Development of melting temperature-based SYBR Green I polymerase chain reaction methods for multiplex genetically modified organism detection

2003 ◽  
Vol 323 (2) ◽  
pp. 164-170 ◽  
Author(s):  
Marta Hernández ◽  
David Rodrı́guez-Lázaro ◽  
Teresa Esteve ◽  
Salomé Prat ◽  
Maria Pla
Keyword(s):  
Polymerase Chain Reaction ◽  
Melting Temperature ◽  
Genetically Modified ◽  
Genetically Modified Organism ◽  
Sybr Green ◽  
Sybr Green I ◽  
Chain Reaction ◽  
Genetically Modified Organism Detection ◽  
Polymerase Chain

A Duplex SYBR Green I-based Real-time Polymerase Chain Reaction Assay for Rapid Detection of Canine Kobuvirus and Canine Circovirus

2021 ◽  
Author(s):  
Yang Pan ◽  
Jing Chen ◽  
Junhuang Wu ◽  
Yongxia Wang ◽  
Junwei Zou ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Rapid Detection ◽  
Simultaneous Detection ◽  
Sybr Green ◽  
Sybr Green I ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain ◽  
Canine Kobuvirus

Abstract Background: Canine Kobuvirus (CaKoV) and Canine Circovirus (CaCV) are viruses that infect dogs causing diarrheal symptoms that are very similar. However, there is no clinical method to detect a co-infection of these two viruses.Results: In this study, a duplex SYBR Green I-based quantitative real-time polymerase chain reaction (PCR) assay for the rapid and simultaneous detection of CaKoV and CaCV was established. CaKoV and CaCV were distinguished by their different melting temperature which was 86℃ for CaKoV and 78℃ for CaCV. The assay was highly specific, with no cross-reactivity with other common canine viruses and demonstrated high sensitivity. The detection limits of CaKoV and CaCV were 8.924 × 101 copies/μL and 3.841 × 101 copies/μL, respectively. The highest intra- and inter-assay Ct value variation coefficients (CV) of CaKoV were 0.40% and 0.96%, respectively. For CaCV, the highest intra- and inter-assay Ct value variation coefficients were 0.26% and 0.70%, respectively. In 57 clinical samples, positive detection rates of CaKoV and CaCV were 8.77% (7/57) and 15.79% (9/57), respectively. The co-infection rate was 7.02% (4/57). Conclusions: The duplex SYBR Green I-based real-time PCR assay established in this study is a fast, efficient, and sensitive method for the simultaneous detection of the two viruses and provides a powerful tool for the rapid detection of CaKoV and CaCV in clinical practice.

Detection of the Escherichia coli pathogenic geneeaewith three real-time polymerase chain reaction methods

2007 ◽  
Vol 53 (3) ◽  
pp. 398-403 ◽  
Author(s):  
Joanne  Karen McCrea ◽  
Chenyi Liu ◽  
Lai-King Ng ◽  
Gehua Wang
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Detection Limit ◽  
Real Time ◽  
Turnaround Time ◽  
Sybr Green ◽  
Sybr Green I ◽  
Chain Reaction ◽  
Culture Techniques ◽  
Polymerase Chain

Several real-time polymerase chain reaction (PCR) methods are currently available to rapidly detect the presence of a specific DNA sequence. When used for detection of pathogenic organisms, the turnaround time for PCR-based methods is much lower than for traditional culture techniques. This study compared the sensitivity of three real-time PCR methods when detecting the Escherichia coli pathogenic gene eae to determine which method is most effective in identifying very low levels of the organism. The three methods were used to detect the eae gene over a range of DNA concentrations. The differences in sensitivity were statistically significant (p < 0.05), and SYBR Green I PCR was found to have the lowest detection limit of the three; LUX primers had the highest detection limit. Therefore, using a defined DNA concentration for detecting the eae gene, SYBR Green I is the best alternative.

Evaluation of gold nanoparticles as the additive in real-time polymerase chain reaction with SYBR Green I dye

2008 ◽  
Vol 19 (25) ◽  
pp. 255101 ◽  
Author(s):  
Wenchao Yang ◽  
Lijuan Mi ◽  
Xueyan Cao ◽  
Xiaodong Zhang ◽  
Chunhai Fan ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Gold Nanoparticles ◽  
Real Time ◽  
Sybr Green ◽  
Sybr Green I ◽  
Chain Reaction ◽  
Polymerase Chain
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