scholarly journals A Droplet Digital PCR Assay to Detect SARS-CoV-2 RNA

2020 ◽  
Author(s):  
Martin J. Romeo ◽  
Christian P. DiPaola ◽  
Cassidy Mentus ◽  
Cynthia D. Timmers
Keyword(s):  
Limit Of Detection ◽  
Digital Pcr ◽  
The United States ◽  
Cross Reactivity ◽  
Droplet Digital Pcr ◽  
Molecular Testing ◽  
Guidance Document ◽  
Respiratory Pathogens ◽  
Limit Of Quantification ◽  
United States Food

AbstractWe describe a quantitative droplet digital PCR (ddPCR) assay for detection of SARS-CoV-2 viral ribonucleic acid (RNA) in total RNA extracted from human sputum. This method was validated using the guidance of the United States Food and Drug Administration’s Accelerated Emergency Use Authorization (EUA) Template for SARS-CoV-2 that Causes Coronavirus Disease (COVID-19) Molecular Testing of Respiratory Speciment in CLIA Certified High-Complexity Laboratories. Though our laboratory is not CLIA certified, this method met all criteria specified by the guidance document with a Limit of Detection (LOD) of 0.25 copies/μL in the final ddPCR (at least 19/20 replicates reactive), which we consider to be a Lower Limit of Quantification (LLOQ); inclusivity of all known annotated SARS-CoV-2 genomes; no cross-reactivity with other respiratory pathogens; and reactivity of all contrived positives at or above the LOD.

scholarly journals Quantitative Analysis of Polycyclic Aromatic Hydrocarbons (PAHs) Cited by the United States Food and Drug Administration

2013 ◽  
Vol 25 (6) ◽  
pp. 607-616
Author(s):  
W Guthery ◽  
MJ Taylor
Keyword(s):  
Polycyclic Aromatic Hydrocarbons ◽  
Aromatic Hydrocarbons ◽  
Limit Of Detection ◽  
The United States ◽  
Limit Of Quantification ◽  
Total Particulate Matter ◽  
United States Food ◽  
Polycyclic Aromatic ◽  
Cigarette Mainstream Smoke ◽  
Health Canada

AbstractThe yields of 16 polycyclic aromatic hydrocarbons (PAHs) were determined from cigarette mainstream smoke condensate extracts using Gas Chromatography- Tandem Mass Spectrometry (GC-MS/MS). The method has been validated for ISO and Health Canada Intense (HCI) smoking protocols. Quantifiable levels (ISO means 0.16 to 365 ng/cig; HCI means 0.33 to 1595 ng/cig; n = 30) of 15 PAHs were found in the Kentucky reference cigarette K3R4F. The coefficient of variance (CV) was derived from ten determinations each run in triplicate. The CV range was 8.7% to 24.8% (ISO) and 6.6% to 24.3% (HCI). The limit of detection (LOD) based on empirical precision was ≤ 0.06 ng/cig (ISO) and ≤ 0.20 ng/cig (HCI) for all components except naphthalene (2.89 and 9.62 ng/cig, respectively). The yields from 5 unspecified branded cigarettes (Samples A-E) and 2 other reference cigarettes, K1R5F and the CORESTA monitor CM7, were determined under ISO smoking conditions. The same 15 PAHs were detected as in the K3R4F; however, cigarettes with lower yields of total particulate matter (TPM) were found to contain significantly less PAHs. One component was measured below the limit of quantification (LOQ) in Sample E and 2 components were < LOQ in the K1R5F.

scholarly journals Applying Standard Clinical Chemistry Assay Validation to Droplet Digital PCR Quantitative Liquid Biopsy Testing

2018 ◽  
Vol 64 (12) ◽  
pp. 1732-1742 ◽  
Author(s):  
Dragana Milosevic ◽  
John R Mills ◽  
Michael B Campion ◽  
Noemi Vidal-Folch ◽  
Jesse S Voss ◽  
...  
Keyword(s):  
Dynamic Range ◽  
Clinical Laboratory ◽  
Limit Of Detection ◽  
Clinical Chemistry ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Performance Criteria ◽  
Limit Of Quantification ◽  
Assay Validation ◽  
Copy Numbers

