scholarly journals RT-qPCR Diagnostics: The “Drosten” Sars-Cov-2 Assay Paradigm

2021 ◽  
Author(s):  
Stephen Bustin ◽  
Sara Kirvell ◽  
Jim F Huggett ◽  
Tania Nolan
Keyword(s):  
Genomic Sequence ◽  
Public Awareness ◽  
Quantitative Polymerase Chain Reaction ◽  
Analytical Sensitivity ◽  
Polymerase Gene ◽  
Chain Reaction ◽  
Rna Quantification ◽  
Viral Genes ◽  
Polymerase Chain ◽  
Rna Polymerase Gene

The reverse transcription quantitative polymerase chain reaction (RT-qPCR) is an established tool for the diagnosis of RNA pathogens. Its potential for automation has caused it to be used as a presence/absence diagnostic tool even when RNA quantification is not required. This technology has been pushed to the forefront of public awareness by the COVID-19 pandemic, as its global application has enabled rapid and analytically sensitive mass testing, with the first test targeting three viral genes published within days of the publication of the SARS-CoV-2 genomic sequence. One of those, targeting the RNA-dependent RNA polymerase gene, has been heavily criticised for supposed scientific flaws at the molecular and methodological level and this criticism has been extrapolated to doubts about the validity of RT-qPCR for COVID-19 testing in general. We have analysed this assay in detail and our findings reveal some limitations, but also highlight the robustness of the RT-qPCR methodology for SARS-CoV-2 detection. Whilst our data show that some errors can be tolerated, it is always prudent to confirm that primer and probe sequences complement their intended target, since when errors do occur, they may result in a reduction in the analytical sensitivity. However, in this case it is unlikely that a mismatch will result in poor specificity or significant number of false positive SARS-CoV-2 diagnoses, especially as this is routinely checked by diagnostic laboratories as part of their quality assurance.

scholarly journals Rt-Qpcr Diagnostics: The “Drosten” Sars-Cov-2 Assay Paradigm

2021 ◽  
Author(s):  
Stephen Bustin ◽  
Sara Kirvell ◽  
Jim F Huggett ◽  
Tania Nolan
Keyword(s):  
Genomic Sequence ◽  
Public Awareness ◽  
Quantitative Polymerase Chain Reaction ◽  
Analytical Sensitivity ◽  
Polymerase Gene ◽  
Chain Reaction ◽  
Rna Quantification ◽  
Viral Genes ◽  
Polymerase Chain ◽  
Rna Polymerase Gene

The reverse transcription quantitative polymerase chain reaction (RT-qPCR) is an established tool for the diagnosis of RNA pathogens. Its potential for automation has caused it to be used as a presence/absence diagnostic tool even when RNA quantification is not required. This technology has been pushed to the forefront of public awareness by the COVID-19 pandemic, as its global application has enabled rapid and analytically sensitive mass testing, with the first test targeting three viral genes published within days of the publication of the SARS-CoV-2 genomic sequence. One of those, targeting the RNA-dependent RNA polymerase gene, has been heavily criticised for supposed scientific flaws at the molecular and methodological level and this criticism has been extrapolated to doubts about the validity of RT-qPCR for COVID-19 testing in general. We have analysed this assay in detail and our findings reveal some limitations, but also highlight the robustness of the RT-qPCR methodology for SARS-CoV-2 detection. Whilst our data show that some errors can be tolerated, it is always prudent to confirm that primer and probe sequences complement their intended target as when errors do occur, they may result in a reduction in the analytical sensitivity. However, in this case it is unlikely that a mismatch will result in poor specificity or significant number of false positive SARS-CoV-2 diagnoses, especially as this is routinely checked by diagnostic laboratories as part of their quality assurance.

