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.

scholarly journals Analytical Sensitivity of the Abbott BinaxNOW COVID-19 Ag Card

2020 ◽  
Vol 59 (3) ◽  
Author(s):  
Garrett A. Perchetti ◽  
Meei-Li Huang ◽  
Margaret G. Mills ◽  
Keith R. Jerome ◽  
Alexander L. Greninger
Keyword(s):  
Reverse Transcription ◽  
Limit Of Detection ◽  
Digital Pcr ◽  
Detection Methods ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Qrt Pcr ◽  
Viral Transport Medium ◽  
Antigen Tests ◽  
Antigen Testing

ABSTRACT Multiple rapid antigen (Ag) tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have recently received emergency-use authorization (EUA) from the U.S. Food and Drug Administration (FDA). Although less sensitive than molecular detection methods, rapid antigen testing offers the potential for inexpensive, quick, decentralized testing. Robust analytical sensitivity data in comparison to reverse transcription-quantitative PCR (qRT-PCR) are currently lacking for many rapid antigen tests. Here, we evaluated the analytical sensitivity of the Abbott BinaxNOW COVID-19 Ag card using SARS-CoV-2-positive clinical specimens quantified by reverse transcription-droplet digital PCR (RT-ddPCR) and multiple FDA EUA qRT-PCR platforms using RNA standards. Initial and confirmatory limits of detection for the BinaxNOW COVID-19 Ag card were determined to be equivalent to 4.04 × 104 to 8.06 × 104 copies/swab. We further confirmed this limit of detection with 72 additional clinical samples positive for SARS-CoV-2 in either phosphate-buffered saline or viral transport medium. One hundred percent of samples with viral loads of >40,000 copies/swab were detected by rapid antigen testing. These data indicate that the BinaxNOW COVID-19 Ag card has an analytical sensitivity approximately equivalent to a generic qRT-PCR cycle threshold (CT) value of 29 to 30.

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

10.3791/62295 ◽  
2021 ◽  
Author(s):  
Raphael Nyaruaba ◽  
Xiaohong Li ◽  
Caroline Mwaliko ◽  
Changchang Li ◽  
Matilu Mwau ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Detection And Quantification

scholarly journals Accurate Detection of Methicillin-Resistant Staphylococcus aureus in Mixtures by Use of Single-Bacterium Duplex Droplet Digital PCR

2017 ◽  
Vol 55 (10) ◽  
pp. 2946-2955 ◽  
Author(s):  
Jun Luo ◽  
Junhua Li ◽  
Hang Yang ◽  
Junping Yu ◽  
Hongping Wei
Keyword(s):  
Staphylococcus Aureus ◽  
Limit Of Detection ◽  
Direct Method ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Clinical Samples ◽  
Methicillin Resistant ◽  
Content Type ◽  
Accurate Detection ◽  
Nasal Swabs

ABSTRACT Accurate and rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) is needed to screen MRSA carriers and improve treatment. The current widely used duplex PCR methods are not able to differentiate MRSA from coexisting methicillin-susceptible S. aureus (MSSA) or other methicillin-resistant staphylococci. 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 droplet digital PCR (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 analyzed 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. Initial 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.

Quantification of BCR-ABL1 Fusion Transcripts in Patients With Acute B Lymphoblastic Leukemia by Multiplex Droplet Digital PCR

2019 ◽  
Vol 152 (Supplement_1) ◽  
pp. S135-S135
Author(s):  
Ryan Martinez ◽  
Davis Nennig ◽  
Pawel Mroz
Keyword(s):  
Cell Line ◽  
Residual Disease ◽  
Limit Of Detection ◽  
Lymphoblastic Leukemia ◽  
Digital Pcr ◽  
Single Step ◽  
Droplet Digital Pcr ◽  
Myelogenous Leukemia ◽  
Qrt Pcr ◽  
Automated Methods

Abstract Droplet digital PCR (ddPCR) is a novel PCR platform that provides quantification of amplification targets by massive sample partitioning and analysis in a single sample. These results are independent of a calibration curve and may increase detection when compared to quantitative RT-PCR (qRT-PCR). The t(9;22)(BCR-ABL1) translocation is associated with chronic myelogenous leukemia (CML) and a subset of acute B lymphoblastic leukemias (B-ALLs). Several studies have demonstrated the clinical utility of quantifying BCR-ABL1 fusion transcript levels for detecting minimal residual disease (MRD). qRT-PCR is a well-established method for MRD monitoring but has inherent limitations with regard to lower limit of detection and quantification (LOD and LOQ). Here we report the results of a pilot project to compare and evaluate ddPCR and qRT-PCR in detecting BCR-ABL1 e1a2 transcripts for MRD monitoring in B-ALL. We developed and optimized a single-step, multiplexed ddPCR assay for detecting BCR-ABL1 e1a2 and ABL1 e10 from RNA. Droplets were prepared using a QX100 or QX200 droplet generator (Bio-Rad, Hercules, California), and PCR products were run on a QX100 droplet reader and analyzed with QuantaSoft software. Method comparison of droplet generation was assessed between manual and automated methods, finding automated methods gave an 8.2% increase in total droplets generated (15,082 vs 13,940). Next, assay performance was evaluated using the Invivoscribe BCR-ABL1 e1a2 RNA dilution set and t(9;22)-positive SUPB15 cell line. ddPCR demonstrates a limit of detection to 10–6 dilution in the Invoscribe kit and a minimum input of 10–3 to 10–4 ng of RNA from the SUPB15 cell line. Future studies will include multiplexed ddPCR in BCR-ABL1-positive B-ALL patients at time points day 0 (diagnostic sample) and days 33 and 78 postinduction for MRD determination. The performance of ddPCR will be compared with standard qPCR along with clinicopathologic correlation to determine if ddPCR can improve MRD and clinical outcomes of patients with t(9;22)+ B-ALL.