Abstract BACKGROUND Droplet digital PCR (ddPCR) is an emerging technology for quantitative cell-free DNA oncology applications. However, assay performance criteria must be established in a standardized manner to harness this potential. We reasoned that standard protocols used in clinical chemistry assay validation should be able to fill this need. METHODS We validated KRAS, EGFR, and BRAF quantitative ddPCR assays based on the Clinical Laboratory Improvement Act regulations for laboratory-developed tests in clinical chemistry and the matching Clinical and Laboratory Standards Institute guidelines. This included evaluation of limit of the blank (LOB), limit of detection (LOD), limit of quantification (LOQ), intraassay and interassay imprecision, analytical range, dilution linearity, accuracy (including comparison with orthogonal platforms), reference range study, interference, and stability studies. RESULTS For the ddPCR assays, the LOB was 4 mutant copies, LODs were 12 to 22 copies, and LOQs were 35 to 64 copies. The upper limit of the dynamic range was 30000 copies, and dilutions were linear down to the LOQs with good accuracy of spike recovery of Horizon reference material. Method comparisons with next-generation sequencing and an alternative ddPCR platform showed complete qualitative agreement and quantitative concordance, with slopes of 0.73 to 0.97 and R2s of 0.83 to 0.99. No substantial interferences were discovered. Wild-type copy numbers in plasma ranged from 462 to 6169/mL in healthy individuals. CONCLUSIONS Standard clinical chemistry assay validation protocols can be applied to quantitative ddPCR assays. This should facilitate comparison of the performance of different assays and allow establishment of minimal significant change thresholds in monitoring applications.

scholarly journals Development and Clinical Validation of a Droplet Digital PCR Method for Detection of Acinetobacter baumannii and Klebsiella pneumonia in Patients with Suspected Bloodstream Infections

2021 ◽  
Author(s):  
Yang Zheng ◽  
Jun Jin ◽  
Ziqiang Shao ◽  
Jingquan Liu ◽  
Run Zhang ◽  
...  
Keyword(s):  
Blood Culture ◽  
Acinetobacter Baumannii ◽  
Limit Of Detection ◽  
Early Stage ◽  
Bloodstream Infections ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Klebsiella Pneumonia ◽  
Clinical Validation ◽  
Blood Samples

The relatively long turnaround time and low sensitivity of traditional blood culture may delay the effective antibiotic therapy in patients with bloodstream infection (BSI). To reduce the morbidity and mortality of BSI, a rapid and sensitive pathogen detection method is urgently required. Acinetobacter baumannii and Klebsiella pneumonia are two major microorganisms responsible for BSI. Here we reported a novel droplet digital PCR (ddPCR) method that can detect A. baumannii and K. pneumonia in whole blood samples within 4 h, with a specificity of 100% for each strain and limit of detection at 0.93 copies/microliter for A. baumannii and 0.27 copies/microliter for K. pneumonia. Clinical validation in 170 patients with suspected BSIs showed that, compared with blood culture that reported 4 (2.4%) A. baumannii cases and 7 (4.1%) K. pneumonia cases, ddPCR detected 23 (13.5%) A. baumannii cases, 26 (15.3%) K. pneumonia cases, and 4 (2.4%) dual infection cases, including the 11 positive patients reported by blood culture. In addition, the positive patients reported by ddPCR alone (n = 42) had significantly lower serum concentrations of procalcitonin and lactate, SOFA and APACHE II scores, and 28-day mortality than those reported by both blood culture and ddPCR (n = 11), suggesting that patients with less severe manifestations can potentially benefit from the guidance of ddPCR results. In conclusion, our study suggests that ddPCR represents a sensitive and rapid method to identify causal pathogens in blood samples and to guide the treatment decisions in the early stage of BSI.

