

Abstract 4137939: Mosaic loss of Y chromosome is associated with increased all-cause mortality in patients with ST-segment elevation myocardial infarction

Introduction: Mosaic loss of Y chromosome (LOY), a common subtype of somatic mosaicism, is characterized as loss of the entire Y chromosome in a mosaic manner and increases with age. Accumulating evidence has manifested the positive link between LOY and shorter life expectations in elderly men as well as common age-associated diseases, such as cancer and cardiovascular diseases. Experimental studies also demonstrated that hematopoietic LOY in mice induced cardiac fibrosis and led to increased mortality. However, little is known about LOY and prognosis of ST-segment elevation myocardial infarction (STEMI). Hypothesis: We hypothesized that LOY may be associated with lower survival rate of patients with STEMI. Aims: Investigating the impact of LOY in peripheral blood cells on prognosis of patients with STEMI. Methods: This study prospectively enrolled 928 males and 272 females undergoing primary percutaneous coronary intervention for STEMI. LOY was measured in 928 males using droplet digital polymerase chain reaction technique. Results: During a median follow-up of 3.99 years, 93 males (10.0%) and 45 females (16.5%) died. After adjusting for traditional cardiovascular risk factors, the risk of all-cause mortality in males with LOY ≥ 18% (the 90th percentile of LOY) increased to 2.45 (95% confidence intervals [CI]: 1.16–5.15, P = 0.018), 2.11 (95% CI: 1.11–4.01, P = 0.024), and 1.88 (95% CI: 1.02–3.45, P = 0.043) times during 1-, 2-, and 3-year follow-up, respectively, compared to males with LOY < 18%. Among males without prior MI, those with LOY ≥ 18% had an increased risk of all-cause mortality compared to those with LOY < 18% (adjusted hazard ratio 1.93, 95% CI: 1.01–3.67; P = 0.045); this was not observed for males with prior MI. Female patients had a 1.71-fold (95%CI: 1.04–2.81; P = 0.035) increased risk of all-cause mortality than males with LOY < 18%, whereas the risk was similar to males with LOY ≥ 18%. Conclusion: LOY was associated with an increased risk of all-cause mortality in male STEMI patients, particularly in those without prior MI. Detecting LOY in STEMI patients could help further refine sex differences in prognosis and assist in identifying male patients with better prognoses.

Detecting Dirofilaria immitis: Current Practices and Novel Diagnostic Methods

The nematode Dirofilaria immitis is responsible for a vector-borne disease affecting canines and humans worldwide, known as cardiopulmonary dirofilariasis. An accurate and early diagnosis is of the utmost importance for effective disease management. While traditional microscopy-based methods remain invaluable, they have inherent limitations. Serological tests, in particular ELISA and immunochromatographic tests, are employed due to their capacity to detect D. immitis antigens, offering ease of use and diagnostic accuracy. The advent of molecular methods has the potential to enhance routine diagnostic approaches, with polymerase chain reaction (PCR) and real-time PCR (qPCR) becoming the most prevalent techniques. Despite not yet being integrated into routine diagnostics, which are predominantly based on the Knott’s test and serological methods, these techniques offer significant benefits in the context of scientific research. This article proceeds to examine the potential of advanced techniques, such as high-resolution melting qPCR (HRM-qPCR), loop-mediated isothermal amplification (LAMP), droplet digital PCR (ddPCR), and microRNA (miRNA) detection, which are capable of enhanced sensitivity and early detection. The following work provides an in-depth analysis of the various diagnostic methods, emphasising the necessity of the continuous improvement and adaptation of these tools to effectively combat D. immitis. The findings underscore the importance of integrating these advanced methods into routine practice to improve detection rates and outcomes for infected animals.

Beyond the mix: Advancements in simultaneous detection and quantification of human, dog, and cat DNA.

Animal-related crimes have increased with an increase in the number of pets worldwide, underscoring the importance of animal-related biological evidence at crime scenes. Evidence obtained in cases involving dogs and cats often includes a mixture of human and animal DNA. In this study, we developed a method using droplet digital polymerase chain reaction (ddPCR) to simultaneously identify and quantitatively detect human, dog, and cat DNA in mixed samples. HLA-DRA was chosen as a human-specific marker, OR6D7 as a dog-specific marker, and FLAI-K as a cat-specific marker. The species specificity of each target was confirmed using 14 control DNA samples from 11 mammals and 3 birds. Sensitivity tests determined the limit of detection to be 0.0008 ng/μL for human DNA and 0.00061 ng/μL for dog and cat DNA. In the mixture test, each DNA sample was independently and accurately detected in samples containing trace amounts of all three types of DNA. This study demonstrated the effectiveness of applying ddPCR to forensic case samples from dog- and cat-related incidents. We have presented a reliable method for the accurate identification and quantification of human, dog, and cat DNA simultaneously, offering possibilities for advancements in forensic DNA analysis and related fields.

