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.

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 Developing multiplex ddPCR assays for SARS-CoV-2 detection based on probe mix and amplitude based multiplexing

2020 ◽  
Author(s):  
Raphael Nyaruaba ◽  
Changchang Li ◽  
Caroline Mwaliko ◽  
Matilu Mwau ◽  
Nelson Odiwour ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Digital Pcr ◽  
Multiplex Assay ◽  
Small Sample ◽  
Droplet Digital Pcr ◽  
Rt Pcr ◽  
Multiplex Assays ◽  
Reverse Transcription Pcr ◽  
Sample Throughput

AbstractMultiplexing has been highlighted to save on costs, increase sample throughput, and maximize on the number of targets that can be sensitively detected within a small sample. With the ongoing SARS-CoV-2 pandemic, different articles have been published highlighting the superiority of droplet digital PCR (ddPCR) over the gold reverse transcription PCR (RT-PCR) in SARS-CoV-2 detection. However, few studies have been reported on developing multiplex ddPCR assays for SARS-CoV-2 detection and their performance. In this study, we developed simplex (1 target), duplex (2 targets), triplex probe mix (3 targets), and fourplex (4 targets) assays based on a two color ddPCR system for SARS-CoV-2 detection. Results showed that the fourplex assay had the similar limits of detection and accuracy to the lower multiplex assays. Analyzing 94 clinical isolates demonstrated that the ddPCR triplex probe mix assay had better sensitivity than the RT-qPCR assay. Additionally, the ddPCR multiplex assay showed that remdesivir could inhibit the growth of SARS-CoV-2 in vitro while another testing drug couldn’t. Conclusively, our research shows that developing multiplex ddPCR assays is possible by combing probe mix and amplitude based multiplexing, which will help in developing multiplexed ddPCR assays for different SARS-CoV-2 applications.

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

Development of a sensitive and reliable reverse transcription droplet digital PCR assay for the detection of citrus yellow vein clearing virus

2018 ◽  
Vol 164 (3) ◽  
pp. 691-697 ◽  
Author(s):  
Yingjie Liu ◽  
Yingli Wang ◽  
Qin Wang ◽  
Yanhui Zhang ◽  
Wanxia Shen ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Yellow Vein ◽  
Pcr Assay ◽  
Vein Clearing

Rapid diagnosis of enterovirus infection by a new one-step reverse transcription-PCR assay.

1997 ◽  
Vol 35 (4) ◽  
pp. 976-977 ◽  
Author(s):  
H H Kessler ◽  
B Santner ◽  
H Rabenau ◽  
A Berger ◽  
A Vince ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Rapid Diagnosis ◽  
Enterovirus Infection ◽  
Pcr Assay ◽  
Reverse Transcription Pcr ◽  
One Step

Simultaneous Detection of Four Foodborne Viruses in Food Samples Using a One-Step Multiplex Reverse Transcription PCR

2018 ◽  
Vol 28 (2) ◽  
pp. 210-217 ◽  
Author(s):  
Shin-Young Lee ◽  
Mi-Ju Kim ◽  
Hyun-Joong Kim ◽  
KwangCheol Casey Jeong ◽  
Hae-Yeong Kim
Keyword(s):  
Reverse Transcription ◽  
Simultaneous Detection ◽  
Food Samples ◽  
Foodborne Viruses ◽  
Reverse Transcription Pcr ◽  
One Step

scholarly journals Development and application of one-step multiplex reverse transcription PCR for simultaneous detection of five diarrheal viruses in adult cattle

2012 ◽  
Vol 157 (6) ◽  
pp. 1063-1069 ◽  
Author(s):  
Masaharu Fukuda ◽  
Kazufumi Kuga ◽  
Ayako Miyazaki ◽  
Tohru Suzuki ◽  
Keito Tasei ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Simultaneous Detection ◽  
Adult Cattle ◽  
Reverse Transcription Pcr ◽  
One Step
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