qPCR. The quantitative PCR (qPCR) assay targets two genes in CMV simultaneously, the UL55 and UL123 genes, to increase the likelihood of accurately detecting all possible clinical variants. Both targets are detected using the same fluorophore (6-carboxyfluorescein [FAM]), and the stan- dard numbers are set to account for the dual amplification of each CMV template copy. The following primer and TaqMan probe sets (14) were mixed with 2 ⫻ ABI fast mix and 15 l of template DNA for a final reaction volume of 30 l: UL55 forward, TGG GCG AGG ACA ACG AA; UL55 reverse, TGA GGC TGG GAA GCT GAC AT; UL55probe, FAM-TGG GCA ACC ACC GCA CTG AGG-TAM (tetramethylrhodamine azide); UL123exon-4F, TCC CGC TTA TCC TCR GGT ACA; UL123exon-4R, TGA GCC TTT CGA GGA SAT GAA; UL123exon4, FAM-TCT CAT ACA TGC TCT GCA TAG TTA GCC CAA TAC A-TAM. The reactions were run on a StepOnePlus system (Applied Biosystems, Benicia, CA) using the following thermocycler parameters: 95°C for 20 s followed by 45 cycles of 95°C for 3 s and 60°C for 30 s. A three-point standard curve was included in all runs. Data were analyzed with StepOnePlus (version 2.1) analysis software, and quantitation of virus was presented as copies/ml of plasma. ddPCR. DropletdigitalPCR (ddPCR) has been reported to be less affected by target sequence polymorphisms than qPCR (10), and there- fore the assay used a single primer-probe set targeting UL55. Both UL55
ddPCR is a recent technology known for its high sensi- tivity (Pekin et al., 2011). The great sensitivity of the technique is due to the extreme dilution, and hence sub- division of the droplet sample consisting of an oil-based emulsion generated by the QX200™ AutoDG™ Droplet™ Digital™ PCR System. If the dilution is correct within each of the aliquots, a single amplification reaction will be performed, increasing the method sensitivity (Corbi- sier et al., 2015). For this reason, ddPCR was used in this study as an alternative approach for the identification of fetal sex at very early gestation weeks, where conven- tional qPCR was unable to discriminate male from fe- male fetuses. In particular, cffDNAs were extracted from 29 maternal plasma samples at very early gestational ages (4.5-12 weeks) using the QIAamp ® DSP Virus Spin Kit (Qiagen) and analyzed by qPCR and ddPCR for the sex determination (Fig. 1).
The QX200™ DropletDigital™ PCR System (Bio-Rad) was used for analyses. The ddPCR reaction mixture consisted of 1x ddPCR Supermix for Probes (Bio-Rad), 900 nM of each primer, 250 nM of the probe, and approximately 30 ng bisulfite-converted DNA template, in a final volume of 22 μl. Droplets were generated by the QX200 Droplet Generator (Bio-Rad), using 20 μl of the ddPCR mixture and 70 μl droplet generation oil (Bio-Rad). Samples were transferred to a 96-well PCR plate (Bio-Rad) and sealed in the PX1 PCR Plate Sealer (Bio-Rad). The PCR was performed in a T100 Thermal Cycler (Bio-Rad; see Additional file 1: Table S2 for PCR cycling conditions). The fluorescence signals were measured by the QX200 Droplet Reader (Bio-Rad). For each experiment, the following control samples were included: two methylation- positive controls (commercially available in vitro methyl- ated DNA (IVD); Zymo Research), one methylation- negative control (bisulfite-treated DNA from normal blood of healthy donors), one non-bisulfite-converted IVD sam- ple, and a non-template control (NTC; water). All analyses were performed according to the digital MIQE guidelines (Additional file 2) .
Injectable drugs manufactured in E. coli must be tested for host residual DNA (hr DNA) impurity in ensuring drug purity and safety. Because of low allowa- ble hr DNA as impurity, highly sensitive methods are needed. DropletdigitalPCR (ddPCR) is a new method where the reaction is partitioned into about 20,000 nanoliter-sized droplets and each droplet acts as individual PCR reac- tion. After completion of end-point PCR, droplets are analyzed for fluores- cence and categorized as positive or negative and DNA quantified using Pois- son statistics. Here we describe development of a direct E. coli hr DNA dd PCR method where the drug is directly added to the ddPCR reaction. We show that the ddPCR method has acceptable precision and high accuracy, works with different biologic drugs, and compared to qPCR shows higher to- lerance of drug matrices. The method does not require DNA extraction or standard curves for quantification of hr DNA in unknown samples.
