Evaluation of Droplet Digital PCR Assay for the Diagnosis of Candidemia in Blood Samples.
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ABSTRACT:
SUBMITTER: Chen B
PROVIDER: S-EPMC8480469 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundThe reduced amounts of Mycobacterium leprae (M. leprae) among paucibacillary (PB) patients reflect the need to further optimize methods for leprosy diagnosis. An increasing number of reports have shown that droplet digital polymerase chain reaction (ddPCR) is a promising tool for diagnosis of infectious disease among samples with low copy number. To date, no publications have investigated the utility of ddPCR in the detection of M. leprae. The aim of this study was to develop and evaluate a ddPCR assay for the diagnosis of PB leprosy.MethodologyThe two most sensitive DNA targets for detection of M. leprae were selected from electronic databases for assessment of sensitivity and specificity by quantitative polymerase chain reaction (qPCR) and ddPCR. Control patients (n = 59) suffering from other dermatological diseases were used to define the cut-off of the duplex ddPCR assay. For comparative evaluation, qPCR and ddPCR assays were performed in 44 PB patients and 68 multibacillary (MB) patients.Principal findingsM. leprae-specific repetitive element (RLEP) and groEL (encoding the 65 kDa molecular chaperone GroEL) were used to develop the ddPCR assay by systematically analyzing specificity and sensitivity. Based on the defined cut-off value, the ddPCR assay showed greater sensitivity in detecting M. leprae DNA in PB patients compared with qPCR (79.5% vs 36.4%), while both assays have a 100% sensitivity in MB patients.Conclusions/significanceWe developed and evaluated a duplex ddPCR assay for leprosy diagnosis in skin biopsy samples from leprosy patients. While still costly, ddPCR might be a promising diagnostic tool for detection of PB leprosy.
| S-EPMC6438576 | biostudies-literature
Project description:Moths in the genus Helicoverpa are some of the most important agricultural pests in the world. Two species, H. armigera (Hübner) and H. zea (Boddie), cause the majority of damage to crops and millions of dollars are spent annually on control of these pests. The recent introduction of H. armigera into the New World has prompted extensive survey efforts for this species in the United States. Surveys are conducted using bucket traps baited with H. armigera pheromone, and, because the same pheromone compounds attract both species, these traps often capture large numbers of the native H. zea. Adult H. armigera and H. zea are very similar and can only be separated morphologically by minor differences in the genitalia. Thus, a time consuming genitalic dissection by a trained specialist is necessary to reliably identify either species, and every specimen must be dissected. Several molecular methods are available for differentiating and identifying H. armigera and H. zea, including two recently developed rapid protocols using real-time PCR. However, none of the published methods are capable of screening specimens in large batches. Here we detail a droplet digital PCR (ddPCR) assay that is capable of detecting a single H. armigera in a background of up to 999 H. zea. The assay has been tested using bulk extractions of 1,000 legs from actual trap samples and is effective even when using poor quality samples. This study provides an efficient, rapid, reproducible, and scalable method for processing H. armigera survey trap samples in the U.S. and demonstrates the potential for applying ddPCR technology to screen and diagnose invasive species.
| S-EPMC5451079 | biostudies-literature
Project description:Aujeszky's disease, caused by pseudorabies virus (PRV), has damaged the economy of the Chinese swine industry. A large number of PRV gene-deleted vaccines have been constructed based on deletion of the glycoprotein E ( gE) gene combined with other virulence-related gene deletions, such as thymidine kinase ( TK), whereas PRV wild-type strains contain an intact gE gene. We developed a sensitive duplex droplet digital PCR (ddPCR) assay to rapidly detect PRV wild-type isolates and gE gene-deleted viral vaccines. We compared this assay with a TaqMan real-time PCR (qPCR) using the same primers and probes. Both assays exhibited good linearity and repeatability; however, ddPCR maintained linearity at extremely low concentrations, whereas qPCR did not. Based on positive results for both gE and gB, the detection limit of ddPCR was found to be 4.75 copies/µL in contrast of 76 copies/µL for qPCR, showing that ddPCR provided a 16-fold improvement in sensitivity. In addition, no nonspecific amplification was shown in specificity testing, and the PRV wild-type was distinguished from a gE-deleted strain. The ddPCR was more sensitive when analyzing clinical serum samples. Thus, ddPCR may become an appropriate detection platform for PRV.
