taqman qpcr assay
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2021 ◽  
Author(s):  
Silvia Turco ◽  
Giorgia Bastianelli ◽  
Carmen Morales‐Rodrìguez ◽  
Andrea Vannini ◽  
Angelo Mazzaglia

Genes ◽  
2021 ◽  
Vol 12 (5) ◽  
pp. 676
Author(s):  
Takehito Sugasawa ◽  
Shin-ichiro Fujita ◽  
Tomoaki Kuji ◽  
Noriyo Ishibashi ◽  
Kenshirou Tamai ◽  
...  

Plasma cell-free DNA (cfDNA) is frequently analyzed using liquid biopsy to investigate cancer markers. We hypothesized that this concept might be applicable in exercise physiology. Here, we aimed to identify specific cfDNA (spcfDNA) sequences in the plasma of healthy humans using next-generation sequencing (NGS) and clearly define the dynamics regarding spcfDNA-fragment levels upon extreme exercises, such as running a full marathon. NGS analysis was performed using cfDNA of pooled plasma collected from healthy participants. We confirmed that the TaqMan-qPCR assay had high sensitivity and found that the spcfDNA sequence abundance was 16,600-fold higher than that in a normal genomic region. We then used the TaqMan-qPCR assay to investigate the dynamics of spcfDNA-fragment levels upon running a full marathon. The spcfDNA fragment levels were significantly increased post-marathon. Furthermore, spcfDNA fragment levels were strongly correlated with white blood cell and plasma myoglobin concentrations. These results suggest the spcfDNA fragments identified in this study were highly sensitive as markers of extreme physical stress. The findings of this study may provide new insights into exercise physiology and genome biology in humans.


Author(s):  
Takehito Sugasawa ◽  
Shin-ichiro Fujita ◽  
Tomoaki Kuji ◽  
Noriyo Ishibashi ◽  
Kenshirou Tamai ◽  
...  

Plasma cell-free DNA (cfDNA) is frequently analyzed using liquid biopsy to investigate cancer markers. Accordingly, we hypothesized this concept could be applied to the field of exercise physiology. Here, we aimed to identify specific cfDNA (spcfDNA) sequences in the plasma of non-treated human participants using next generation sequencing (NGS) and to clearly define the dynamics regarding the amounts of spcfDNA-fragments upon extreme exercise, such as running a full marathon. NGS analysis was performed using cfDNA of pooled plasma collected from non-treated participants. We confirmed the TaqMan-qPCR assay had a high sensitivity and found the spcfDNA sequence abundance was 16,600-fold higher than a normal genomic region. We then used the TaqMan-qPCR assay to investigate the dynamics of the levels of spcfDNA-fragments upon running a full marathon. Quantities of the spcfDNA fragments were significantly increased post marathon. Furthermore, the amounts of spcfDNA fragments strongly correlated with the numbers of white blood cells and plasma myoglobin concentrations. These results suggest the spcfDNA fragments identified in this study were highly sensitive response markers to extreme physical stress. The findings of this study may provide new insights into exercise physiology and genome biology on the human.


Plant Disease ◽  
2021 ◽  
Author(s):  
Sharifa G. Crandall ◽  
Marina Ramon ◽  
Alyssa Burkhardt ◽  
Julian Camilo Bello Rodriguez ◽  
Nanci Adair ◽  
...  

The ability to detect and quantify aerially dispersed plant pathogens is essential for developing effective disease control measures and epidemiological models that optimize the timing for control . There is an acute need for managing the downy mildew pathogens infecting cucurbits and hop incited by members of the genus Pseudoperonospora (P. cubensis clade 1 and 2 isolates and P. humuli, respectively). A highly specific multiplex TaqMan qPCR assay targeting unique sequences in the pathogens’ mitochondrial genomes was developed that enables detection of all three taxa in a single multiplexed amplification. An internal control included in the reaction evaluated if results were influenced by PCR inhibitors that can make it through the DNA extraction process. Reliable quantification of inoculum as low as three sporangia in a sample was observed. The multiplexed assay was tested with DNA extracted from purified sporangia, infected plant tissue and environmental samples collected on impaction spore traps samplers. The ability to accurately detect and simultaneously quantify all three pathogens in a single multiplexed amplification should improve management options for controlling the diseases they cause.