scholarly journals Multiple Hotspot Mutations Scanning by Single Droplet Digital PCR

2018 ◽  
Vol 64 (2) ◽  
pp. 317-328 ◽  
Author(s):  
Charles Decraene ◽  
Amanda B Silveira ◽  
François-Clément Bidard ◽  
Audrey Vallée ◽  
Marc Michel ◽  
...  
Keyword(s):  
Liquid Biopsy ◽  
Limit Of Detection ◽  
Clinical Importance ◽  
Digital Pcr ◽  
Circulating Tumor Dna ◽  
Droplet Digital Pcr ◽  
Egfr Mutations ◽  
Detection Accuracy ◽  
Exon 2 ◽  
Exon 19

Abstract BACKGROUND Progress in the liquid biopsy field, combined with the development of droplet digital PCR (ddPCR), has enabled noninvasive monitoring of mutations with high detection accuracy. However, current assays detect a restricted number of mutations per reaction. ddPCR is a recognized method for detecting alterations previously characterized in tumor tissues, but its use as a discovery tool when the mutation is unknown a priori remains limited. METHODS We established 2 ddPCR assays detecting all genomic alterations within KRAS exon 2 and EGFR exon 19 mutation hotspots, which are of clinical importance in colorectal and lung cancer, with use of a unique pair of TaqMan® oligoprobes. The KRAS assay scanned for the 7 most common mutations in codons 12/13 but also all other mutations found in that region. The EGFR assay screened for all in-frame deletions of exon 19, which are frequent EGFR-activating events. RESULTS The KRAS and EGFR assays were highly specific and both reached a limit of detection of &lt;0.1% in mutant allele frequency. We further validated their performance on multiple plasma and formalin-fixed and paraffin-embedded tumor samples harboring a panel of different KRAS or EGFR mutations. CONCLUSIONS This method presents the advantage of detecting a higher number of mutations with single-reaction ddPCRs while consuming a minimum of patient sample. This is particularly useful in the context of liquid biopsy because the amount of circulating tumor DNA is often low. This method should be useful as a discovery tool when the tumor tissue is unavailable or to monitor disease during therapy.

scholarly journals Stability-Indicating Photochemical Method for the Assay of Thiamine by Spectrophotometry

2018 ◽  
Vol 2018 ◽  
pp. 1-7 ◽  
Author(s):  
Iqbal Ahmad ◽  
Syed Haider Abbas ◽  
Zubair Anwar ◽  
Muhammad Ali Sheraz ◽  
Sofia Ahmed ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
The United States ◽  
Limit Of Quantification ◽  
Chromatography Method ◽  
Stability Indicating ◽  
Low Concentrations ◽  
Photochemical Method ◽  
Significant Difference ◽  
Liquid Chromatography Method ◽  
States Pharmacopeia

A stability-indicating photochemical method has been developed for the assay of thiamine (TH) salts in aqueous solution and in fresh and aged vitamin preparations. It is based on the photooxidation of TH by UV irradiation to form thiochrome (TC) in alkaline solution. The TC : TH ratio under controlled conditions of light intensity, temperature, pH, exposure time, and irradiation distance is constant and can be used to determine the concentration of UV irradiated TH solutions. TC, on extraction with isobutanol from the photodegraded solution of TH, has been determined by the UV spectrophotometric method at 370 nm. It exhibits a high intensity of absorption in the UV region that can be used for the assay of even low concentrations of TH. Under optimum conditions, Beer’s law is obeyed in the concentration range of 0.20–2.00 mg/100 ml (R2 = 09998). The limit of detection (LOD) and limit of quantification (LOQ) are 0.0076 and 0.0231 mg/100 ml, respectively. The method has been validated and applied to aqueous solutions and vitamin preparations. The results have statistically been compared with the United States Pharmacopeia liquid chromatography method. It has been found that there is no significant difference between the two methods at 95% confidence level.

scholarly journals Implementation and Validation of an Analytical Method for Lincomycin Determination in Feathers and Edible Tissues of Broiler Chickens by Liquid Chromatography Tandem Mass Spectrometry