scholarly journals Development and Application of a Real-Time Reverse-Transcription PCR and Droplet Digital PCR Assays for the Direct Detection of Potato mop top virus in Soil

2020 ◽  
Vol 110 (1) ◽  
pp. 58-67 ◽  
Author(s):  
Binod Pandey ◽  
Ipsita Mallik ◽  
Neil C. Gudmestad
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Direct Detection ◽  
Digital Pcr ◽  
Soil Samples ◽  
Droplet Digital Pcr ◽  
Minimal Amount ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Potato Mop Top Virus

Potato mop top virus (PMTV) is a continuing threat to potato production throughout the world. It has the potential to persist in the soil for long periods in the sporosori of its vector Spongospora subterranea f. sp. subterranea, which is as an important source for PMTV infection and dissemination. In this study, we used real-time quantitative reverse-transcription PCR (qRT-PCR) and reverse-transcription droplet digital PCR (RT-ddPCR) assays of the total RNA extracted directly from the soil to develop a simple, fast, and sensitive method to detect PMTV in soil samples using a specific primer with high efficiency despite a minimal amount of viral RNA. The designed primers are resilient in the presence of various PCR inhibitors in the soil when RNA is extracted. Both assays detected PMTV in all soil types used and supported the detection of <10 PMTV copies µl−1 in the RNA sample. With qRT-PCR, detection was linear, with amplification efficiencies ranging from 93.3 to 105.3% for silt loam, loamy sand, sand, and sandy loam in various experiments with R2 > 0.99. Furthermore, the RT-ddPCR assay also demonstrated a high degree of linearity (R2 > 0.99 and P < 0.0001) with the RNA extracted from the soil samples representing different textures and physiochemical characteristics that were artificially spiked with infested S. subterranea f. sp. subterranea sporosori. Additionally, both assays successfully detected PMTV in different types of naturally infested soil with PMTV carrying S. subterranea f. sp. subterranea sporosori levels ranging from 6.2 × 102 g−1 to 1.2 × 106 g−1 in soils with pH ranging from 4.9 to 7.5 and organic matter ranging from 0.9 to 5.1%, demonstrating the potential to detect PMTV in a wide variety of soils. To our knowledge, this is the first report of the development of real-time PCR and ddPCR methods for the direct detection of a soilborne virus in soil.

scholarly journals Investigation of SARS-CoV-2 on Ocular Surface of Coronavirus Disease 2019 Patients Using One-Step Reverse-Transcription Droplet Digital PCR

2021 ◽  
Vol Volume 14 ◽  
pp. 5395-5401
Author(s):  
Xian Zhang ◽  
Liting Chen ◽  
Gaoxiang Wang ◽  
Liwen Chen ◽  
Lifang Huang ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Ocular Surface ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
One Step

scholarly journals Validation of a One-Step Reverse Transcription-Droplet Digital PCR (RT-ddPCR) Approach to Detect and Quantify SARS-CoV-2 RNA in Nasopharyngeal Swabs

2021 ◽  
Vol 2021 ◽  
pp. 1-6
Author(s):  
Catia Mio ◽  
Adriana Cifù ◽  
Stefania Marzinotto ◽  
Barbara Marcon ◽  
Corrado Pipan ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
University Hospital ◽  
Reverse Transcription Pcr ◽  
Containment Measures ◽  
One Step ◽  
Molecular Biology Method ◽  
Sensitivity Specificity ◽  
The University

Background. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has rapidly spread worldwide from the beginning of 2020. Quantitative reverse transcription-PCR (RT-qPCR) is, to this day, the preferred methodology for viral RNA detection, even if not without problems. To overcome some of the limitations still existing for the detection and quantification of nucleic acids in various applications, the use of one-step reverse transcription-droplet digital PCR (RT-ddPCR) has been established. The purpose of this study was, then, to evaluate the efficacy of ddPCR for the detection of SARS-CoV-2 RNA in nasopharyngeal swabs, optimizing the detection of low-viral load-burdened samples. Methods. The RT-ddPCR workflow was validated for sensitivity, specificity, linearity, reproducibility, and precision using samples from 90 COVID-19-infected patients referred to the Department of Laboratory Medicine of the University Hospital of Udine (Italy). Results. The present study shows that RT-ddPCR allows the detection of as low as 10.3 copies of a SARS-COV-2 E-gene per sample with a higher level of accuracy and precision, especially at low concentration. Conclusion. During the postpeak phase of the SARS-CoV-2 pandemic, it is essential to rely on a highly robust molecular biology method to identify infected subjects, whether they have symptoms or not, in order to prepare appropriate containment measures.

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