2020 ◽  
Author(s):  
Pria N Ghosh ◽  
Ruhan Verster ◽  
Thomas Sewell ◽  
Simon O’Hanlon ◽  
Lola M Brookes ◽  
...  

AbstractThe ability to detect and monitor infectious disease in a phylogenetically informative manner is critical for their management. Phylogenetically informative diagnostic tests enable patterns of pathogen introduction or changes in the distribution of genotypes to be measured, enabling research into the ecology of the pathogen. Batrachochytrium dendrobatidis (Bd), a causative agent of chytridiomycosis in amphibian populations, emerged worldwide in the 21st century and is composed of six lineages which are display varying levels of virulence in their hosts.Research into the distribution, ecology and pathogenicity of these lineages has been hampered by an inability to type lineage efficiently. Here, we describe a lineage-specific TaqMan qPCR assay that differentiates the two lineages of Bd most commonly associated with chytridiomycosis: BdGPL and BdCAPE. We demonstrate how this assay can be used for the surveillance of wild populations of amphibians in Southern Africa using skin swabs, tissue samples and cultured isolates.


Genes ◽  
2020 ◽  
Vol 11 (7) ◽  
pp. 750
Author(s):  
Takehito Sugasawa ◽  
Kai Aoki ◽  
Kouki Yanazawa ◽  
Kazuhiro Takekoshi

The World Anti-Doping Agency has prohibited gene doping in the context of progress in gene therapy. There is a risk that the augmentation of genes using plasmids could be applied for gene doping. However, no gold standard method to detect this has been established. Here, we aimed to develop a method to detect multiple transgene fragments as proof of gene doping. Firstly, gene delivery model mice as a mimic of gene doping were created by injecting firefly luciferase plasmid with polyethylenimine (PEI) into the abdominal cavity. The results confirmed successful establishment of the model, with sufficient luminescence upon in vivo imaging. Next, multiple transgene fragments in the model were detected in plasma cell-free (cf)DNA, blood-cell-fraction DNA, and stool DNA using the TaqMan- quantitative real-time PCR(qPCR) assay, with the highest levels in plasma cfDNA. Using just a single drop of whole blood from the model, we also attempted long-term detection. The results showed that multiple transgene fragments were detected until 11 days. These findings indicate that the combination of plasma cfDNA or just one drop of whole blood with TaqMan-qPCR assay is feasible to detect plasmid-PEI-based gene doping. Our findings could accelerate the development of methods for detecting gene doping in humans.


Author(s):  
Takeito Sugasawa ◽  
Kai Aoki ◽  
Koki Yanazawa ◽  
Ryo Hagino ◽  
Shinsuke Tamai ◽  
...  

The World Anti-Doping Agency has prohibited gene doping in the context of progress in gene therapy. There is a risk that the artificial regulation of genes using plasmids could be applied for gene doping. However, no gold standard method to detect this has been established. Here, we aimed to develop a method to detect multiple transgene fragments as proof of gene doping. First, gene delivery model mice as a mimic of gene doping were created by injecting firefly luciferase plasmid with polyethylenimine (PEI) into the abdominal cavity. The results confirmed successful establishment of the model, with sufficient luminescence upon in vivo imaging. Next, multiple transgene fragments in the model were detected in plasma cell-free (cf)DNA, blood-cell-fraction DNA, and stool DNA using the TaqMan-qPCR assay, with the highest levels in plasma cfDNA. Using just a single drop of whole blood from the model, we also attempted long-term detection. The results showed that multiple transgene fragments were detected until 11 days. These findings indicate that the combination of plasma cfDNA or just one drop of whole blood with TaqMan-qPCR assay is feasible to detect plasmid-PEI-based gene doping. Our findings could accelerate the development of methods for detecting gene doping in humans.


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