2020
DOI: 10.1080/14737159.2021.1865807
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Developing multiplex ddPCR assays for SARS-CoV-2 detection based on probe mix and amplitude based multiplexing

Abstract: Introduction: With the ongoing SARS-CoV-2 pandemic, different articles have been published highlighting the superiority of droplet digital PCR (ddPCR) over the gold-standard 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. This study shows steps on how to develop different ddPCR SAR-CoV-2 assays including higher order multiplex assays for SARS-CoV-2 detection and antiviral scr… Show more

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Cited by 41 publications
(46 citation statements)
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“…To ensure the stability of sample partitioning and reduce the amount of reagent used, the reagent volume selected for further experiments was 3.4 μL. This chip may cause about a 30% liquid loss, which is less than the reagent loss on a commercial dPCR platform (around 35% liquid loss) [ 27 , 28 ]. Moreover, by optimizing the structure of the chip, such as by increasing the number of reaction chambers in one branch and reducing the flow resistance ratio between the reaction chamber and buffer chamber, the liquid loss could be further reduced in the proposed chip.…”
Section: Resultsmentioning
confidence: 99%
“…To ensure the stability of sample partitioning and reduce the amount of reagent used, the reagent volume selected for further experiments was 3.4 μL. This chip may cause about a 30% liquid loss, which is less than the reagent loss on a commercial dPCR platform (around 35% liquid loss) [ 27 , 28 ]. Moreover, by optimizing the structure of the chip, such as by increasing the number of reaction chambers in one branch and reducing the flow resistance ratio between the reaction chamber and buffer chamber, the liquid loss could be further reduced in the proposed chip.…”
Section: Resultsmentioning
confidence: 99%
“…The gold standard RT-qPCR based detection method plays a key role in the global fight against COVID-19 by providing a reliable tool for early detection and timely isolation of individuals infected with SARS-CoV-2 ( Nyaruaba et al, 2020 ). Increasing understanding of the ways through which SARS-CoV-2 virus is transmitted to humans, animals, environments, and goods packaging, especially cold chain goods, will help reduce and control the spread of SARS-CoV-2.…”
Section: Discussionmentioning
confidence: 99%
“…The copy numbers of the viral genes and the endogenous RNase P were absolutely quantified in the ddPCR system. To co-amplify the viral genes and endogenous control (RNase P gene) in the same reaction, we applied duplex TaqMan probes for the ddPCR and labeled them with 6-FAM and VIC fluorescent signals respectively [16]. In brief, 20 µL of a PCR reaction mix that contains 10 µL of 2× ddPCR Supermix for Probes (No dUTP), 2 µL of a primer/probe mixture (final concentration of each primer: approximately 900 nM; hydrolysis probe: approximately 250 nM), 3 µL of 1:20 diluted cDNA, and 5 µL of RNase-DNase-free water was prepared.…”
Section: Endogenous Control and Absolute Quantification By Ddpcrmentioning
confidence: 99%