2019 ◽  
Vol 2019 ◽  
pp. 1-8 ◽  
Author(s):  
Aldo Maddaleno ◽  
Ekaterina Pokrant ◽  
Francisca Yanten ◽  
Betty San Martin ◽  
Javiera Cornejo
Keyword(s):  
Broiler Chickens ◽  
Limit Of Detection ◽  
Coefficient Of Determination ◽  
Guidance Document ◽  
Limit Of Quantification ◽  
Liquid Chromatograph ◽  
Analytical Methodology ◽  
Extraction Solvent ◽  
Liquid Chromatography Tandem Mass ◽  
Edible Tissues

Recent studies have detected different antimicrobial residues in broiler chicken feathers, where they persisted for longer periods of time and at greater concentrations than in edible tissues. However, until today, lincomycin behaviour in this nonedible tissue has not been assessed yet. Considering this, an analytical methodology to detect and quantify this antibiotic concentration in feathers, muscle, and liver tissues from broiler chickens was implemented and in-house validated. The methodology will allow the determination of the bioaccumulation of this highly persistent antibiotic in feathers of treated birds. For this purpose, 98% lincomycin and 95% lincomycin D3 standards were used. Methanol was selected as the extraction solvent, and Chromabond® Florisil® cartridges were used for the clean-up stage. The separation of analytes was performed through the analytical column SunFire C18 with a running time of 4 minutes, and the instrumental analysis was performed through an LC-MS/MS, with a liquid chromatograph Agilent® 1290 Infinity, coupled to an AB SCIEX® API 5500 mass spectrometer. An internal protocol for an in-house validation was designed based on recommendations from Commission Decision 2002/657/EC and the Guidance document on the estimation of limit of detection and limit of quantification for measurements in the field of contaminants in feed and food. The average retention time for lincomycin was 2.255 min (for quantifier ion, 126.0). The calibration curves showed a coefficient of determination (r2) greater than 0.99 for all matrices, while recovery levels ranged between 98% and 101%. The limit of detection (LOD) calculated was of 19, 22, and 10 μg·kg−1, and the limit of quantification (LOQ) was of 62, 73, and 34 μg·kg−1 in feathers, muscle, and liver, respectively. This method detects lincomycin in the studied matrices, confidently and accurately, as it is required for designing analytical studies of drug residues in edible and nonedible tissues, such as feathers.

scholarly journals Development of SARS-CoV-2 packaged RNA reference material for nucleic acid testing

2021 ◽  
Author(s):  
Sang-Soo Lee ◽  
Seil Kim ◽  
Hee Min Yoo ◽  
Da-Hye Lee ◽  
Young-Kyung Bae
Keyword(s):  
Nucleic Acid ◽  
Copy Number ◽  
Target Gene ◽  
Target Genes ◽  
Digital Pcr ◽  
Number Concentration ◽  
Droplet Digital Pcr ◽  
Molecular Testing ◽  
Testing Laboratories ◽  
Viral Target

AbstractNucleic acid tests to detect the SARS-CoV-2 virus have been performed worldwide since the beginning of the COVID-19 pandemic. For the quality assessment of testing laboratories and the performance evaluation of molecular diagnosis products, reference materials (RMs) are required. In this work, we report the production of a lentiviral SARS-CoV-2 RM containing approximately 12 kilobases of its genome including common diagnostics targets such as RdRp, N, E, and S genes. The RM was measured with multiple assays using two different digital PCR platforms. To measure the homogeneity and stability of the lentiviral SARS-CoV-2 RM, reverse transcription droplet digital PCR (RT-ddPCR) was used with in-house duplex assays. The copy number concentration of each target gene in the extracted RNA solution was then converted to that of the RM solution. Their copy number values are measured to be from 1.5 × 105 to 2.0 × 105 copies/mL. The RM has a between-bottle homogeneity of 4.80–8.23% and is stable at 4 °C for 1 week and at −70 °C for 6 months. The lentiviral SARS-CoV-2 RM closely mimics real samples that undergo identical pre-analytical processes for SARS-CoV-2 molecular testing. By offering accurate reference values for the absolute copy number of viral target genes, the developed RM can be used to improve the reliability of SARS-CoV-2 molecular testing.

