Droplet Digital PCR Method Research Articles

Three-dimensional linkage analysis with digital PCR for genome integrity and identity of recombinant adeno-associated virus

Recombinant adeno-associated virus (rAAV) has emerged as the vector of choice for in vivo gene delivery, with numerous clinical trials underway for the treatment of various human diseases. Utilizing rAAV in gene therapy requires a highly precise quantification method to determine the viral genome titer and further establish the optimal therapeutic dosage for a rAAV product. The conventional single-channel droplet digital PCR (1D ddPCR) method offers only partial information regarding the viral vector genome titer, lacking insights into its integrity. In our pursuit of further advancing rAAV analysis, we have developed a novel 3D ddPCR assay with advanced 3D linkage analysis. We have designed the three amplicon sites targeting both ends of the viral genome, as well as the center of key therapeutic gene of interest (GOI). This study aims to offer a more comprehensive and insightful assessment of rAAV products which includes not only quantity of viral genome titer but also the quality, distinguishing between partial ones and intact full-length viral genomes with the right GOI. Importantly, due to the random partitioning property of a digital PCR system, the 3D linkage analysis of rAAV viral genome requires a proper mathematical model to identify the true linked DNA molecules (full-length/intact DNA) from the population of false/unlinked DNA molecules (fragmented/partial DNA). We therefore have developed an AAV 3D linkage analysis workflow to characterize genomic integrity and intact titer for rAAV gene therapy products. In this study, we focus on evaluating our 3D linkage mathematical model by performing DNA mixing experiments and a case study using multiple rAAV samples. Particularly, we rigorously tested our algorithms by conducting experiments involving the mixing of seven DNA fragments to represent various AAV viral genome populations, including 3 single partials, 3 double partials, and 1 full-length genomes. Across all 37 tested scenarios, we validated the accuracy of our workflow’s output for the percentages of 3D linkage by comparing to the known percentages of input DNA. Consequently, our comprehensive AAV analytical package not only offers insights into viral genome titer but also provides valuable information on its integrity and identity. This cost-effective approach, akin to the setup of traditional 1D or 2D dPCR, holds the potential to advance the application of rAAV in cell and gene therapy for the treatment of human diseases.

Development and validation of a multiplex chip-based droplet digital PCR method for detecting CNVs in 7q11.2 and 22q11.2 regions.

Copy number variations (CNVs) in the 7q11.2 and 22q11.2 chromosomal regions are major contributors to genetic disorders such as Williams-Beuren syndrome and 22q11.2 deletion/duplication syndromes. These disorders are characterized by facial anomalies, growth retardation, intellectual disabilities, and lethal cardiovascular abnormalities. Despite the development and clinical application of various rapid molecular tests, each has significant limitations. We developed a multiplex chip-based droplet digital PCR (ddPCR) method for detecting microdeletions and microduplications in the 7q11.2 and 22q11.2 regions. We evaluated its linearity and reproducibility and tested it on 100 clinical patients with congenital heart defects to further verify its clinical applicability. We successfully developed a method capable of simultaneously detecting four types of CNVs in a single assay, demonstrating high accuracy and reproducibility. Compared to traditional methods, our approach depicted 100% concordance for positive and negative results. Additionally, our method accurately quantified target gene concentrations, allowing for precise evaluation of CNVs in the target regions. This study introduces a rapid, technically straightforward, and efficient quantitative chip-based ddPCR method for the detailed classification of CNVs in the 7q11.2 and 22q11.2 regions. Our findings indicated that chip-based ddPCR can be seamlessly implemented as a first-line screening tool in routine diagnostics.

Development of a droplet digital PCR method for the detection of Ureaplasma urealyticum

Development of a droplet digital PCR method for the detection of Ureaplasma urealyticum

Development of the duplex droplet digital PCR for quantitative monitoring of mixed infections of Meloidogyne incognita and M. enterolobii.