ABSTRACT We utilized pulsed-ﬁeld gel electrophoresis (PFGE) to purify high- molecular-weight DNA from HIV-infected cells. This puriﬁcation, in combination with our previously described dropletdigitalPCR (ddPCR) assay, was used to develop a method to quantify proviral integrated HIV DNA free of lower-molecular-weight spe- cies of HIV DNA. Episomal 2-long-terminal-repeat (2-LTR) circles were completely cleared from HIV DNA samples. Technical replicates of the complete assay, starting with the same specimens, resulted in no statistical differences in quantiﬁcation of in- tegrated HIV gag sequences in cellular DNA from cells from HIV-infected subjects af- ter prolonged treatment with antiretroviral therapy (ART). The PFGE ddPCR assay was compared to the Alu-gag quantitative PCR (qPCR) assay, the most widely used assay to measure proviral integrated HIV DNA. Spearman’s rho nonparametric corre- lation determined PFGE ddPCR results to be positively correlated with Alu-gag qPCR results (r ⫽ 0.7052; P ⫽ 0.0273). In summary, PFGE ddPCR is a sensitive, reproduc- ible, and robust method to measure proviral integrated HIV DNA and is theoretically more accurate than previously described assays, because it is a direct measure of in- tegrated HIV DNA.
According to the SEVA nomenclature, replication sys- tems take up the second position in the three digit code (e.g. 2 in ‘pSEVA123 ′ ), while the first and the third repre- sent the antibiotic resistance and the cargo genes, respec- tively. Here, we used the Kanamycin resistance gene and a styA-EGFP styB (AEB) expression cassette as a cargo module that was already described previously in stud- ies using Pseudomonas [7, 20]. This cassette consists of a styrene monooxygenase (styA) in frame fused to an EGFP reporter and an FAD cofactor reductase (styB). The origi- nal purpose of this construct is the conversion of styrene to styrene oxide and it was used here as a realistic model of a biotechnological process. However, problems related to low PCN and plasmid loss in a part of the population were the starting point for the systematic investigation of plasmid stability in this study. Our aim was to determine the PCN of standard plasmid vectors by highly accurate DropletDigitalPCR (ddPCR), and to reveal relationships between copy number and the degree of heterogeneity as determined by EGFP fluorescence. While fluorescence can be measured on the level of single cells using flow cytometry, determination of PCN by ddPCR requires a larger cell sample. The method applied here employed cell sorting followed by ddPCR and was previously tested with cell numbers ranging from 1 to 10,000 . It appeared that the PCN of a sub-population was constant, regardless how many cells were used, but cell numbers of 1 or 10 showed very high variation and were not suitable. We therefore decided to use 1000 sorted cells of a single, selected sub-population to determine PCN, a number that yielded the best performance in ddPCR (low varia- tion and optimal droplet occupation) .
Karlovich et al also revealed that the sensitivity and specific- ity of BEAMing was 73.3% and 50.0%. 34 Hence, the ddPCR Figure 2 Meta-analysis of (A) sensitivity, (B) specificity, (C) positive likelihood ratio, (D) negative likelihood ratio, (E) diagnostic odds ratio, (F) and sROC curve for dropletdigitalPCR for diagnosing epidermal growth factor receptor T790M mutation in circulating tumor Dna.
ABSTRACT Chronic infection with Helicobacter pylori causes peptic ulcers and stom- ach cancer in a subset of infected individuals. While standard eradication therapy in- cludes multiple antibiotics, treatment failure due to resistance is an increasing clini- cal problem. Accurate assessment of H. pylori antimicrobial resistance has been limited by slow growth and sampling of few isolates per subject. We established a method to simultaneously quantify H. pylori clarithromycin-resistant (mutant) and -susceptible (wild-type) 23S rRNA gene alleles in both stomach and stool samples using dropletdigitalPCR (ddPCR). In 49 subjects, we assessed the performance of these assays alongside clarithromycin MIC testing of up to 16 H. pylori isolates per subject and included both cancer (25 subjects) and noncancer (24 subjects) cases. Gastric ddPCR and H. pylori culture showed agreement with urea breath test (UBT) detection of infection in 94% and 88% of subjects, respectively, while stool ddPCR showed agreement with UBT in 92% of subjects. Based on MIC testing of 43 culture-positive cases, 20 subjects had only susceptible isolates, 14 had a mix of susceptible and resistant isolates, and 9 had only resistant isolates. ddPCR of gastric samples indicated that 21 subjects had only wild-type alleles, 13 had a mixed genotype, and 9 had only mutant alleles. Stool ddPCR detected mutant al- leles in four subjects for which mutant alleles were not detected by stomach ddPCR, and no resistant isolates were cultured. Our results indicate that ddPCR detects H. pylori clarithromycin resistance-associated genotypes, especially in the context of heteroresistance.
