Sensitive and accurate quantification of human malaria parasites using droplet digital PCR (ddPCR).
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ABSTRACT:
SUBMITTER: Koepfli C
PROVIDER: S-EPMC5159915 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundUreaplasma spp. are associated with various infectious diseases in females, but there is still limited evidence regarding whether they are related to nonspecific cervicitis. The aim of this study was to develop and evaluate a digital droplet PCR (ddPCR) assay for the detection and quantification of Ureaplasma spp. in cervical swabs.MethodsA total of 267 non-specific cervicitis (NSC) patients and 195 asymptomatic females were included in this study. We produced standard curves for Ureaplasma spp. to evaluate the analytical performance of the ddPCR assay. Then, we detected and quantified the bacterial load of Ureaplasma spp. in cervical swabs.ResultsThe prevalences of U. parvum were 37.8% (101/267) and 29.7% (58/195), U. urealyticum were 9.0% (24/267) and 8.7% (17/195) in the NSC group and control group, respectively. In addition, the median copy number of U. parvum was 2.5 × 104 copies/ml (n = 101) in the NSC group and 9.2 × 103 copies/ml (n = 58) in the control group. The U. parvum load in the NSC group was significantly higher than that in the asymptomatic individuals (P < 0.001). whereas the median load of U. urealyticum was 8.4 × 103 copies/ml (n = 24) and 1.4 × 103 (n = 17) copies/ml in the two groups, respectively, , the difference was not statistically significant (P = 0.450).ConclusionsOur study is the first to develop a droplet digital PCR (ddPCR) method for the detection and quantification of Ureaplasma spp. in clinical samples, and the method has excellent analytical performance and a wide range of clinical application prospects.
| S-EPMC8359095 | biostudies-literature
Project description:BackgroundSome mutations in the receptor binding domain of the SARS-CoV-2 Spike protein are associated with increased transmission or substantial reductions in vaccine efficacy, including in recently described Omicron subvariants. The changing frequencies of these mutations combined with their differing susceptibility to available therapies have posed significant problems for clinicians and public health professionals.ObjectiveTo develop an assay capable of rapidly and accurately identifying variants including Omicron in clinical specimens to enable case tracking and/or selection of appropriate clinical treatment.Study designUsing three duplex RT-ddPCR reactions targeting four amino acids, we tested 419 positive clinical specimens from February to December 2021 during a period of rapidly shifting variant prevalences and compared genotyping results to genome sequences for each sample, determining the sensitivity and specificity of the assay for each variant.ResultsMutation determinations for 99.7% of detected samples agree with NGS data for those samples, and are accurate despite wide variation in RNA concentration and potential confounding factors like transport medium, presence of additional respiratory viruses, and additional mutations in primer and probe sequences. The assay accurately identified the first 15 Omicron variants in our laboratory including the first Omicron in Washington State and discriminated against S-gene dropout Delta specimen.ConclusionWe describe an accurate, precise, and specific RT-ddPCR assay for variant detection that remains robust despite being designed prior the emergence of Delta and Omicron variants. The assay can quickly identify mutations in current and past SARS-CoV-2 variants, and can be adapted to future mutations.
| S-EPMC9212762 | biostudies-literature
Project description:Detection of bovine and porcine in gelatin-based products is important as species fraud and product mislabeling may have a detrimental impact on customers who have health, ethical, and religious concerns about animal products. The duplex droplet digital PCR (ddPCR) assay using double-quenched probes has been developed for quantification and detection of porcine and bovine DNA in gelatin capsules. A DNA mixture derived from gelatin was found to have a limit of detection as low as 0.001 ng/µl for porcine samples and 0.01 ng/µl for bovine samples. DNA from 12 other distinct species was tested with the bovine and porcine probes, showing high specificity for this method. The test was validated using fifty-five commercial supplement and pharmaceutical capsules, of which 17 were positive for bovine and/or porcine DNA. This study shows that the duplex ddPCR is reliable for routine analysis in the identification of bovine and porcine origins for gelatin capsules.Supplementary informationThe online version contains supplementary material available at 10.1007/s10068-022-01204-x.