Root-knot nematodes (RKN, Meloidogyne spp.) are economically significant pests that cause immense damage to a wide range of crops. Among them, M. incognita and M. enterolobii are of particular concern, as their high virulence and broad host range. RKN are challenging for detection due to their subterranean lifestyle underground. Also, the mixed infection of nematodes in field crops complicates the need for more accurate diagnostic and quantification technologies. To address this challenge, we developed and optimized a novel duplex droplet digital PCR (ddPCR) method, using primer/probe sets targeting M. incognita and M. enterolobii, to simultaneously identify and quantify both species within a single assay. The innovative ddPCR diagnostic demonstrated excellent performance in terms of sensitivity, precision and reproductivity when quantifying the eggs and soil samples containing juveniles of both species. Moreover, the application of the duplex ddPCR method enables the monitoring of population dynamics of M. incognita and M. enterolobii under competitive environmental conditions. Our results indicated that the reproduction factor of M. incognita possibly inhibited when in mixed populations of M. enterolobii. In this study, we first applied duplex ddPCR technique for differentiating mixed infections of M. incognita and M. enterolobii, offering a valuable tool for species detection and quantification. It enables the monitoring of population dynamics for both species, which is crucial for providing theoretical guidance for the implementation of timely and effective control measures. © 2024 Society of Chemical Industry.

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.

A droplet digital PCR method for the detection of scale drop disease virus in yellowfin seabream (Acanthopagrus latus).

In this study, a ddPCR method for the detection of scale drop disease virus (SDDV) in yellowfin seabream (Acanthopagrus latus) was established based on Real-time fluorescence quantitative PCR detection methods and principles. The reaction conditions were optimized, and the sensitivity, specificity, accuracy, and reproducibility were assessed. The results showed that threshold line position was determined to be 1900 by the ddPCR method; the optimum annealing temperature for SDDV detection by the ddPCR method was 60°C; the limit of detection was 1.4-1.7 copies/μL; the results of specific detection of other common viruses, except for SDDV specific amplification, were all negative; and the relative standard deviation (RSD) for the reproducibility validation was 0.77%. The samples of yellowfin seabream (Acanthopagrus latus) liver, spleen, kidney, heart, intestine, brain, blood, muscle, skin and ascites with three replicates, respectively, were tested using the ddPCR method, and the results were consistent with clinical findings. The ddPCR method established in this study has the advantages of high sensitivity, high specificity, good reproducibility and simple steps for the quantitative detection of SDDV, which could be used for the nucleic acid detection of clinical SDDV samples, and provided a new quantitative method for the diagnosis of yellowfin seabream SDDV in the early stage of pathogenesis.

A droplet digital PCR assay to detect Chinese rice-field eels rhabdovirus.

Chinese rice-field eels rhabdovirus (CrERV) causes haemorrhagic disease in Chinese rice-field eels (Monopterus albus), leading to significant mortality and economic losses. Sensitive detection of CrERV nucleic acids is essential to control the spread of this pathogen and to treat infected individuals. Herein, we developed an efficient and sensitive droplet digital PCR (ddPCR) method to rapidly detect and quantify CrERV. The ddPCR assay optimal conditions were an annealing temperature of 53°C, and primer and probe concentrations of 0.5 and 0.25 μM, respectively. The assay had a diagnostic sensitivity of 0.23 copies/μL, and was highly specific, showing no cross reactivity with other viruses (infectious haematopoietic necrosis virus, grass carp reovirus, spring viremia of carp virus, largemouth bass ranavirus, carp edema virus, Chinese giant salamander iridovirus, and white spot syndrome virus). Real-time quantitative PCR testing of 30 Chinese rice-field eels samples detected CrERV in 7 samples (23.3%), whereas ddPCR detected CrERV in 12 samples (40%), demonstrating its higher sensitivity. Thus, ddPCR represents an advanced method to absolutely quantify CrERV in infected fish with low virus concentrations, providing a valuable tool to manage the spread and impact of CrERV.

Discovery unbalanced DNA mixtures and evaluation mixing ratio via a droplet digital PCR method.