Raw poultry and poultry products are a significant source of zoonotic bacterial pathogen transmission; thus the sensitive detection of major zoonotic pathogens (Salmonella spp., Campylobacter jejuni, and Listeria monocytogenes) is a vital food safety issue. Recently, third generation PCR technology, known as dropletdigitalPCR (ddPCR) has been devel- oped to be more accurate and sensitive to detect genetic targets than current quantification methods, but this technology has not been tested within an industrial setting. There is an on-going study within our laboratory is investigating the effects of sampling times and sampling methods on the cultural and molecular (via qPCR) quantification of dominant zoonotic pathogens within a poultry processing facility. This presents a unique opportunity to compare the quantification resulted from this emerging, third generation technology to traditional quantification methods currently employed by the poultry industry. The results show that ddPCR detected pathogen-specific genes from more pathogen:sampling time combina- tions than either the qPCR or culturing methods from the final scalder and chiller tanks at three stages of processing (Start, Mid, and End). In fact, both ddPCR and qPCR substantially outperformed culture methods commonly used in poultry processing food safety-related studies, with Salmonella recovered only from the Mid and End sampling times from the scalder tank. While neither C. jejuni nor L. monocytogenes were recovered culturally, ddPCR was able to detect their respective genes commonly throughout the processing day in both the scalder and chiller water samples. Addition- ally, the use of unfiltered processing water provided significantly greater detection of bacterial and pathogen-specific gene abundances than did an analysis of larger volumes of filtered water. Considering the ddPCR-derived concentrations of the bacterial pathogens were consistent with what was previously found culturally in commercial poultry processing operations, ddPCR represented a significant advancement in poultry processing zoonotic pathogen quantification.
Health care-associated infections with methicillin-resistant Staphylococcus aureus (MRSA) contribute to significant hospitaliza- tion costs. We report here a dropletdigitalPCR (ddPCR) assay, which is a next-generation emulsion-based endpoint PCR assay for high-precision MRSA analysis. Reference cultures of MRSA, methicillin-susceptible S. aureus (MSSA), and confounders were included as controls. Copan swabs were used to sample cultures and collect specimens for analysis from patients at a large teach- ing hospital. Swab extraction and cell lysis were accomplished using magnetic-driven agitation of silica beads. Quantitative PCR (qPCR) (Roche Light Cycler 480) and ddPCR (Bio-Rad QX100 dropletdigitalPCR system) assays were used to detect genes for the staphylococcal protein SA0140 (SA) and the methicillin resistance (mecA) gene employing standard TaqMan chemistries. Both qPCR and ddPCR assays correctly identified culture controls for MRSA (76), MSSA (12), and confounder organisms (36) with 100% sensitivity and specificity. Analysis of the clinical samples (211 negative and 186 positive) collected during a study of MRSA nasal carriage allowed direct comparison of the qPCR and ddPCR assays to the Cepheid MRSA GeneXpert assay. A total of 397 clinical samples were examined in this study. Cepheid MRSA GeneXpert values were used to define negative and positive samples. Both the qPCR and ddPCR assays were in good agreement with the reference assay. The sensitivities for the qPCR and ddPCR assays were 96.8% (95% confidence interval [CI], 93.1 to 98.5%) and 96.8% (95% CI, 93.1 to 98.5%), respectively. Both the qPCR and ddPCR assays had specificities of 91.9% (95% CI, 87.5 to 94.9%) for qPCR and 91.0% (95% CI, 86.4 to 94.2%) for ddPCR technology.
ABSTRACT Accurate and rapid identiﬁcation of methicillin-resistant Staphylococcus aureus (MRSA) is needed to screen MRSA carriers and improve treatment. The cur- rent widely used duplex PCR methods are not able to differentiate MRSA from coex- isting methicillin-susceptible S. aureus (MSSA) or other methicillin-resistant staphylo- cocci. In this study, we aimed to develop a direct method for accurate and rapid detection of MRSA in clinical samples from open environments, such as nasal swabs. The new molecular assay is based on detecting the cooccurrence of nuc and mecA markers in a single bacterial cell by utilizing dropletdigitalPCR (ddPCR) with the chimeric lysin ClyH for cell lysis. The method consists of (i) dispersion of an intact single bacterium into nanoliter droplets, (ii) temperature-controlled release of genomic DNA (gDNA) by ClyH at 37°C, and (iii) ampliﬁcation and detection of the markers (nuc and mecA) using standard TaqMan chemistries with ddPCR. Results were ana- lyzed based on MRSA index ratios used for indicating the presence of the duplex- positive markers in droplets. The method was able to achieve an absolute limit of detection (LOD) of 2,900 CFU/ml for MRSA in nasal swabs spiked with excess amounts of Escherichia coli, MSSA, and other mecA-positive bacteria within 4 h. Ini- tial testing of 104 nasal swabs showed that the method had 100% agreement with the standard culture method, while the normal duplex qPCR method had only about 87.5% agreement. The single-bacterium duplex ddPCR assay is rapid and powerful for more accurate detection of MRSA directly from clinical specimens.