scholarly journals A single droplet digital PCR for ESR1 activating mutations detection in plasma

2019 ◽  
Author(s):  
Emmanuelle Jeannot ◽  
Lauren Darrigues ◽  
Marc Michel ◽  
Marc-Henri Stern ◽  
Jean-Yves Pierga ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Estrogen Receptor ◽  
Metastatic Breast Cancer ◽  
Limit Of Detection ◽  
Growth Factor Receptor ◽  
Metastatic Breast ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Plasma Samples ◽  
Activating Mutations

AbstractBackgroundActivating mutations in the estrogen receptor 1 (ESR1) gene are recurrent mechanisms of acquired resistance to aromatase inhibitors (AI), and may be the target of other selective estrogen receptor down-regulators. To assess the clinical utility of monitoring ESR1 resistant mutations, a droplet digital PCR (ddPCR)-based assay compatible with body fluids is ideal due to its cost-effectiveness and quick turnaround.MethodsWe designed a multiplex ddPCR, which combines a drop-off assay, targeting the clustered hotspot mutations found in exon 8, with another pair of probes interrogating the E380Q mutation in exon 5. We assessed its sensitivity in vitro using synthetic oligonucleotides, harboring E380Q, L536R, Y537C, Y537N, Y537S or D538G mutations. Validation of the assay was performed on plasma samples from a prospective study and compared to next generation sequencing (NGS) data.ResultsThe multiplex ESR1-ddPCR showed a high sensitivity with a limit of detection ranging from 0.07 to 0.19% in mutant allele frequency depending on the mutation tested. The screening of plasma samples from patients with AI-resistant metastatic breast cancer identified ESR1 mutations in 29% of them with perfect concordance (and higher sensitivity) to NGS data obtained in parallel. Additionally, this test identifies patients harboring polyclonal alterations. Furthermore, the monitoring of ctDNA using this technique during treatment follow-up predicts the radiological response to palbociclib-fulvestrant.ConclusionThe multiplex ESR1-ddPCR detects, in a single reaction, the most frequent ESR1 activating mutations and is compatible with plasma samples. This method is thus suitable for real-time ESR1 mutation monitoring in large cohorts of patients.Statement of translational relevanceExons 5 and 8 mutations in ESR1 are recurrent mechanisms of resistance to aromatase inhibitors (AI) in estrogen receptor (ER)-positive metastatic breast cancer and may be targeted by selective ER down-regulators. We implemented a novel droplet digital PCR, which allows for the detection of the most frequent ESR1 mutations in circulating cell-free DNA. In prospectively collected plasma samples, ESR1 mutations were found in 29% of AI-resistant patients, with excellent concordance and higher sensitivity to next generation sequencing. Moreover, circulating ESR1 mutations appear to be reliable markers for ctDNA monitoring in order to predict treatment response. Ultimately, the short turnaround time, high sensitivity and limited cost of the ESR1-ddPCR are compatible with repeated samplings to detect the onset of resistance to AI before the radiological progression. This opens a window of opportunity to develop new clinical strategies for breast cancer hormone therapy, as tested in an ongoing phase 3 trial.List of abbreviationsAIAromatase InhibitorcfDNACell-free DNActDNACirculating tumor DNAddPCRDroplet digital PCRER+ HER2-MBCER+ HER2-negative Metastatic Breast CancerEREstrogen ReceptorER+Estrogen Receptor positiveLOBLimit of blankLODLimit of detectionMAFMutant Allele FrequencyPBMCPeripheral blood mononuclear cellsPDProgressive diseaseSDStandard deviationToPTime of progressionWTWild typeHuman genesESR1: Estrogen Receptor 1HER2: Human Epidermal Growth Factor Receptor 2EGFR: Epithelial Growth Factor ReceptorKRAS: KRAS proto-oncogene, GTPaseBRAF: B-Raf Proto-Oncogene, Serine/Threonine kinase