| S-EPMC10082860 | biostudies-literature
Project description:Droplet digital polymerase chain reaction (ddPCR) is a partial PCR based on water-oil emulsion droplet technology. It is a highly sensitive method for detecting and delineating minor alleles from complex backgrounds and provides absolute quantification of DNA targets. The ddPCR technology has been applied for detection of many pathogens. Here the sensitive assay utilizing ddPCR for detection and quantification of Plasmodium species was investigated. The assay was developed for two levels of detection, genus specific for all Plasmodium species and for specific Plasmodium species detection. The ddPCR assay was developed based on primers and probes specific to the Plasmodium genus 18S rRNA gene. Using ddPCR for ultra-sensitive P. falciparum assessment, the lower level of detection from concentrated DNA obtained from a high volume (1 mL) blood sample was 11 parasites/mL. For species identification, in particular for samples with mixed infections, a duplex reaction was developed for detection and quantification P. falciparum/ P. vivax and P. malariae/ P. ovale. Amplification of each Plasmodium species in the duplex reaction showed equal sensitivity to singleplex single species detection. The duplex ddPCR assay had higher sensitivity to identify minor species in 32 subpatent parasitaemia samples from Cambodia, and performed better than real-time PCR. The ddPCR assay shows high sensitivity to assess very low parasitaemia of all human Plasmodium species. This provides a useful research tool for studying the role of the asymptomatic parasite reservoir for transmission in regions aiming for malaria elimination.
| S-EPMC5396971 | biostudies-literature
Project description:Quantitative viral load assays have transformed our understanding of viral diseases. They hold similar potential to advance COVID-19 control and prevention, but SARS-CoV-2 viral load tests are not yet widely available. SARS-CoV-2 molecular diagnostic tests, which typically employ real-time RT-PCR, yield semiquantitative results only. Droplet digital RT-PCR (RT-ddPCR) offers an attractive platform for SARS-CoV-2 RNA quantification. Eight primer/probe sets originally developed for real-time RT-PCR-based SARS-CoV-2 diagnostic tests were evaluated for use in RT-ddPCR; three were identified as the most efficient, precise, and sensitive for RT-ddPCR-based SARS-CoV-2 RNA quantification. For example, the analytical efficiency for the E-Sarbeco primer/probe set was approximately 83%, whereas assay precision, measured as the coefficient of variation, was approximately 2% at 1000 input copies/reaction. Lower limits of quantification and detection for this primer/probe set were 18.6 and 4.4 input SARS-CoV-2 RNA copies/reaction, respectively. SARS-CoV-2 RNA viral loads in a convenience panel of 48 COVID-19-positive diagnostic specimens spanned a 6.2log10 range, confirming substantial viral load variation in vivo. RT-ddPCR-derived SARS-CoV-2 E gene copy numbers were further calibrated against cycle threshold values from a commercial real-time RT-PCR diagnostic platform. This log-linear relationship can be used to mathematically derive SARS-CoV-2 RNA copy numbers from cycle threshold values, allowing the wealth of available diagnostic test data to be harnessed to address foundational questions in SARS-CoV-2 biology.
| S-EPMC8164350 | biostudies-literature
Project description:Droplet digital PCR provides superior accuracy in nucleic acid quantitation. The requirement of microfluidics to generate and analyze the emulsions, however, is a barrier to its adoption, particularly in low resource or clinical settings. Here, we report a novel method to prepare ddPCR droplets by vortexing and readout the results by bulk analysis of recovered amplicons. We demonstrate the approach by accurately quantitating SARS-CoV-2 sequences using entirely bulk processing and no microfluidics. Our approach for quantitating reactions should extend to all digital assays that generate amplicons, including digital PCR and LAMP conducted in droplets, microchambers, or nanoliter wells. More broadly, our approach combines important attributes of ddPCR, including enhanced accuracy and robustness to inhibition, with the high-volume sample processing ability of quantitative PCR.