scholarly journals RT-qPCR Diagnostics: The “Drosten” SARS-CoV-2 Assay Paradigm

2021 ◽  
Vol 22 (16) ◽  
pp. 8702
Author(s):  
Stephen Bustin ◽  
Sara Kirvell ◽  
Jim F. Huggett ◽  
Tania Nolan
Keyword(s):  
Genomic Sequence ◽  
Public Awareness ◽  
Quantitative Polymerase Chain Reaction ◽  
Analytical Sensitivity ◽  
Polymerase Gene ◽  
Chain Reaction ◽  
Rna Quantification ◽  
Viral Genes ◽  
Polymerase Chain ◽  
Rna Polymerase Gene

The reverse transcription quantitative polymerase chain reaction (RT-qPCR) is an established tool for the diagnosis of RNA pathogens. Its potential for automation has caused it to be used as a presence/absence diagnostic tool even when RNA quantification is not required. This technology has been pushed to the forefront of public awareness by the COVID-19 pandemic, as its global application has enabled rapid and analytically sensitive mass testing, with the first assays targeting three viral genes published within days of the publication of the SARS-CoV-2 genomic sequence. One of those, targeting the RNA-dependent RNA polymerase gene, has been heavily criticised for supposed scientific flaws at the molecular and methodological level, and this criticism has been extrapolated to doubts about the validity of RT-qPCR for COVID-19 testing in general. We have analysed this assay in detail, and our findings reveal some limitations but also highlight the robustness of the RT-qPCR methodology for SARS-CoV-2 detection. Nevertheless, whilst our data show that some errors can be tolerated, it is always prudent to confirm that the primer and probe sequences complement their intended target, since, when errors do occur, they may result in a reduction in the analytical sensitivity. However, in this case, it is unlikely that a mismatch will result in poor specificity or a significant number of false-positive SARS-CoV-2 diagnoses, especially as this is routinely checked by diagnostic laboratories as part of their quality assurance.

scholarly journals Field evaluation of a quantitative polymerase chain reaction assay for Mycoplasma hyorhinis

2014 ◽  
Vol 26 (6) ◽  
pp. 755-760 ◽  
Author(s):  
Maria J. Clavijo ◽  
Simone Oliveira ◽  
Jeffrey Zimmerman ◽  
Aaron Rendahl ◽  
Albert Rovira
Keyword(s):  
Polymerase Chain Reaction ◽  
Polymerase Chain Reaction Assay ◽  
Quantitative Polymerase Chain Reaction ◽  
Systemic Disease ◽  
Analytical Sensitivity ◽  
Upper Respiratory Tract ◽  
Chain Reaction ◽  
Mycoplasma Hyorhinis ◽  
Field Samples ◽  
Polymerase Chain

Mycoplasma hyorhinis has emerged as an important cause of systemic disease in nursery pigs. However, this bacterium can also be found in the upper respiratory tract of healthy swine. The current study describes the development of a quantitative polymerase chain reaction assay for the detection of M. hyorhinis and the evaluation of the assay in both disease diagnosis and disease surveillance using a large number of field samples. The analytical sensitivity was estimated to be 12 genome equivalents/μl. The assay was highly specific, detecting all 25 M. hyorhinis isolates tested and none of the 19 nontarget species tested. Assay repeatability was evaluated by testing different matrices spiked with known amounts of M. hyorhinis. Overall, assessment of the repeatability of the assay showed suitable precision within and between runs for all matrices. The coefficient of variation ranged from 10% to 24%. Mycoplasma hyorhinis DNA was detected in 48% of samples (pericardium, pleura, joints, nasal cavity, and lungs) from pigs with systemic disease. Mycoplasma hyorhinis was detected in nasal (92%) and oropharyngeal swabs (66%), as well as in oral fluids (100%). Potential uses of this tool involve the characterization of the prevalence of this pathogen in swine herds as well as bacterial quantification to evaluate intervention efficacy.

scholarly journals Validation of a Triplex Quantitative Polymerase Chain Reaction Assay for Detection and Quantification of Traditional Protein Sources, Pisum sativum L. and Glycine max (L.) Merr., in Protein Powder Mixtures