Small amounts of DNA from a perpetrator collected during crime-scene investigations can be masked by large amounts of DNA from the victim. These samples can provide important information for the perpetrator's conviction. Short tandem repeat (STR) detection system is not sensitive enough to detect trace amounts of minor components in unbalanced mixed DNA. We developed a system using droplet digital polymerase chain reaction (ddPCR) capable of discovering trace components and accurately determining the ratio of mixed DNA in extremely unbalanced mixtures. The non-recombining regions of the X chromosome and Y chromosome were quantified in the DNA of male and female mixtures using duplex ddPCR. Absolute quantification of low-abundance portions of trace samples and unbalanced mixtures was done using different mixing ratios. The ddPCR system could be used to detect low-abundance samples with < 5 copies of DNA components in an extremely unbalanced mixture at a mixing ratio of 10000:1. The high sensitivity and specificity of the system could identify the mixing ratio of mixed DNA accurately. A ddPCR system was developed for evaluation of mixed samples of male DNA and female DNA. Our system could detect DNA quantities as low as 5 copies in extremely unbalanced mixed samples with good specificity and applicability. This method could assist forensic investigators in avoiding the omission of important physical evidence, and evaluating the ratio of mixed male/female trace samples.

Different ploidy-level hybrids derived from female common carp × male topmouth culter

Distant hybridization is an effective strategy for creating polyploids in fish genetic breeding. In this study, the cross of common carp (Cyprinus carpio, 2n = 100, COC, ♀) and topmouth culter (Culter alburnus, 2n = 48, TC, ♂), which belonged to different subfamilies, was conducted and obtained surviving hybrid offspring (CT hybrids). Based on the measurement of DNA content and chromosomal numbers, three ploidy levels of CT hybrids were identified: diploid hybrids (2n = 74, 2nCT), triploid hybrids (3n = 124, 3nCT), and tetraploid hybrids (4n = 148, 4nCT). Analyses of countable traits and measurable traits indicated a significant similarity between CT hybrids and COC in comparison to TC. The genetic variation analysis of 5S rDNA and HoxD10a gene in CT hybrids revealed the retention of all COC genotypes and the partial elimination of TC genotypes, as well as the generation of novel variant genotypes (derived from TC genotypes) and recombinant genotypes (derived from parental genotypes), which suggested that in CT hybrids, the paternal subgenome exhibited greater instability than the maternal subgenome. In addition, the droplet digital PCR (ddPCR) method, combining allele-specific probes targeting the EPHX2 gene, was employed for the first time to investigate the genome composition of hybrids. The results showed that the ratios of COC-allele number to TC-allele number were approximately 1 in 2nCT, 2 in 3nCT, and 1.5 in 4nCT, respectively, which confirmed the hybridization origins for each type and effectively distinguished between the three CT hybrids. The obtainment of CT hybrids harbors potential benefits and application in aquaculture and provides an ideal model for understanding the influence of hybridization and polyploidization on the genomic composition of fish species.

Sensitivity analysis of RT-qPCR and RT-ddPCR for SARS-CoV-2 detection with mutations on N1 and E primer-probe region.

The emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has resulted in a growing number of mutations in its genome, presenting new challenges for the diagnosis of SARS-CoV-2 using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and droplet digital PCR (RT-ddPCR) methods. There is an urgent need to develop refined methods for modifying primers and probes to improve the detection of these emerging variants. In this study, our focus was on the SNVs that have emerged in the CDC-N1 and Charité-E primer-probe regions. Our research has confirmed that the presence of these SNVs in the primer or probe region can significantly affect the results of coronavirus disease 2019 tests. we have developed and validated a modified detection method that can provide higher sensitivity and specificity. This study emphasizes the importance of refining the primer-probe sets to ensure the diagnostic accuracy of RT-qPCR and RT-ddPCR detection.

Development of a multiplex droplet digital PCR method for detection and monitoring of Mycobacterium tuberculosis and drug-resistant tuberculosis

BackgroundThe 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.MethodsA 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. We collected a total of 18 MDR-TB residual samples referred for PCR analysis. Total DNA was extracted from the samples and subjected to the quadruplex ddPCR assay. Their results were compared to those of known resistance phenotypes.ResultsThe analytical sensitivity and specificity of the multiplex ddPCR assay for detecting INH, RIF, EMB, FQ, and SM resistance-causing mutations ranged from 71.43 to 100% and 94.12–100%, respectively. Follow-up sample results showed that the quadruplex ddPCR assay was sensitive enough to detect IS6110 and other mutations even after onset of treatment.ConclusionsWe developed a sensitive and accurate multiplex ddPCR assay that can detect the presence of tuberculosis quantitatively and resistance-conveying mutations concurrently. This tool could aid clinicians in the diagnosis and treatment monitoring of tuberculosis.