Detection of circulating tumor cells in patients with lung cancer using a rare cell sorter: a pilot study

BackgroundWe developed a Rare Cell Sorter (RCS) for collecting single cell including circulating tumor cells (CTCs). This single-institution pilot study evaluated the ability of this device to detect tumor-like cells in patients with lung cancer and confirmed their genuineness based on the epidermal growth factor receptor (EGFR) mutation concordance with tissue samples.MethodsThis study included patients treated for lung cancer from September 2021 to August 2022 in University of Tsukuba Hospital. Peripheral blood samples were obtained before surgery or during periodic medical checks for patients treated with drugs. We used the RCS to capture cells based on size. The cells were stained, and the Hoechst-positive, CD45-negative, and epithelial celladhesion molecule (EpCAM)- positive cells were defined as CTCs, were collected. The presumptive CTCs were counted and tested using digital droplet polymerase chain reaction for EGFR mutations and compared with the tissue EGFR status to check concordance.ResultsEighteen patients were included in this study and CTCs were detected in 6 patients (33%). The CTCs from three patients showed EGFR mutation, and the EGFR mutation status of CTCs concorded with that of tissue samples in 83% of the cases (5/6). Only one CTC showed a different status from the tissue, and the concordance rate of EGFR status between CTCs and the tissue was 96% (24/25).ConclusionThe ability of the RCS to detect CTCs in patients with lung cancer was demonstrated based on the concordance of EGFR status in this pilot study. This novel hybrid method of CTC recovery using the RCS has the potential to recover a wide range of CTCs regardless of EpCAM. Further validation through a large-scale study is needed.

Human pathogen nucleic acids in wastewater solids from 191 wastewater treatment plants in the United States

We measured concentrations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants, influenza A and B viruses, respiratory syncytial virus, human metapneumovirus, enterovirus D68, human parainfluenza types 1, 2, 3, 4a, and 4b in aggregate, norovirus genotype II, rotavirus, Candida auris, hepatitis A virus, human adenovirus, mpox virus, H5 influenza A virus, and pepper mild mottle virus nucleic acids in wastewater solids prospectively at 191 wastewater treatment plants in 40 states across the United States plus Washington DC. Measurements were made two to seven times per week from 1 January 2022 to 30 June 2024, depending on wastewater treatment plant staff availability. Measurements were made using droplet digital (reverse-transcription–) polymerase chain reaction (ddRT-PCR) following best practices for making environmental molecular biology measurements. These data can be used to better understand disease occurrence in communities contributing to the wastewater.

Development of a droplet digital PCR assay for the detection of BK polyomavirus.

The objective of this study was to establish a more sensitive and specific diagnostic method for detecting plasma BK polyomavirus (BKPyV) DNA load in patients after renal transplantation using droplet digital polymerase chain reaction (ddPCR) and to validate the methodology. The linear range, lower limit of detection, accuracy, precision, and specificity of the detection system were evaluated by using the WHO BKPyV standard (7.2 log10 IU/mL) as a reference, in accordance with the relevant documents of the Clinical and Laboratory Standards Institute. Plasma samples were collected from 74 renal transplantation patients with urinary BKPyV-DNA levels exceeding 7 log10 copies/mL. Quantitative PCR (qPCR) and ddPCR were performed, and their diagnostic efficacy for BKPyV-DNA in the diagnosis of BK polyomavirus-associated nephropathy was evaluated using a receiver operating characteristic (ROC) curve. The coefficients of variation for the repeated detection of BKPyV standard DNA were 2.55 and 4.71 at concentrations of 6.2 and 3.2 log10 IU/mL, respectively. The linear range was 2.2-6.2 log10 IU/mL, and the lowest detection limit was 100 IU/mL. By utilizing histopathological examination of renal biopsy as the gold standard for BKPyV diagnosis, the area under the ROC curve of 74 post-transplantation plasma samples detected by the ddPCR system was found to be 0.875 (95% CI: 0.797-0.953, P < 0.01). The optimal threshold was 512.86 copies/mL, with a sensitivity of 90.0% and a specificity of 67.6%. In comparison, the area under the ROC curve for qPCR was 0.668 (95% CI: 0.583-0.752, P < 0.01), with an optimal threshold of 11,481.54 copies/mL, a sensitivity of 35.0%, and a specificity of 100.0%. Pairwise comparison (Delong test) of the ROC curves of the two systems showed a significant difference in the area under the curve, with a difference of 0.207 and a P-value <0.01. The BKPyV nucleic acid detection system, based on ddPCR, is appropriate for the regular monitoring of the BK polyomavirus, specifically in plasma samples containing low viral DNA loads while it provides the benefits of both absolute quantification and high sensitivity.IMPORTANCEIt was previously believed that droplet digital polymerase chain reaction had limitations, including high cost, limited throughput, and cumbersome operation, which hindered its widespread application in clinical practice. However, the current fully automated digital PCR platform, combined with streamlined operations, can detect 96 samples at once, and the entire process can be completed within an hour, laying a solid foundation for its extensive use.

