Spatially isolated reactions in a complex array: using magnetic beads to purify and quantify nucleic acids with digital and quantitative real-time PCR in thousands of parallel microwells

Lab on a Chip ◽  
2020 ◽  
Vol 20 (10) ◽  
pp. 1771-1779 ◽  
Author(s):  
W. Hampton Henley ◽  
Nathan A. Siegfried ◽  
J. Michael Ramsey
Keyword(s):  
Nucleic Acid ◽  
Sample Preparation ◽  
Nucleic Acids ◽  
Real Time ◽  
Real Time Pcr ◽  
Magnetic Beads ◽  
Digital Pcr ◽  
Quantitative Real Time Pcr

Encoded beads carrying primer pairs for nucleic acid targets are used for sample preparation and multiplexed-in-space digital PCR quantification.

Reverse transcriptase droplet digital PCR vs reverse transcriptase quantitative real‐time PCR for serum HBV RNA quantification

2020 ◽  
Vol 92 (12) ◽  
pp. 3365-3372 ◽  
Author(s):  
Umaporn Limothai ◽  
Natthaya Chuaypen ◽  
Kittiyod Poovorawan ◽  
Watcharasak Chotiyaputta ◽  
Tawesak Tanwandee ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Real Time ◽  
Real Time Pcr ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Quantitative Real Time Pcr ◽  
Rna Quantification

scholarly journals Earlier relapse detection after allogeneic haematopoietic stem cell transplantation by chimerism assays: Digital PCR versus quantitative real-time PCR of insertion/deletion polymorphisms

PLoS ONE ◽  
2019 ◽  
Vol 14 (2) ◽  
pp. e0212708 ◽  
Author(s):  
Jennifer Valero-Garcia ◽  
María del Carmen González-Espinosa ◽  
Manuel Barrios ◽  
Greta Carmona-Antoñanzas ◽  
Javier García-Planells ◽  
...  
Keyword(s):  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Real Time ◽  
Cell Transplantation ◽  
Real Time Pcr ◽  
Haematopoietic Stem Cell Transplantation ◽  
Digital Pcr ◽  
Haematopoietic Stem Cell ◽  
Quantitative Real Time Pcr

Locked nucleic acids for optimizing displacement probes for quantitative real-time PCR

2006 ◽  
Vol 348 (2) ◽  
pp. 294-299 ◽  
Author(s):  
Brett Kennedy ◽  
Khalil Arar ◽  
Valin Reja ◽  
Robert J. Henry
Keyword(s):  
Nucleic Acids ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Real Time Pcr ◽  
Locked Nucleic Acids

scholarly journals Library of Prefabricated Locked Nucleic Acid Hydrolysis Probes Facilitates Rapid Development of Reverse-Transcription Quantitative Real-Time PCR Assays for Detection of Novel Influenza A/H1N1/09 Virus

2009 ◽  
Vol 55 (12) ◽  
pp. 2218-2222 ◽  
Author(s):  
Jürgen J Wenzel ◽  
Heiko Walch ◽  
Markus Bollwein ◽  
Hans Helmut Niller ◽  
Waltraud Ankenbauer ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Reverse Transcription ◽  
Real Time Pcr ◽  
Influenza A ◽  
Rapid Development ◽  
Locked Nucleic Acid ◽  
Quantitative Real Time Pcr ◽  
Influenza A H1n1 ◽  
Novel Influenza A

Abstract Background: The emergence of a novel pandemic human strain of influenza A (H1N1/09) has clearly demonstrated the need for flexible tools enabling the rapid development of new diagnostic methods. Methods: We designed a set of reverse-transcription quantitative real-time PCR (RT-qPCR) assays based on the Universal ProbeLibrary (UPL)—a collection of 165 presynthesized, fluorescence-labeled locked nucleic acid (LNA) hydrolysis probes—specifically to detect the novel influenza A virus. We evaluated candidate primer/UPL-probe pairs with 28 novel influenza A/H1N1/09 patient samples of European and Mexican origin. Results: Of 14 assays in the hemagglutinin (HA) and neuraminidase (NA) genes, 12 detected viral nucleic acids from diluted patient samples without need for further optimization. We characterized the diagnostic specificity of the 2 best-performing assays with a set of samples comprising various influenza virus strains of human and animal origin that showed no cross-reactivity. The diagnostic sensitivity of these 2 primer/probe combinations was in the range of 100–1000 genomic copies/mL. In comparison to a reference assay recommended by the German health authorities, the analytical sensitivities and specificities of the assays were equivalent. Conclusions: Facing the emergence of novel influenza A/H1N1/09, we were able to develop, within 2 days, a set of sensitive and specific RT-qPCR assays for the laboratory diagnosis of suspected cases. H1N1/09 served as a model to show the feasibility of the UPL approach for the expedited development of new diagnostic assays to detect emerging pathogens.