A droplet digital PCR method (ddPCR) for sensitive detection and quantification of Carassius auratus herpesvirus (CaHV)

Carassius auratus herpesvirus (CaHV) is a pathogen isolated from crucian carp (Carassius auratus) associated with high mortality. A diagnosis method that can detect the virus at an early stage, specifically and accurately, is an urgent requirement for the prevention of CaHV transmission. In the present study, a droplet digital PCR (ddPCR) method based on the tumor necrosis factor receptor (TNFR) gene was established to detect and quantify CaHV DNA with high specificity and no cross-reactions with other aquatic viruses. Skin mucus samples were collected from infected crucian carp from Day 1–8 after infection, and positive amplification was detected on the first day by ddPCR (0.54 copies/μL), whereas the presence of CaHV was not detected by routine PCR until Day 6. Tissue DNA was then collected from the head kidney of 20 fishes which were injected with CaHV and died during the experiment. The five negative samples checked by routine PCR were detected by ddPCR and real-time PCR (qPCR), respectively. The results showed that the positive detection rate of ddPCR (100%) was higher than that of qPCR (40%). The detection limit of the ddPCR was found to be 0.52 copies/μL, which was much lower than the 50.12 copies/μL determined by qPCR. Overall, ddPCR offers a highly promising diagnosis method for the absolute quantification of CaHV in carrier fish and samples from the skin mucus and head kidney with low viral concentrations.

Abstract 3699: Identifying novel resistance biomarkers in circulating tumor cell-expressed transcriptomes of metastatic castration-resistant prostate cancer patients treated with androgen receptor signaling inhibitors

Abstract Introduction: For individuals with metastatic castration-resistant prostate cancer (mCRPC), therapeutic options include androgen receptor signaling inhibitors (ARSIs) like abiraterone or enzalutamide, as well as taxane-based chemotherapy. Despite this, establishing the best treatment approach is still debated, with varied responses to treatments observed. Notably, around 30%-40% of mCRPC patients either do not benefit from ARSI therapy or quickly become resistant. PSMA PET/CT demonstrates prognostic significance in forecasting responses to ARSI treatments, underscoring its utility in managing mCRPC. Nevertheless, the effectiveness of blood test-measured PSMA expression levels in diagnosing and treating prostate cancer remains uncertain. Consequently, there is a crucial demand for predictive biomarkers, particularly those indicating therapeutic resistance, to customize treatments according to the unique tumor biology of individual patients. Materials and Methods: We recruited 21 healthy donors and 60 ARSI-treated metastatic castration-resistive prostate cancer patients. We isolate the circulating tumor cells (CTCs) using lateral magnetophoretic microfluidic technology. The EpCAM-labeled CTCs were specifically isolated by microfluidic technology and analyzed for cell enumeration and transcriptomic analysis. Prostate cancer and epithelial-related six genes (PSMA, PSA, AR, AR-V7, EpCAM, and KRT19) were detected by droplet digital PCR (ddPCR) method. Results: In ARSI-treated groups, the connection between transcript expression and CTC count is differentiated by the presence or absence of a PSA response. Specifically, 41.7% (25/60) of the ARSI group exhibited a PSA non-response, defined as less than a 50% reduction from the baseline level. These patients demonstrated elevated PSA, PSMA, and AR-V7 mRNA expression levels. Kaplan-Meier survival analysis indicated that patients with high PSMA, PSA, and AR-V7 expressions and a higher CTC count experienced poorer PSA-PFS, radiological-PFS, and overall survival (OS) than those with lower expression levels. This trend was echoed in the findings of the proportional hazards model. Notably, patients with higher PSMA mRNA expression faced significantly lower PSA-PFS (HR: 2.28, 95% CI: 1.20-4.33, p = 0.012), radiological-PFS (HR: 4.75, 95% CI: 2.27-9.93, p &amp;lt; 0.001), and OS (HR: 6.99, 95% CI: 2.93-16.73, p &amp;lt; 0.001) than those with lower expression. The Cox model presented analogous results for CTC count and PSA mRNA levels. However, other transcripts, such as AR, KRT19, and EpCAM, did not correlate significantly with the response to ARSI treatment. Conclusions: The study's conclusion revealed that PSMA, PSA, and AR-V7 are biomarkers resistant to ARSI treatment among the CTC-derived transcriptomic genes. Citation Format: Hyungseok Cho, Jiwon Cha, Ki-Ho Han, Jae-Seung Chung. Identifying novel resistance biomarkers in circulating tumor cell-expressed transcriptomes of metastatic castration-resistant prostate cancer patients treated with androgen receptor signaling inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3699.