Mutant KRAS in Circulating Tumor DNA as a Biomarker in Localized Pancreatic Cancer in Patients Treated with Neoadjuvant Chemotherapy.

The primary objective was to determine the prognostic significance of circulating tumor DNA (ctDNA) in patients receiving neoadjuvant chemotherapy (NAC) for localized pancreatic ductal adenocarcinoma (PDAC) using digital droplet polymerase chain reaction (ddPCR). Increasingly, ctDNA is being used for clinical decision-making in a variety of solid malignancies. However, the detection and prognostic value of KRAS ctDNA as assessed by ddPCR during NAC has yet to be characterized. Patients with localized PDAC eligible to receive NAC were prospectively enrolled. Peripheral blood samples were obtained at diagnosis, after NAC, and after resection and analyzed for ctDNA using ddPCR. Log-rank tests and Cox proportional hazards model were used to assess for association with OS. 84 patients were included in the analysis. Mutant KRAS ctDNA was detected in 49.3% of patients at diagnosis, 69.6% of patients after NAC, and 69.7% of patients after resection, respectively. There were 15 (17.9%) patients that cleared mutational ctDNA over the course of treatment. Clearance of ctDNA during NAC was associated with improved overall survival (OS) (18.4 mo. vs NR, P<0.05). Detection of mutant KRAS G12V after NAC and resection was associated with shorter OS (18.0 versus NR months, P<0.031). Detection of the KRAS G12V mutation after resection was associated with reduced OS (aHR 36.75, 95% CI 2.93-461.38). Throughout treatment, KRAS ctDNA is detectable by ddPCR in patients with localized PDAC treated with NAC. Detection of mutant KRAS G12V after resection was associated with reduced OS.

Quantification of Adeno-Associated Viral Genomes in Purified Vector Samples by Digital Droplet Polymerase Chain Reaction.

Adeno-associated virus (AAV) is a non-pathogenic virus used as a delivery vehicle to transfer therapeutic genes into patients. Accurate quantification of AAV genome copy number in vector preparations is essential for bioprocess optimization and dosage calculation in both preclinical and clinical studies of AAV-based gene therapy products. Currently, a consensus protocol for AAV viral genome titration is lacking. Here, we present a digital droplet PCR (dd_PCR) protocol for the quantification of viral genomes in purified vector samples. Samples are treated with DNase I to eliminate unpackaged contaminant DNA. DNase-treated samples are then mixed with an appropriate primer-probe set (designed according to the target AAV genome) and PCR reagents, and then loaded into a droplet generator. The prepared droplets are transferred into a PCR plate, where PCR amplification is performed and analyzed. The viral genome titer is calculated based on the concentration (copies/µL), accounting for sample dilutions. A successful measurement shows a clear separation of the positive and negative droplet clouds, has at least 10,000 accepted droplets, shows a value between 10 copies/µL and 10,000 copies/µL, and has a coefficient of variation (CV) between repeats lower than 20%. Reliable viral genome titration will aid in the development of safe and effective AAV-based gene therapy products.

Use of Nuclear Factor of Activated T Cell-Regulated Gene Expression for Monitoring Immunosuppression with Extended-Release Tacrolimus after Liver Transplantation-A Proof of Concept.

