A Convenient and Sensitive Method for Deoxynucleoside Triphosphate Quantification by the Combination of Rolling Circle Amplification and Quantitative Polymerase Chain Reaction

2021 ◽  
Author(s):  
Hsin-Yen Wang ◽  
Peng Hsin ◽  
Chang-Yu Huang ◽  
Zee-Fen Chang
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Rolling Circle Amplification ◽  
Chain Reaction ◽  
Rolling Circle ◽  
Deoxynucleoside Triphosphate ◽  
Polymerase Chain ◽  
Sensitive Method

Amplification of Padlock Probes for DNA Diagnostics by Cascade Rolling Circle Amplification or the Polymerase Chain Reaction

1999 ◽  
Vol 123 (12) ◽  
pp. 1170-1176 ◽  
Author(s):  
David C. Thomas ◽  
Glenn A. Nardone ◽  
Sandra K. Randall
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Polymerase ◽  
Rolling Circle Amplification ◽  
Strand Displacement ◽  
Chain Reaction ◽  
Dna Diagnostics ◽  
Gene Target ◽  
Rolling Circle ◽  
Padlock Probes ◽  
Polymerase Chain

Abstract Context.—Padlock probes are highly specific reagents for DNA diagnostics that can discriminate gene sequences with single base mutations. When the 3′ and 5′ terminal regions of the oligonucleotide probes are juxtaposed on a target DNA sequence, they can be circularized by enzymatic ligation and become topologically locked to the target. However, to be useful in solution-based diagnostics, the sensitivity of padlock probes must be markedly enhanced. Objective.—To describe two methods for geometric amplification of circularized padlock probes. Design.—Cascade rolling circle amplification is an isothermal system that uses generic primers and a DNA polymerase with strong strand displacement activity to amplify circularized probes by a mechanism combining rolling circle replication and strand displacement synthesis. One of the primers was designed as an energy transfer–labeled primer, which generates a fluorescence signal only when incorporated into the amplified product, enabling a direct means of detection. Results.—Using pUC19 as a model target to circularize an 89-base probe, a 10 billion–fold amplification was achieved with Bst DNA polymerase (large fragment) within 1 hour starting with as few as 10 probe molecules. The polymerase chain reaction was also used to amplify ligated padlock probes in a rare target detection system. In mixing experiments containing both normal and mutant p53 or c-Ki-ras2 gene target sequences, mutant targets were easily detected in the presence of a 500-fold excess of normal target copies. Conclusion.—These results indicate that padlock probes can be amplified to the high levels required for solution-based DNA diagnostics.

scholarly journals Comparison of RT-qPCR results of different gene targets for SARS-CoV-2 in asymptomatic individuals during COVID-19 pandemic

2021 ◽  
Vol 5 (s1) ◽  
pp. 26-31
Author(s):  
Gulten Tuncel ◽  
Mahmut Cerkez Ergoren ◽  
Buket Baddal ◽  
Pinar Tulay ◽  
Ayse Arikan ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcriptase ◽  
Quantitative Polymerase Chain Reaction ◽  
Viral Transmission ◽  
Chain Reaction ◽  
Northern Cyprus ◽  
Polymerase Chain ◽  
Asymptomatic Individuals ◽  
Higher Sensitivity ◽  
Sensitive Method

Abstract A reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is regarded as the most sensitive method available and is being used for screening procedure for all incoming passengers to Northern Cyprus for SARS-CoV-2. This study investigated the compatibility of two different RT-qPCR methodologies Diagnovital® and Bio-Speedy® by re-analyzing the previously confirmed positive samples. A total of 43 previously confirmed positive samples were re-analyzed by two different commercially available SARS-CoV-2 RT-qPCR kits. Only 23.5% of positive samples detected by Diagnovital® RT-qPCR kit were detected by Bio-Speedy® detection kit. In conclusion, adoption of Diagnovital® RT-qPCR kit detecting two regions of SARS-CoV-2 genome in our laboratories enabled the detection of SARS-CoV-2 in asymptomatic cases with higher sensitivity and contributed to the prevention of viral transmission within the country. The timely detection of infection in asymptomatic individuals may be the key to a successful fight against the COVID- 19 pandemic.

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.

scholarly journals Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
One Step ◽  
Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

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