Comparison of the NeuMoDX, Diasorin Simplexa, Cepheid and Roche CDC SARS-CoV 2 EUA assays using nasopharyngeal/nasal swabs in universal transport media (UTM) and sputum and tracheal aspirates

2020 ◽  
Author(s):  
Robert Tibbetts ◽  
Kathy Callahan ◽  
Kareem Rofoo ◽  
Richard J. Zarbo ◽  
Linoj Samuel
Keyword(s):  
The United States ◽  
World Health ◽  
Molecular Testing ◽  
Additional Specimen ◽  
Significant Difference ◽  
Nasal Swabs ◽  
Henry Ford ◽  
United States Food ◽  
Health Organization ◽  
Tracheal Aspirates

AbstractIn March 2019 the outbreak of SARS-CoV 2 was officially defined as a pandemic by the World Health Organization and shortly after, the United States Food and Drug Administration (FDA) granted Emergency Use Authorization (EUA) to the Centers for Disease Control (CDC) for reverse transcription polymerase chain reaction (rtPCR) molecular testing for the detection of the SARS-CoV-2 virus from NP swabs. Since then, EUA with relaxed regulations were granted to numerous manufacturers and clinical microbiology laboratories to implement in-house testing assays with nasopharyngeal swabs (NP) and subsequently additional specimen types. Because of supply chain shortages leading to competition for reagents, sustaining any significant volume of testing soon became problematic. As a countermeasure, within several weeks the Henry Ford Microbiology Laboratory validated 4 different rtPCR assays and multiple specimen types using NeuMoDX, Diasorin Simplexa, Cepheid and Roche platforms. The purpose of this study was to analyze the analytic sensitivity of these rtPCR assays with NP/nasal swabs and sputum/tracheal aspirates. Qualitative analytic agreement between the 4 platforms for NP/nasal swabs ranged 95% - 100% overall with no statistically significant difference in threshold cT values. Similar results were obtained with the sputum/tracheal aspirates. These data demonstrate the high accuracy and reproducibility in detection of SARS-CoV 2 between the rtPCR assays performed on 4 different platforms with numerous specimen types.

scholarly journals Application of One-Step Reverse Transcription Droplet Digital PCR for Dengue Virus Detection and Quantification in Clinical Specimens

Diagnostics ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 639
Author(s):  
Dumrong Mairiang ◽  
Adisak Songjaeng ◽  
Prachya Hansuealueang ◽  
Yuwares Malila ◽  
Paphavee Lertsethtakarn ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Lower Limit ◽  
Reverse Transcription ◽  
Limit Of Detection ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Clinical Samples ◽  
Qrt Pcr ◽  
One Step ◽  
Detection And Quantification

Detection and quantification of viruses in laboratory and clinical samples are standard assays in dengue virus (DENV) studies. The quantitative reverse transcription polymerase chain reaction (qRT-PCR) is considered to be the standard for DENV detection and quantification due to its high sensitivity. However, qRT-PCR offers only quantification relative to a standard curve and consists of several “in-house” components resulting in interlaboratory variations. We developed and optimized a protocol for applying one-step RT-droplet digital PCR (RT-ddPCR) for DENV detection and quantification. The lower limit of detection (LLOD95) and the lower limit of quantification (LLOQ) for RT-ddPCR were estimated to be 1.851 log10-copies/reaction and 2.337 log10-copies/reaction, respectively. The sensitivity of RT-ddPCR was found to be superior to qRT-PCR (94.87% vs. 90.38%, p = 0.039) while no false positives were detected. Quantification of DENV in clinical samples was independently performed in three laboratories showing interlaboratory variations with biases <0.5 log10-copies/mL. The RT-ddPCR protocol presented here could help harmonize DENV quantification results and improve findings in the field such as identifying a DENV titer threshold correlating with disease severity.

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