There is a narrow therapeutic window for immunosuppression using calcineurin inhibitors. Drug trough levels do not reflect immunosuppression and should be replaced by pharmacodynamic monitoring. This prospective cohort study was designed to evaluate the effect of an extended-release formulation of tacrolimus (LCP Tac) on the nuclear factor of activated T cell-regulated gene expression (NFAT-RGE). The expression of interleukin-2, interferon-γ, granulocyte-macrophage colony-stimulating factor, and three reference genes was measured. Samples from 23 patients at defined time points in the first year after liver transplantation were analyzed using a droplet digital polymerase chain reaction. All samples were within the targeted trough levels of LCP Tac, and their LCP Tac peak levels and residual NFAT-RGE showed a strong inverse correlation (r = -0.8). Most importantly, there was an individual immunosuppressive response to the LCP Tac. The mean individual trough effect of LCP Tac on the three target genes when all time points were pooled was 33% (26-56%) in patients without infection and 81% (53-95%) in those with infection (p < 0.011). The mean individual peak effect was 48% (44-64%) in patients without infection and 91% (90-94%) in those with infection (p < 0.001). Thus, tailored immunosuppression based on residual NFAT-RGE could prevent infections associated with over-immunosuppression early after liver transplantation.

Insights into Porphyromonas somerae in Bladder Cancer Patients: Urinary Detection by ddPCR.

To date, the increased awareness of the impact of microbes on human health has promoted scientific interest in microbiome studies for diagnostic and therapeutic purposes, revealing correlations between specific taxa and cancer. In particular, numerous species of Porphyromonas have been associated with several types of tumors. Previously, we studied the urobiome using Next-Generation Sequencing (NGS), and found an increase in Porphyromonas somerae in first morning urine of subjects affected by bladder cancer (BCa). Here, we aimed to confirm the presence of P. somerae in BCa patients by using droplet digital Polymerase Chain Reaction (ddPCR), testing a cohort of 102 male subjects over 50 years. Our findings showed a significant increase in P. somerae in the urine of the BCa group within both ddPCR and NGS, and a correlation between the two methods was observed at a statistical level. Moreover, P. somerae's identification with ddPCR confirmed a significant association between this bacterium and the presence of BCa, highlighting its potential role as a biomarker. This allows us to propose the ddPCR as a suitable method for first-stage BCa screening and follow-up.

KRAS mutations detection methodology: from RFLP to CRISPR/Cas based methods.

In personalized cancer medicine, the identification of KRAS mutations is essential for making treatment decisions and improving patient outcomes. This work presents a comprehensive review of the current approaches for detection of KRAS mutations in different cancers. We highlight the value of fast and reliable KRAS mutations discovery and the effectiveness of molecular testing for selecting individuals who might benefit from targeted therapy. We provide an overview of various methods and tools available for detecting KRAS mutations, such as digital droplet PCR, next-generation sequencing (NGS), and polymerase chain reaction (PCR). We also address the difficulties and limitations in the identification of KRAS mutations, namely tumor heterogeneity and the emergence of resistance mechanisms. This article aims to guide clinicians in KRAS mutation identification.

Noninvasive, microbiome-based diagnosis of inflammatory bowel disease.

Despite recent progress in our understanding of the association between the gut microbiome and inflammatory bowel disease (IBD), the role of microbiome biomarkers in IBD diagnosis remains underexplored. Here we developed a microbiome-based diagnostic test for IBD. By utilization of metagenomic data from 5,979 fecal samples with and without IBD from different geographies and ethnicities, we identified microbiota alterations in IBD and selected ten and nine bacterial species for construction of diagnostic models for ulcerative colitis and Crohn's disease, respectively. These diagnostic models achieved areas under the curve >0.90 for distinguishing IBD from controls in the discovery cohort, and maintained satisfactory performance in transethnic validation cohorts from eight populations. We further developed a multiplex droplet digital polymerase chain reaction test targeting selected IBD-associated bacterial species, and models based on this test showed numerically higher performance than fecal calprotectin in discriminating ulcerative colitis and Crohn's disease from controls. Here we discovered universal IBD-associated bacteria and show the potential applicability of a multibacteria biomarker panel as a noninvasive tool for IBD diagnosis.