Surveillance of SARS-CoV-2 in wastewater by quantitative PCR and digital PCR: a case study in Shijiazhuang city, Hebei province, China

ABSTRACT In this study, we reported the first long-term monitoring of SARS-CoV-2 in wastewater in Mainland China from November 2021 to October 2023. The city of Shijiazhuang was employed for this case study. We developed a triple reverse transcription droplet digital PCR (RT-ddPCR) method using triple primer-probes for simultaneous detection of the N1 gene, E gene, and Pepper mild mottle virus (PMMoV) to achieve accurate quantification of SARS-CoV-2 RNA in wastewater. Both the RT-ddPCR method and the commercial multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) method were implemented for the detection of SARS-CoV-2 in wastewater in Shijiazhuang City over a 24-month period. Results showed that SARS-CoV-2 was detected for the first time in the wastewater of Shijiazhuang City on 10 November 2022. The peak of COVID-19 cases occurred in the middle of December 2022, when the concentration of SARS-CoV-2 in the wastewater was highest. The trend of virus concentration increases and decreases forming a “long-tailed” shape in the COVID-19 outbreak and recession cycle. The results indicated that both multiplex RT-ddPCR and RT-qPCR are effective in detecting SARS-CoV-2 in wastewater, but RT-ddPCR is capable of detecting low concentrations of SARS-CoV-2 in wastewater which is more efficient. The SARS-CoV-2 abundance in wastewater is correlated to clinical data, outlining the public health utility of this work. Highlights First long-term monitoring of SARS-CoV-2 in wastewater in Mainland China COVID-19 outbreak was tracked in Shijiazhuang City from outbreak to containment Wastewater was monitored simultaneously using RT-ddPCR and RT-qPCR methods Triple primer-probe RT-ddPCR detects N1 and E genes of SARS-CoV-2 and PMMoV

Utilizing droplet digital polymerase chain reaction for siRNA quantitation in rodent plasma and tissue via stem-loop reverse transcription.

Background: siRNA is a promising therapeutic modality highlighted by several US FDA approvals since 2018, with many more oligonucleotide assetsin clinical development. To support siRNA discovery and development, robust and sensitive quantitative platforms for bioanalysis must be established to assess pharmacokinetic/pharmacodynamic relationships and toxicology. Droplet digital PCR offers improved sensitivity and throughput, as well as reduced susceptibility to matrix effects, compared with other analytical platforms. Methodology: The authors developed a stem-loop reverse transcription droplet digital PCR method to measure siRNA in mouse plasma and liver extract using bioanalytical method qualification guidelines. Conclusion: This newly developed assay has been demonstrated to be a superior alternative to other platforms, with the added benefit of greater sensitivity, with dynamic range from 390 to 400,000 copies/reaction and readiness for FDA investigational new drug-enabling applications.

The Prognostic Value of Somatic Mutations of Epigenetic Regulation Genes in Acute Myeloid Leukemias in Real-World Clinical Practice: Results of an Observational Non-Interventional Prospective Interregional Study