scholarly journals Quantification of BK Virus Standards by Quantitative Real-Time PCR and Droplet Digital PCR Is Confounded by Multiple Virus Populations in the WHO BKV International Standard

2017 ◽  
Vol 63 (3) ◽  
pp. 761-769 ◽  
Author(s):  
Allen C Bateman ◽  
Alexander L Greninger ◽  
Ederlyn E Atienza ◽  
Ajit P Limaye ◽  
Keith R Jerome ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Real Time ◽  
Real Time Pcr ◽  
Digital Pcr ◽  
Bk Virus ◽  
Droplet Digital Pcr ◽  
Quantitative Real Time Pcr ◽  
International Standard ◽  
Multiple Virus ◽  
Generation Sequencing

Abstract BACKGROUND The WHO recently released a BK virus (BKV) international standard. This study evaluated the WHO international standard and commercially available BKV standards by quantitative real-time PCR (qPCR) and droplet digital PCR (ddPCR). METHODS WHO, Exact Diagnostics, Acrometrix, and Zeptometrix BKV standards were tested by qPCR and ddPCR. Two preparations of NIST BKV clones were also tested. Nucleic acid was extracted with the Roche MP96 and MPLC, followed by quantification in duplicate. To resolve discrepancies, we sequenced the WHO and NIST materials. RESULTS Manufacturers' expected copies/mL were close to WHO IU/mL: linear regression of qPCR data revealed 1.12 Exact copies/IU, 0.76 Acrometrix copies/IU, and 0.70 Zeptometrix copies/IU. For ddPCR, similar concentrations were measured when either the VP1 region or the T region was targeted, and concentrations were almost 2-fold higher when both regions were targeted simultaneously. ddPCR results for the VP1 and T regions were similar for all commercial standards, but targeting the T region of the WHO standard led to a 4-fold lower result than the VP1 region. Next-generation sequencing revealed no primer or probe mismatches. However, large differences in coverage across the WHO standard and junctional reads were observed, indicating subpopulations of the WHO standard with deletions in the T region. CONCLUSIONS BKV standards showed concordance among providers, but the WHO standard contains subpopulations of viruses with various deletions in the T region. PCR results will vary depending on which region of the WHO standard is targeted.

Quantitative Real-Time PCR and Digital PCR to Evaluate Residual Quantity of HAV in Experimentally Depurated Mussels

2021 ◽  
Author(s):  
Maria Grazia Amoroso ◽  
Denise Di Concilio ◽  
Antonio Luca Langellotti ◽  
Anna Martello ◽  
Barbara Cioffi ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Digital Pcr ◽  
Quantitative Real Time Pcr

Development of droplet digital PCR assays to quantify genes involved in nitrification and denitrification, comparison with quantitative real-time PCR and validation of assays in vineyard soil

2020 ◽  
pp. 1-14
Author(s):  
Tanja M. Voegel ◽  
Melissa M. Larrabee ◽  
Louise M. Nelson
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Environmental Samples ◽  
Soil Bacteria ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Quantitative Real Time Pcr ◽  
Soil Dna ◽  
Soil C ◽  
Total Bacteria

Quantifying genes in soil is important to relate the abundance of soil bacteria to biogeochemical cycles. Quantitative real-time PCR is widely used for quantification, but its use with environmental samples is limited by poor reaction efficiencies or by PCR inhibition through co-purified soil substances. Droplet digital PCR (ddPCR) is a technology for absolute, sensitive quantification of genes. This study optimized eight ddPCR assays to quantify total bacteria and archaea as well as the nitrification (bacterial and archaeal amoA) and denitrification (nirS, nirK, nosZI, nosZII) genes involved in the generation or reduction of the greenhouse gas nitrous oxide. Detection and quantification thresholds were compared with those of quantitative real-time PCR and were equal to, or improved, in ddPCR. To validate the assays using environmental samples, soil DNA was isolated from two vineyards in the Okanagan valley in British Columbia, Canada, over the 2017 growing season. Soil properties related to the observed gene abundances were determined. Total bacteria, nirK, and nosZII increased with time and the soil C/N ratio and NH4+-N concentration affected total archaea and archaeal amoA negatively. The results, compared with those of other studies, showed that ddPCR is a valid alternative to qPCR to quantify genes involved in nitrification or denitrification.

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