Plasma EGFR mutation ctDNA dynamics in patients with advanced EGFR-mutated NSCLC treated with Icotinib: phase 2 multicenter trial result

Plasma epidermal growth factor receptor mutation (EGFRm) circulating tumor DNA (ctDNA) dynamics exhibit promise in predicting outcomes in patients with EGFRm-advanced non-small cell lung cancer (NSCLC). However, there remains limited trial-level data on integrating ctDNA monitoring into clinical practice. We performed a prospective, multicenter trial to investigate the relationship between EGFRm ctDNA dynamic changes and clinical outcomes in NSCLC patients with EGFRm. Ninety-eight treatment-naive EGFRm-advanced NSCLC patients were recruited and administered icotinib until disease progression. Plasma samples were collected at baseline and four weeks after icotinib administration. ctDNA was analyzed using a droplet-digital polymerase chain reaction. At baseline, 71.4% of patients had detectable EGFRm ctDNA. Among them, 45.9% of patients’ ctDNA became undetectable within four weeks of treatment. These patients demonstrated significantly longer progression-free survival (PFS) and overall survival (OS) than those with detectable ctDNA after treatment (P = 0.004 and < 0.001, respectively) and were comparable to those with undetectable ctDNA at both baseline and four weeks. ctDNA detectable at four weeks emerged as a poor independent risk factor for PFS and OS. Patients whose ctDNA became undetectable after treatment had outcomes similar to those with initially undetectable ctDNA, underscoring the predictive value of ctDNA dynamics in treatment efficacy.Registry and the Registration No. of the study/trial: ChiCTR-DDD-17013131. Date of registration: 2017-10-27.

Evaluation of Molecular Assays for Diagnosis of Amoebic Liver Abscess in India with Bayesian Latent Class Analysis.

Amoebic liver abscess (ALA) is the most common extra-intestinal complication of Entamoeba histolytica, accounting for 50,000 deaths annually, and is endemic in South Asia. Diagnosis based on microscopic examination is insensitive, and serological assays are not discerning of current infections in endemic settings with high exposure. For a rapid and confirmatory laboratory diagnosis of ALA, the performance of a polymerase chain reaction (PCR), quantitative real time PCR (qPCR), digital droplet PCR (ddPCR), and a loop-mediated isothermal amplification (LAMP) assay that detects E. histolytica DNA in liver abscess pus, and a lectin antigen detection ELISA were evaluated against clinical diagnosis (based on predefined criteria) as the gold standard. Owing to the lack of a laboratory gold standard, a Bayesian latent class analysis approach was also used to determine sensitivity and specificity of these assays. In the latent class analysis, qPCR and ddPCR showed the highest sensitivity (98% and 98.1%) and specificity (both 96.6%), and although clinical diagnosis had a comparable sensitivity to qPCR and ddPCR (95.2%), poorer specificity (64.3%) was seen. Kappa agreement analysis showed that qPCR and ddPCR had a perfect agreement of 1 followed by an agreement of 0.76 (95% CI: 0.64-0.88) with PCR. Considering the performance characteristics and relative ease of setting up qPCR as well as the wide availability of qPCR equipment needed, this would be the most optimal assay for rapid, confirmatory, molecular diagnosis of ALA in the tertiary care laboratory setting in India, whereas further optimization of LAMP or antibody-based detection is required for use at smaller or secondary hospitals.

Water and Electrolyte Content in Hypertension in the Skin (WHYSKI) in Primary Aldosteronism.

Primary aldosteronism (PA), the most common curable salt-dependent form of arterial hypertension, features renal K+ loss and enhanced Na+ reabsorption. We investigated whether the electrolyte, water, and TonEBP (tonicity-responsive enhancer binding protein)/NFAT5 (nuclear factor of activated T cells 5) content is altered in the skin of patients with PA and corrected by surgical cure. We obtained skin biopsies from 80 subjects: 49 consecutive patients with PA, optimally treated with a mineralocorticoid receptor antagonist; 6 essential hypertensives; and 25 normotensive controls. We measured Na+, K+, water content with atomic absorption spectroscopy after ashing, and NFAT5 mRNA with digital droplet polymerase chain reaction. The patients with PA were retested after adrenalectomy. We discovered a higher dry weight of the skin biopsy specimen at surgery than at follow-up (P<0.001) and a direct correlation with electrolyte and water content (all P<0.01), indicating the need for dry weight adjustment of electrolyte and water data. Surgical cure of PA markedly increased skin dry weight-adjusted K+ (from 1.14±0.1 to 2.81±0.27 µg/mg; P<0.001) and water content (from 2.92±1.4 to 3.85±0.23 mg/mg; P<0.001), but left dry weight-adjusted skin Na+ content unaffected. In patients with PA, NFAT5 mRNA was higher (P=0.031) than in normotensive controls and decreased after surgery (P=0.035). Despite mineralocorticoid receptor antagonist treatment ensuring normokalemia, the patients with PA had a skin cell K+ depletion that was corrected by adrenalectomy. The activated NFAT5/TonEBP pathway during mineralocorticoid receptor antagonist administration suggests enhanced skin Na+ lymphatic drainage and can explain the lack of overt skin Na+ accumulation in patients with PA. Its deactivation after surgical cure can account for the lack of skin Na+ decrease postadrenalectomy. URL: https://www.clinicaltrials.gov; Unique identifier: NCT06090617.