Several platforms of ddPCR have been developed by different manufacturers, such as Fluidigm, Sysmex Inostics (BEAMing DigitalPCR), Bio-Rad Laboratories, or RainDance Technologies. Some of them have already been tested for detection of KRAS mutations producing different results [12–20]. The present study is aimed at evaluating the sensitivity and reproducibility of a new dropletdigitalPCR system (Bio-Rad QX-200 platform) for detection of KRAS G12V mutation in samples of plasma where this mutation has previously been confirmed. This particular mutation was chosen because it has been associated with a worse progression in our series of patients, showing a markedly poor clinical outcome, high rate of post-operative complications, and short time of survival.
limitations such as performance variation in and between assays  and artificial qPCR data resulting from sam- ples with low target concentration but high levels of impurity . These limitations can be overcome by dropletdigitalPCR (ddPCR) as it has been shown to produce more precise and reproducible results when compared to qPCR . In the QX200 ddPCR (Bio- Rad, USA), a single PCR reaction is divided into approxi- mately 20,000 droplets which are treated as individual reactions. Each reaction contains the relevant forward and reverse primer, the probes to detect the target gene and template DNA molecule.
DropletdigitalPCR (ddPCR) can be used to detect low frequency mutations in oncogene- driven lung cancer. The range of KRAS point mutations observed in NSCLC necessitates a multiplex approach to efficient mutation detection in circulating DNA. Here we report the design and optimisation of three discriminatory ddPCR multiplex assays investigating nine different KRAS mutations using PrimePCR ™ ddPCR ™ Mutation Assays and the Bio-Rad QX100 system. Together these mutations account for 95% of the nucleotide changes found in KRAS in human cancer. Multiplex reactions were optimised on genomic DNA extracted from KRAS mutant cell lines and tested on DNA extracted from fixed tumour tissue from a cohort of lung cancer patients without prior knowledge of the specific KRAS genotype. The multiplex ddPCR assays had a limit of detection of better than 1 mutant KRAS molecule in 2,000 wild-type KRAS molecules, which compared favourably with a limit of detection of 1 in 50 for next generation sequencing and 1 in 10 for Sanger sequencing. Multiplex ddPCR assays thus provide a highly efficient methodology to identify KRAS mutations in lung adenocarcinoma.
Cell-free DNA (cfDNA) analysis may provide a non-invasive diagnostic approach for lung adenocarcinoma patients. Recently, dropletdigitalPCR (ddPCR) has been developed as a highly sensitive detection method for a low mutant allele percentage. The ddPCR detection limit for epithelial growth factor receptor (EGFR) mutations was evaluated using cell lines, NCI-H1975 for EGFR L858R point mutation and PC-9 for EGFR E746-A750del. Subsequently, detection of EGFR mutations by ddPCR was performed in tumor DNA (tDNA) and cfDNA samples of 19 lung adenocarcinoma patients whose tumor biopsies were already evaluated for EGFR mutations by clamp PCR (13 of L858R, 3 of E746-750del, and 3 of EGFR negative). In 12 cases, immunohistochemical analysis was performed to quantify the number of EGFR L858R-positive cells rate. EGFR point mutation or deletion were detected in 16 tumor DNA samples. In the measurable cfDNA sam- ples, the rate of detection by ddPCR in cfDNA was 61.5% (8/13) for L858R and 100% (3/3) for E746- A750del. A relative correlation was found between the allele fraction (AF) of tDNA and the number of EGFR L858R-positive cells rate. No correlation was found between the AF of tDNA and AF of cfDNA. In our study, cfDNA mutation detection was not associated with clinicopathological features, but cases with high AF of cfDNA did have metastatic lesions. Our study shows that ddPCR enables cfDNA analysis for EGFR L858R and E746-A750del, with a high detection rate. Therefore, cfDNA analysis using ddPCR may complement to tumor biopsy and is beneficial for precision medicine in lung adenocarcinoma patients.