Aim. To assess the rate of DNMT3A, IDH1, IDH2, and ASXL1 gene mutations and their effect on the prognosis both as isolated findings and in combination with well-known chromosomal aberrations and gene mutations in newly diagnosed acute myeloid leukemia (AML) patients from some regions of the Russian Federation.&#x0D; Materials &amp; Methods. The study enrolled 83 patients with newly diagnosed AML from 22 regions of the Russian Federation, who underwent molecular genetic examination for detecting IDH1 (R132), IDH2 (R140), ASXL1, and DNMT3A gene mutations with droplet digital PCR and Sanger sequencing methods.&#x0D; Results. The mutation rate in DNMT3A was 16.7 %, in IDH1 (R132) it was 6 %, in IDH2 (R140) it was 9.6 %, and in ASXL1 it was 6 %. The R140 mutation in IDH2 correlated with the older age of patients. The mutations in IDH1 (R132), IDH2 (R140), and DNMT3A showed a significant association with mutated NPM1. The mutations in IDH1 (R132), IDH2 (R140) were reported to occur significantly more often in patients with normal karyotype. The IDH1 (R132) and IDH2 (R140) mutations appeared to have a favorable effect on AML prognosis, which is most likely to be associated with a high rate of their compatibility with NPM1 mutation. The mutated type of DNMT3A had a negative effect on overall survival of patients with NPM1 mutation. The mutation in ASXL1 also appeared to be an unfavorable prognostic factor for overall survival of patients with wild type NPM1.&#x0D; Conclusion. A high rate of mutation occurrence in epigenetic regulation genes as well as the prognostic potential of these mutations in AML necessitate the need for determining the mutation status of DNMT3A, IDH1, IDH2, and ASXL1 in the context of primary diagnosis in real-world clinical practice.

Development and Application of Droplet Digital PCR Assay for the Detection of Watermelon Silver Mottle Virus and Melon Yellow Spot Virus

Watermelon silver mottle virus (WSMoV) and melon yellow spot virus (MYSV) (Tospoviridae, Orthotospovirus) are responsible for silver mottle mosaic and yellow spot symptoms, posing threats to melon (Cucumis melo), watermelon (Citrullus lanatus), and cucumber and leading to significant economic losses in China. Early disease detection and monitoring of these two viruses are necessary for disease management, for which a rapid, reliable, and adaptable diagnostic method is required. In this study, using a droplet digital PCR (ddPCR) method, the conserved region of the nucleocapsid gene (N gene) sequence was detected in WSMoV and MYSV. The probes and primers for WSMoV and MYSV did not detect other relevant cucurbit viruses, and the specificity reached 100%. Although both qPCR and ddPCR exhibited good reproducibility, the reproducibility of ddPCR was better than that of qPCR. The reproducibility of ddPCR was proved to be 100%. Moreover, ddPCR exhibited a good linear correlation with varying concentrations of targets. The detection limits of WSMoV and MYSV in ddPCR were 18 and 9 copies/μL and were approximately 12- and 18-times more than those in qPCR, respectively. Finally, 62 samples collected from the field (including infected melon, watermelon, and weeds) were further evaluated for the presence of WSMoV and MYSV. The field samples exhibited 91.94% and 51.61% positivity rates in ddPCR assays for WSMoV and MYSV, respectively; the rates were higher than those in qPCR (59.68% and 43.39%, respectively). The results indicated that ddPCR has a higher accuracy than qPCR. Therefore, ddPCR could be used in the clinical diagnosis of early infections of WSMoV and MYSV. To the best of our knowledge, this is the first study to establish a ddPCR method for the detection of WSMoV and MYSV. The application of this method for differential detection of MYSV and WSMoV will help in understanding the epidemics caused by these two important viruses and provide important information for the early detection, monitoring, and rapid extermination of infection.

Establishment and clinical application of a droplet digital PCR method for the detection of Edwardsiella tarda.

Edwardsiella tarda (E. tarda) can infect humans and a variety of animals, including fish, amphibians, reptiles, birds, and mammals. However, a more highly sensitive, specific, and repeatable test for its detection is lacking. The objective of this study was to develop a highly sensitive, specific, and repeatable droplet digital polymerase chain reaction (ddPCR)-based method for the quantitative detection of E. tarda. The gyrB gene was selected as the target gene, and primers and probe were designed and synthesized. Using E. tarda genomic DNA as templates, the reaction method was optimized to establish a linear relationship with real-time PCR detection methods. The sensitivity, specificity, and repeatability of the method were analyzed, and clinical samples were tested. When the primer and probe concentrations were 900 and 300 nM, respectively, and the annealing temperature was 57°C, the efficiency of the ddPCR amplification reaction was highest and the boundary between positive and negative droplet distribution was clearest. The sensitivity was high, with detection limit being as low as 0.56 copies·μL-1; additionally, and a good linear relationship (R 2 = 0.9962) between ddPCR and real-time PCR detection, within the range of 1-25,000 copies·μL-1, was evident. The repeatability was good, with a detection coefficient of variation of 2.74%. There was no cross-reactivity with 15 other common pathogenic microorganisms in aquatic animals (Streptococcus agalactiae, Streptococcus iniae, Streptococcus suis type 2, Nocardia seriolae, Vibrio parahaemolyticus, Aeromonas sobria, red sea bream iridovirus, decapod iridescent virus 1, enterocytozoon hepatopenaei, carp edema virus, Koi herpesvirus, goldfish hematopoietic necrosis virus, tilapia lake virus, viral nervous necrosis virus, or grass carp reovirus) in positive samples. Among the 48 clinical samples, including Bahaba taipingensis and its live food fish, pond water samples, and routine monitoring samples (Koi), 21 were positive for E. tarda, consistent with the bacterial isolation and identification results. The E. tarda ddPCR detection method has high specificity, sensitivity, and repeatability, can more accurately quantify E. tarda, and provides a useful reference for research related to this bacterium.