2021 ◽  
Vol 12 ◽  
Author(s):  
Adam C. Faller ◽  
Dhivya Shanmughanandhan ◽  
Subramanyam Ragupathy ◽  
Yanjun Zhang ◽  
Zhengfei Lu ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Glycine Max ◽  
Pisum Sativum ◽  
Quantitative Polymerase Chain Reaction ◽  
Qpcr Assay ◽  
Analytical Sensitivity ◽  
Chain Reaction ◽  
Powder Mixtures ◽  
Protein Sources ◽  
Polymerase Chain

Several botanicals have been traditionally used as protein sources, including the leguminous Pisum sativum L. and Glycine max (L.) Merr. While a rich history exists of cultivating these plants for their whole, protein-rich grain, modern use as powdered supplements present a new challenge in material authentication. The absence of clear morphological identifiers of an intact plant and the existence of long, complex supply chains behoove industry to create quick, reliable analytical tools to identify the botanical source of a protein product (many of which contain multiple sources). The utility of molecular tools for plant-based protein powder authentication is gaining traction, but few validated tools exist. Multiplex quantitative polymerase chain reaction (qPCR) can provide an economical means by which sources can be identified and relative proportions quantified. We followed established guidelines for the design, optimization, and validation of qPCR assay, and developed a triplex qPCR assay that can amplify and quantify pea and soy DNA targets, normalized by a calibrator. The assay was evaluated for analytical specificity, analytical sensitivity, efficiency, precision, dynamic range, repeatability, and reproducibility. We tested the quantitative ability of the assay using pea and soy DNA mixtures, finding exceptional quantitative linearity for both targets – 0.9983 (p < 0.0001) for soy and 0.9915 (p < 0.0001) for pea. Ratios based on mass of protein powder were also tested, resulting in non-linear patterns in data that suggested the requirement of further sample preparation optimization or algorithmic correction. Variation in fragment size within different lots of commercial protein powder samples was also analyzed, revealing low SD among lots. Ultimately, this study demonstrated the utility of qPCR in the context of protein powder mixtures and highlighted key considerations to take into account for commercial implementation.

AN IMPROVED METHOD FOR INHIBITOR FREE NUCLEIC ACIDS FROM POLYPHENOL RICH LEAVES OF MUSA SPP

2017 ◽  
Vol 23 (1) ◽  
Author(s):  
N.NANDHA KUMAR ◽  
K. SOURIANATHA SUNDARAM ◽  
D. SUDHAKAR ◽  
K.K. KUMAR
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Proteinase K ◽  
Chain Reaction ◽  
Banana Plant ◽  
High Molecular Weight Dna ◽  
Musa Spp ◽  
Leaf Tissues ◽  
Polymerase Chain

Excessive presence of polysaccharides, polyphenol and secondary metabolites in banana plant affects the quality of DNA and it leads to difficult in isolating good quality of DNA. An optimized modified CTAB protocol for the isolation of high quality and quantity of DNA obtained from banana leaf tissues has been developed. In this protocol a slight increased salt (NaCl) concentration (2.0M) was used in the extraction buffer. Polyvinylpyrrolidone (PVP) and Octanol were used for the removal of polyphenols and polymerase chain reaction (PCR) inhibitors. Proteins like various enzymes were degraded by Proteinase K and removed by centrifugation from plant extract during the isolation process resulting in pure genomic DNA, ready to use in downstream applications including PCR, quantitative polymerase chain reaction (qPCR), ligation, restriction and sequencing. This protocol yielded a high molecular weight DNA isolated from polyphenols rich leaves of Musa spp which was free from contamination and colour. The average yields of total DNA from leaf ranged from 917.4 to 1860.9 ng/ìL. This modified CTAB protocol reported here is less time consuming 4-5h, reproducible and can be used for a broad spectrum of plant species which have polyphenol and polysaccharide compounds.

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