chain reaction (PCR) is not sensitive enough to detect these low amounts of tumor DNA. Real-time PCR slightly im- proves the detection limit (eg, Cobas [Roche, Basel, Switzerland], Therascreen [QIAGEN, Valencia, CA]), but a major improvement in sensitivity was achieved by the development of digital platforms that target speci ﬁ c mu- tations like dropletdigitalPCR (ddPCR) and beads, emulsion, ampli ﬁ cation, and magnetics digitalPCR. 13 This
Background: Breast cancer circulating biomarkers include carcinoembryonic antigen and carbohydrate antigen 15 – 3, which are used for patient follow-up. Since sensitivity and specificity are low, novel and more useful biomarkers are needed. The presence of stable circulating microRNAs (miRNAs) in serum or plasma suggested a promising role for these tiny RNAs as cancer biomarkers. To acquire an absolute concentration of circulating miRNAs and reduce the impact of preanalytical and analytical variables, we used the dropletdigitalPCR (ddPCR) technique.
Retrotransposition of long interspersed element 1 (L1) in human germline and somatic cells contributes to gen- omic variation in human populations and is implicated in tumorigenesis. In this study, we designed dropletdigitalPCR (ddPCR) assays to detect rare L1 insertion events in heterogeneous human genomic DNA samples. Traditional qPCR methods are unable to confidently dis- cern rare target DNA sequences among input DNA as complex as a human genome due to low-chance priming events that cause background signal and lead to false- positive determinations. This effect is exacerbated when the target DNA involves L1 sequence, which occupies approximately 17% of the human genome. Using univer- sal 5’ and 3’ L1 primers and probes in ddPCR, paired with a locus-specific primer near the assayed insertion site, we detected polymorphic L1 5’ and 3’ junctions in genomic DNA from a heterogeneous sample when as few as 0.01% of the cells contained the polymorphic L1. The ability to confidently detect and simultaneously quantify the level of a L1 insertion locus in a mosaic sample, such as tumor biopsy genomic DNA, will allow rapid validation of high-throughput sequencing data on de novo L1 insertions for a given sample, establishment of a minimum of residual disease detection for a cancer cell-specific L1 insertion, or sampling of pools of human genomic DNA for rare L1 allele detection.
DropletdigitalPCR (ddPCR) is one of the latest molecular amplification techniques that offers high precision and sensitivity and detects rare alleles, copy number variations and absolute quantification of DNA [9–11]. Its high sensitivity enables the detection of a mutant allele fraction as low as 0.1% . The basic principle of ddPCR relies on the generation of a large number of partitions in the form of nanoliter-sized droplets, each of which carries out a PCR reaction on one template. PCR-positive and PCR-negative droplets are then counted by a specialized droplet reader to provide absolute quantification of target DNA in a digital form and are then analyzed by software. As an “ultra-sensitive detection method”, ddPCR was applied in the pretreatment of EGFR T790M mutations in non-small cell lung cancer patients . Detection of HER2 amplification in gastric cancer by ddPCR was as effective as immunohistochemistry/ fluorescence in situ hybridization (IHC/FISH) and may become a standard method for analyzing formalin-fixed paraffin-embedded (FFPE) samples . In glioma, ddPCR successfully measured the IDH1 mutations in extracellular vesicles and cerebrospinal fluid .
ddPCR. A laboratory-developed test (LDT) for CMV detection was used, together with a QX100 dropletdigitalPCR system (Bio-Rad, Pleas- anton, CA). The ddPCR reaction mixture consisted of 10 l of a 2 ⫻ ddPCR master mix (Bio-Rad), 2 l of CMV primer/probe mix (artus CMV PCR analyte-specific reagent; Qiagen, Inc., Valencia, CA), and 5 l of sample nucleic acid solution in a final volume of 20 l. The entire reaction mixture was loaded into a disposable plastic cartridge (Bio-Rad) together with 70 l of droplet generation oil (Bio-Rad) and placed in the droplet generator (Bio-Rad). After processing, the droplets generated from each sample were transferred to a 96-well PCR plate (Eppendorf, Germany). PCR amplification was carried out on a T100 thermal cycler (Bio-Rad) using a thermal profile of beginning at 95°C for 10 min, fol- lowed by 40 cycles of 94°C for 30 s and 60°C for 60 s, 1 cycle of 98°C for 10 min, and ending at 12°C. After amplification, the plate was loaded on the droplet reader (Bio-Rad) and the droplets from each well of the plate were read automatically at a rate of 32 wells per hour. ddPCR data were ana- lyzed with QuantaSoft analysis software (Bio-Rad), and the quantification of the target molecule was presented as the number of copies per l of PCR mixture.