scholarly journals A New Sensitive Method for the Detection of Mycoplasmas Using Fluorescence Microscopy

Cells ◽  
2019 ◽  
Vol 8 (12) ◽  
pp. 1510 ◽  
Author(s):  
Anna Ligasová ◽  
Markéta Vydržalová ◽  
Renata Buriánová ◽  
Lenka Brůčková ◽  
Renata Večeřová ◽  
...  

Contamination of cell cultures by mycoplasmas is a very common phenomenon. As they can substantially alter cell metabolism and potentially spread to all cell cultures in laboratory, their early detection is necessary. One of the fastest and cheapest methods of mycoplasma detection relies on the direct staining of mycoplasmas’ DNA by DAPI or Hoechst dyes. Although this method is easy and fast to perform, it suffers from the low signal provided by these dyes compared to the nuclear DNA. Therefore, the reporter cell lines are used for cultivation of mycoplasmas before DAPI or the Hoechst staining step. In the study presented, we have developed and tested a new immunofluorescence assay for the detection of mycoplasmas. The method is based on the enzymatic labeling using DNA polymerase I and modified nucleotides utilizing nicks in the mycoplasmas’ DNA. Modified nucleotides are incorporated into mycoplasmas’ DNA and subsequently visualized by immunofluorescence microscopy. The developed approach is independent of the mycoplasma strain, does not intensely stain nuclear DNA, does not stain other bacteria, and provides higher sensitivity than the approach based on the direct labeling using DAPI or Hoechst dyes.

1994 ◽  
Vol 41 (1) ◽  
pp. 79-86 ◽  
Author(s):  
S Sen ◽  
S Mukhopadhyay ◽  
J Wetzel ◽  
T K Biswas

The mitochondrial DNA (mtDNA) polymerase was isolated from a protease-deficient yeast strain (PY2), and purified about 3000 fold by a column chromatography on phosphocellulose, heparin-agarose, and single-stranded DNA cellulose. The purified polymerase was characterized with respect to optimal nucleotide concentrations, template-primer specificity and sensitivity to some inhibitors. These results were compared with the nuclear DNA polymerase I activity. Both polymerases showed similar requirement of deoxynucleotide concentrations (Km < 1 microM), and highest activity with poly(dA-dT) template. However, the mtDNA polymerase was more sensitive to ddTTP, EtBr and Mn2+ inhibition in comparison to the nuclear DNA polymerase I. The mtDNA polymerase did not need ATP as an energy source for in vitro DNA synthesis. This mtDNA polymerase preparation also showed 3'-->5' exonuclease activity.


2021 ◽  
Vol 10 (1) ◽  
Author(s):  
Ani Saghatelyan ◽  
Hovik Panosyan ◽  
Armen Trchounian ◽  
Nils‐Kåre Birkeland

Biochemistry ◽  
1984 ◽  
Vol 23 (9) ◽  
pp. 2073-2078 ◽  
Author(s):  
Anup K. Hazra ◽  
Sevilla Detera-Wadleigh ◽  
Samuel H. Wilson

2013 ◽  
Vol 4 (1) ◽  
Author(s):  
Johannes Hohlbein ◽  
Louise Aigrain ◽  
Timothy D. Craggs ◽  
Oya Bermek ◽  
Olga Potapova ◽  
...  

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