| S-EPMC6504158 | biostudies-literature
Project description:Bovine paratuberculosis (PTB) is an infectious disease that affects ruminants worldwide and is a burden on the dairy industry. PTB control measures include culling of Mycobacterium avium subsp. paratuberculosis (MAP)-infected animals from the herd and the enhancement of farm-biosecurity measures. Diagnostics tools for the direct detection of MAP are fecal real-time qPCR and bacteriological culture, the last one being considered the gold standard. However, both show limitations for detecting subclinical MAP-infected cattle with low bacterial load in feces and gut tissues. Droplet digital polymerase chain reaction (ddPCR) is a third-generation PCR method that shows high reproducibility for the quantification of low DNA copy numbers. The objective of this study was to design a ddPCR assay to detect and quantify a fragment of the F57 MAP-specific sequence in samples of naturally MAP-infected Holstein cattle. DNA was isolated from whole-blood and fecal samples from control cows with a negative ELISA and qPCR result (N = 75) and from cows with PTB-associated focal (N = 32), multifocal (N = 21), and diffuse lesions (N = 17) in gut tissues. After ddPCR, the DNA extracted from fecal samples of cows with diffuse lesions showed higher mean copies per microliter (13,791.2 copies/μl) than samples from cows with multifocal lesions (78.8 copies/μl), focal lesions (177.1 copies/μl) or control cows (4.8 copies/μl) (P ≤ 0.05). Significant differences in mean DNA copies/μl were also observed in the blood samples from cows with focal lesions (47.7 copies/μl) when compared with cows with multifocal and diffuse lesions; 18.1 and 12.4 copies/μl, respectively. Using a principal component analysis, the results of the fecal ddPCR clustered together with the results of a commercial ELISA for the specific detection of MAP antibodies, fecal and tissue qPCR, and bacteriological culture results. In contrast, blood ddPCR results clustered together with the results of an ELISA for the detection of a biomarker of subclinical PTB, the ABCA13 transporter. Blood ddPCR was the most sensitive tool (sensitivity 71%, specificity 100%) of all the quantitative methods used in the study for the detection of subclinical cows with focal lesions.
| S-EPMC9563315 | biostudies-literature
Project description:In this study, the applicability of droplet digital PCR (ddPCR) for routine analysis in food and feed samples was demonstrated with the quantification of genetically modified organisms (GMOs). Real-time quantitative polymerase chain reaction (qPCR) is currently used for quantitative molecular analysis of the presence of GMOs in products. However, its use is limited for detecting and quantifying very small numbers of DNA targets, as in some complex food and feed matrices. Using ddPCR duplex assay, we have measured the absolute numbers of MON810 transgene and hmg maize reference gene copies in DNA samples. Key performance parameters of the assay were determined. The ddPCR system is shown to offer precise absolute and relative quantification of targets, without the need for calibration curves. The sensitivity (five target DNA copies) of the ddPCR assay compares well with those of individual qPCR assays and of the chamber digital PCR (cdPCR) approach. It offers a dynamic range over four orders of magnitude, greater than that of cdPCR. Moreover, when compared to qPCR, the ddPCR assay showed better repeatability at low target concentrations and a greater tolerance to inhibitors. Finally, ddPCR throughput and cost are advantageous relative to those of qPCR for routine GMO quantification. It is thus concluded that ddPCR technology can be applied for routine quantification of GMOs, or any other domain where quantitative analysis of food and feed samples is needed.
| S-EPMC3642186 | biostudies-literature
Project description:BackgroundThe presence of carbapenemase-producing bacteria (CPB) in animal hosts and along the food chain may result in the development of reservoirs for human infections. Several CPB strains isolated from animals have been reported, suggesting that transmission and dissemination of the corresponding genes between humans and animals may occur. Animal and food samples have complex backgrounds that hinder the detection of CPB present in low concentrations by standard detection procedures.MethodsWe evaluated the possibility of detecting blaKPC, blaVIM, and blaOXA-48-like carbapenemases in 286 animal and food samples (faeces from farm and companion animals, raw meat, bivalve molluscs) by culture-based and standard molecular methods and by ddPCR.ResultsThe proposed ddPCR managed to detect the target genes, also in samples resulting negative to standard methods. While the presence of blaKPC and blaVIM was detected in few samples (~3%), one third of the samples (n = 94/283) carried different variants of blaOXA-48-like genes.ConclusionA specific and sensitive method such as ddPCR could be suitable to evaluate the current veterinarian and environmental situation and to assess the dynamic transmission and persistence of CPB between animals and humans and vice versa.
| S-EPMC9774140 | biostudies-literature
Project description:Objective Bacterial DNA (bactDNA) detection can be used to quickly identify pathogenic bacteria and has been studied on ascitic fluid. We aimed to retrospectively analyze the diagnostic value and applicational prospect of the bactDNA load in spontaneous bacterial peritonitis (SBP). Method We extracted viable bactDNA from ascitic samples of 250 patients with decompensated cirrhosis collected from October 2019 to April 2021 and detected the bactDNA by droplet digital polymerase chain reaction (ddPCR). We used ascitic samples of a baseline cohort of 191 patients to establish diagnostic thresholds for SBP and analyze the patients’ diagnostic performance based on ascites polymorphonuclear (PMN) and clinical manifestation. We performed bactDNA quantification analysis on 13 patients with a PMN less than 250 cells/mm3 but with clinical symptoms. The dynamic changes of the bactDNA load from eight patients (before, during, and after SBP) were analyzed. Results After the removal of free DNA, the bactDNA detected by ddPCR was generally decreased (1.75 vs. 1.5 log copies/µl, P < 0.001). Compared with the traditional culture and PMN count in the SBP diagnosis, the bactDNA showed that the ddPCR sensitivity was 80.5%, specificity was 95.3%, positive predictive value was 82.5%, and negative predictive value was 94.7%, based on clinical composite criteria. In patients with a PMN less than 250 cells/mm3, the bactDNA load of 13 patients with symptoms was significantly higher than those without symptoms (2.7 vs. 1.7 log copies/µl, P < 0.001). The bactDNA in eight patients had SBP that decreased by 1.6 log copies/µl after 48 h of antibiotic treatment and by 1.0 log copies/µl after 3 days of continued treatment. Conclusion BactDNA detection can be used to further enhance the diagnostic efficiency of SBP. Therefore, the application of ddPCR assay not only can be used to discriminate and quantify bacteria but also can be used in the clinical assessment for antibiotics treatment.