| S-EPMC7814815 | biostudies-literature
Project description:BackgroundSimian immunodeficiency virus (SIV)-infected rhesus macaques constitute an excellent model of human HIV infection. Sensitive detection of SIV RNA in cell and tissue samples from infected animals subjected to treatment regimens becomes especially critical in determining which therapeutic attempts are successful, and consequently, which interventions should be prioritized in HIV cure research.ResultsIn this report, we describe the design and testing of a Raindance ddPCR platform-based, sensitive SIV reverse transcription droplet digital PCR (RT-ddPCR) assay by exploring the combinations of various priming conditions and reverse transcriptases, and testing one-step vs. two-step procedures, to eliminate background signal(s) and enable detection and quantification of low level target signals.ConclusionsSimilar reaction conditions and assay validation procedures can be explored for potential development of additional assays for other applications that require sensitive detection of low-level targets in RNA samples.
| S-EPMC7883996 | biostudies-literature
Project description:Atypical porcine pestivirus (APPV) is a recently discovered RNA virus, which mainly caused congenital tremor in piglets. Droplet digital PCR (ddPCR) is an absolute quantitative method that does not rely on the standard curve but has high sensitivity and accuracy. The present study aimed to develop a ddPCR detection assay for APPV. Furthermore, we evaluated the limit of detection, sensitivity, specificity and reproducibility of the ddPCR and real-time quantitative PCR (qPCR) and tested 135 clinical samples to calculate the detection rate of the two methods. The results showed that both methods had a strong linear relationship and quantitative correlation. The ddPCR assay had a minimum detection limit of 0.15 copies/μL for APPV, with a sensitivity 100 times that of qPCR. We tested clinical samples and found that the APPV ddPCR had a 27.4% positive detection rate, noticeably higher than that of the qPCR (14.8%). Additionally, the APPV ddPCR method had excellent repeatability and specificity. In brief, our study provided a novel, feasible and sensitive diagnostic technique to identify and monitor APPV.
| S-EPMC9151951 | biostudies-literature
Project description:INTRODUCTION:For the relative quantification of isoform expression, RT-qPCR has been the gold standard for over a decade. More recently, digital PCR is becoming widely implemented, as it is promised to be more accurate, sensitive and less affected by inhibitors, without the need for standard curves. In this study we evaluated RT-qPCR versus RT-droplet digital PCR (ddPCR) for the relative quantification of isoforms in controls and carriers of the splice site mutation BRCA1 c.212+3A>G, associated with increased expression of several isoforms. MATERIALS AND METHODS:RNA was extracted from EBV cell lines of controls and heterozygous BRCA1 c.212+3A>G carriers. Transcript-specific plasmids were available to determine the efficiency, precision, reproducibility and accuracy of each method. RESULTS:Both ddPCR and RT-qPCR were able to accurately quantify all targets and showed the same LOB, LOD and LOQ; also precision and reproducibility were similar. Both techniques have the same dynamic range and linearity at biologically relevant template concentrations. However, a significantly higher cost and workload was required for ddPCR experiments. CONCLUSIONS:Our study recognizes the potential and validity of digital PCR but shows the value of a highly optimized qPCR for the relative quantification of isoforms. Cost efficiency and simplicity turned out to be better for RT-qPCR.
| S-EPMC5634819 | biostudies-literature
Project description:Accurate and precise nucleic-acid quantification is crucial for clinical and diagnostic decisions, as overestimation or underestimation can lead to misguided treatment of a disease or incorrect labelling of the products. Digital PCR is one of the best tools for absolute nucleic-acid copy-number determination. However, digital PCR needs to be well characterised in terms of accuracy and sources of uncertainty. With droplet digital PCR, discrepancies between the droplet volume assigned by the manufacturer and measured by independent laboratories have already been shown in previous studies. In the present study, we report on the results of an inter-laboratory comparison of different methods for droplet volume determination that is based on optical microscopy imaging and is traceable to the International System of Units. This comparison was conducted on the same DNA material, with the examination of the influence of parameters such as droplet generators, supermixes, operators, inter-cartridge and intra-cartridge variability, and droplet measuring protocol. The mean droplet volume was measured using a QX200™ AutoDG™ Droplet Digital™ PCR system and two QX100™ Droplet Digital™ PCR systems. The data show significant volume differences between these two systems, as well as significant differences in volume when different supermixes are used. We also show that both of these droplet generator systems produce droplets with significantly lower droplet volumes (13.1%, 15.9%, respectively) than stated by the manufacturer and previously measured by other laboratories. This indicates that to ensure precise quantification, the droplet volumes should be assessed for each system.
| S-EPMC5670190 | biostudies-literature