Circulating methylated HOXA9 tumor DNA as a biomarker for mortality in recurrent breast cancer: An exploratory study.

Methylated homeobox A9 (meth-HOXA9) circulating tumor DNA may be a relevant biomarker in breast cancer, although its clinical significance remains unknown. The present exploratory study aimed to investigate the association between meth-HOXA9 and mortality in patients with recurrent breast cancer. The cohort study enrolled 51 patients with breast cancer recurrence from the Department of Oncology, Vejle Hospital, University Hospital of Southern Denmark (Vejle, Denmark). Tissue samples from primary surgery and biopsies, and plasma samples obtained at the time of recurrence were analyzed for meth-HOXA9 using a methylation-specific droplet digital polymerase chain reaction. Using Cox regression, hazard ratios (HRs) for mortality with 95% confidence intervals (CIs) comparing patients with detectable and undetectable meth-HOXA9 in both tumor tissue and plasma were estimated. Among the 50 patients with data on tumor tissue meth-HOXA9, there was no association between meth-HOXA9 in the primary tumor and mortality (HR 1.09, 95% CI 0.47-2.52). A total of 34 patients had data on plasma meth-HOXA9 at the time of recurrence. Detectable plasma meth-HOXA9 was associated with higher mortality (HR 3.95, 95% CI 1.50-10.37). Among the 20 patients with data on both plasma and metastatic tissue meth-HOXA9, meth-HOXA9 was detectable in 90% of metastases and 65% of plasma samples. In conclusion, detectable plasma meth-HOXA9 was significantly associated with higher mortality in recurrent breast cancer; therefore, plasma meth-HOXA9 may prove useful as a prognostic marker in patients with breast cancer.

Development and validation of a droplet digital PCR method for quantifying lentiviral vector infectious titer

Lentiviruses, with their high transduction efficiency and gene expression levels, are widely used as gene delivery vectors in the development of chimeric antigen receptor T cells (CAR-T) and other genetically modified cell therapies. Accurate determination of the lentiviral vector infectious titer is essential to ensure effective transduction and product consistency. In this study, we developed an efficient method for lentiviral vector titration based on digital droplet polymerase chain reaction (ddPCR) technology, enabling absolute quantification of the target gene. Benzonase treatment of non-transduced plasmids substantially shortened the experimental period, reducing cell culture duration from 10-14 days–3 days. The method was rigorously validated by assessing specificity, working range, limit of quantification, precision, accuracy, and robustness. This study demonstrates the feasibility of combining enzymatic digestion with ddPCR to quantify lentiviral vector infectious titer and provides a detailed and readily adaptable methodology for the scientific community.

Enhancing Sensitivity in Detecting Severe Fever With Thrombocytopenia Syndrome Virus: Development of a Reverse Transcription-Droplet Digital Polymerase Chain Reaction.

Severe fever with thrombocytopenia syndrome (SFTS) is a highly fatal disease. Droplet digital polymerase chain reaction (ddPCR) presents unparalleled sensitivity and enables absolute quantification of viral load. In this prospective study, we enrolled 111 patients with SFTS and collected 259 continuous samples. Our findings unveil a robust reverse transcription (RT)-ddPCR method for SFTS with a limit of detection of 2.46 copies/µL (95% CI, 1.50-11.05), surpassing the sensitivity of RT-quantitative polymerase chain reaction at 103.29 copies/µL (95% CI, 79.69-216.35). Longitudinal cohort analysis revealed significantly higher RT-ddPCR detection rates at days 10 to 11, 13 to 14, and ≥15 of the disease course as compared with RT-quantitative polymerase chain reaction (P < .05). Positive RT-ddPCR results were associated with declined platelet and elevated aspartate aminotransferase and lactate dehydrogenase on the same day vs negative RT-ddPCR samples. RT-ddPCR exhibits commendable diagnostic efficacy in SFTS, and it remains detectable in blood samples from patients with an extended disease course. Furthermore, RT-ddPCR correlates with clinical laboratory tests, furnishing valuable reference data for clinical diagnosis.