| S-EPMC9433567 | biostudies-literature
Project description:The prion protein (PRNP) gene encoding prion protein is considered a prerequisite for the occurrence of scrapie disease, and knockout of the PRNP gene in transgenic goat is one effective approach to avoid scrapie. This study aims to establish an event-specific droplet digital polymerase chain reaction (ddPCR) assay to detect and quantify the content of genetically modified (GM) PRNP-knockout goat event KoP1. The developed ddPCR assay presents high specificity, sensitivity, accuracy, precision and wide dynamic range. The limits of detection and quantification were as low as 1.44 and 7.2 haploid genome equivalent (HGE) per reaction, respectively. Furthermore, this assay was successfully applied in quantifying the goat KoP1 GM content in milk, feces and living environmental soil samples. We believe that the developed ddPCR assay has the potential to be used in the evaluation of horizontal gene transfer and the practical risk assessment of GM goat event KoP1 and its derivatives.
| S-EPMC8953510 | biostudies-literature
Project description:IntroductionIt has been proposed that the copy number (CN) variation (CNV) in β-defensin genes (DEFB) on human chromosome 8p23 determines phenotypic differences in inflammatory diseases. However, no method for accurate and easy DEFB CN quantification is yet available.ObjectiveDroplet digital polymerase chain reaction (ddPCR) is a novel method for CNV quantification and has been used for genes such as CCL4L, CCL3L1, AMY1, and HER2. However, to date, no ddPCR assay has been available for DEFB CN determination. In the present study, we aimed to develop and evaluate such a ddPCR assay.MethodsThe assay was designed using DEFB4 and RPP30 as the target and the reference gene, respectively. To evaluate the assay, 283 DNA samples with known CNs previously determined using the multiple ligation-dependent probe amplification (MLPA) method, the current gold standard, were used as standards. To discover the optimal DNA template amount, we tested 80 to 2.5 ng DNA by a serial of one to two dilutions of eight samples. To evaluate the reproducibility of the assay, 31 samples were repeated to calculate the intra- and inter-assay variations. To further validate the reliability of the assay, the CNs of all 283 samples were determined using ddPCR. To compare results with those using quantitative PCR (qPCR), DEFB CNs for 48 samples were determined using qPCR with the same primers and probes.ResultsIn a one-dimensional plot, the positive and negative droplets were clearly separated in both DEFB4 and RPP30 detection channels. In a two-dimensional plot, four populations of droplets were observed. The 20 ng template DNA proved optimal, with either high (80 ng) or low (10, 5, 2.5 ng) template input leading to ambiguous or inaccurate results. For the 31 standard samples, DEFB CNs were accurately determined with small intra- and inter-assay variations (coefficient of variation < 0.04 for both). In the validation cohort, ddPCR provided the correct CN for all 283 samples with high confidence. qPCR measurements for the 48 samples produced noisy data with high uncertainty and low accuracy.ConclusionsddPCR is an accurate, reproducible, easy-to-use, cheap, high-throughput method for DEFB CN determination. ddPCR could be applied to DEFB CN quantification in large-scale case-control studies.
| S-EPMC8320422 | biostudies-literature
Project description:Transposable elements (TEs) constitute approximately half of Bovine genome. They can be a powerful species-specific marker without regression mutations by the structure variation (SV) at the time of genomic evolution. In a previous study, we identified the Hanwoo-specific SV that was generated by a TE-association deletion event using traditional PCR method and Sanger sequencing validation. It could be used as a molecular marker to distinguish different cattle breeds (i.e., Hanwoo vs. Holstein). However, PCR is defective with various final copy quantifications from every sample. Thus, we applied to the droplet digital PCR (ddPCR) platform for accurate quantitative detection of the Hanwoo-specific SV. Although samples have low allele frequency variation within Hanwoo population, ddPCR could perform high sensitive detection with absolute quantification. We aimed to use ddPCR for more accurate quantification than PCR. We suggest that the ddPCR platform is applicable for the quantitative evaluation of molecular markers.
| S-EPMC7120350 | biostudies-literature