Development and evaluation of a triplex droplet digital PCR method for differentiation of M. tuberculosis, M. bovis and BCG.

Tuberculosis, caused by Mycobacterium tuberculosis complex (MTBC), remains a global health concern in both human and animals. However, the absence of rapid, accurate, and highly sensitive detection methods to differentiate the major pathogens of MTBC, including M. tuberculosis, M. bovis, and BCG, poses a potential challenge. In this study, we have established a triplex droplet digital polymerase chain reaction (ddPCR) method employing three types of probe fluorophores, with targets M. tuberculosis (targeting CFP-10-ESAT-6 gene of RD1 and Rv0222 genes of RD4), M. bovis (targeting CFP-10-ESATs-6 gene of RD1), and BCG (targeting Rv3871 and Rv3879c genes of ΔRD1), respectively. Based on optimization of annealing temperature, sensitivity and repeatability, this method demonstrates a lower limit of detection (LOD) as 3.08 copies/reaction for M. tuberculosis, 4.47 copies/reaction for M. bovis and 3.59 copies/reaction for BCG, without cross-reaction to Mannheimia haemolytica, Mycoplasma bovis, Haemophilus parasuis, Escherichia coli, Pasteurella multocida, Ochrobactrum anthropi, Salmonella choleraesuis, Brucella melitensis, and Staphylococcus aureus, and showed repeatability with coefficients of variation (CV) lower than 10%. The method exhibits strong milk sample tolerance, the LOD of detecting in spike milk was 5 × 103 CFU/mL, which sensitivity is ten times higher than the triplex qPCR. 60 clinical DNA samples, including 20 milk, 20 tissue and 20 swab samples, were kept in China Animal Health and Epidemiology Center were tested by the triplex ddPCR and triplex qPCR. The triplex ddPCR presented a higher sensitivity (11.67%, 7/60) than that of the triplex qPCR method (8.33%, 5/60). The positive rates of M. tuberculosis, M. bovis, and BCG were 1.67, 10, and 0% by triplex ddPCR, and 1.67, 6.67, and 0% by triplex qPCR, with coincidence rates of 100, 96.7, and 100%, respectively. Our data demonstrate that the established triplex ddPCR method is a sensitive, specific and rapid method for differentiation and identification of M. tuberculosis, M. bovis, and BCG.

Efficient extraction of adventitious virus nucleic acid using commercially available methods

An essential step in pharmaceutical product development is screening for contamination with adventitious agents, and there is desire to develop highly sensitive assays to detect adventitious viral nucleic acid. This study sought to examine the nucleic acid extraction efficiency of three viral candidates in relevant background matrices using four different extraction methods. Three model adventitious viruses, Minute virus of Mice, Porcine Circovirus, and Feline Leukemia Virus, were diluted within a variety of background matrices relevant to pharmaceutical production methods. Upon extraction, the nucleic acid was quantified using droplet digital PCR methods. Four nucleic acid extraction methods were assessed, including commercially available kits and manual extraction methods. Each method recovered nucleic acid post-extraction for each of the model viruses within the tested background matrices. The silica-column based method recovered a greater amount of viral nucleic acid, compared to the other methods tested. Similar trends were observed when model virus was diluted in bioreactor supernatant, which replicates industry testing conditions and provides details on which extraction methods might be used in Next Generation Sequencing and PCR methods for detecting contamination within pharmaceutical products.