DAPI Staining and Fluorescence Microscopy Techniques for Phytoplasmas

Andrade, Nancy; Arismendi, Nolberto

doi:10.1007/978-1-62703-089-2_10

Cited by 10 publications

(4 citation statements)

References 17 publications

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“…2 mM glutamine microscope slide and mounted with buffered glycerol or phosphate buffered saline (PBS) followed by observation of mycoplasma bodies using a drop of immersion oil on it. Conversely, there is no need to use cover slip and scraper for DAPI staining protocols of floating cells (Schaper and Converse 1985;Andrade and Arismendi 2013). Due to the limitations related to the direct analysis of cell lines by DAPI staining, mycoplasma-free Vero cell line (NCBI C101) was employed as an indicator as well as negative control.…”

Section: Cell Culturessupporting

confidence: 50%

Real-time PCR assay is superior to other methods for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran

et al. 2015

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Mycoplasmas are the most important contaminants of cell cultures throughout the world. They are considered as a major problem in biological studies and biopharmaceutical economic issues. In this study, our aim was to find the best standard technique as a rapid method with high sensitivity, specificity and accuracy for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran. Thirty cell lines suspected to mycoplasma contamination were evaluated by five different techniques including microbial culture, indirect DNA DAPI staining, enzymatic mycoalert Ò assay, conventional PCR and real-time PCR. Five mycoplasma-contaminated cell lines were assigned as positive controls and five mycoplasma-free cell lines as negative controls. The enzymatic method was performed using the mycoalert Ò mycoplasma detection kit. Real-time PCR technique was conducted by PromoKine diagnostic kits. In the conventional PCR method, mycoplasma genus-specific primers were designed to analyze the sequences based on a fixed and common region on 16S ribosomal RNA with PCR product size of 425 bp. Mycoplasma contamination was observed in 60, 56.66, 53.33, 46.66 and 33.33 % of 30 different cell cultures by real-time PCR, PCR, enzymatic mycoalert Ò , indirect DNA DAPI staining and microbial culture methods, respectively. The analysis of the results of the different methods showed that the realtime PCR assay was superior the other methods with the sensitivity, specificity, accuracy, predictive value of positive and negative results of 100 %. These values were 94.44, 100, 96.77, 100 and 92.85 % for the conventional PCR method, respectively. Therefore, this study showed that real-time PCR and PCR assays based on the common sequences in the 16S ribosomal RNA are reliable methods with high sensitivity, specificity and accuracy for detection of mycoplasma contamination in cell cultures and other biological products.

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Section: Cell Culturessupporting

confidence: 50%

Real-time PCR assay is superior to other methods for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran

et al. 2015

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“…The amount of TNF-α in the gingival crevicular fluid of the volunteers after 2-min contact with LH and PH rice gels in comparison with the rice gel base and standard filter blocks for violet (355-425 nm), blue (450-490 nm), and green (515-560 nm) excitation light (21,22). Photograph was taken using Kodak Tri-Xpan 400, Provia Fujichrome 400, Ilford 400 and Kodak Ektachrome 400 films.…”

Section: Tnf-α Releasesupporting

confidence: 43%

<i>In vitro</i> oral epithelium cytotoxicity and <i>in vivo</i> inflammatory inducing effects of anesthetic rice gel

Khongkhunthian

Supanchart

Yotsawimonwat

et al. 2017

DD&T

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“…Healthy and ROLF-infected rice plants were screened by nested PCR. DAPI staining of the rice tissues was performed by following the protocols of phytoplasma study [ 41 , 42 ]. Root and leaf tissues were used for specimen preparations.…”

Section: Methodsmentioning

confidence: 99%

Geographic Distribution, Genetic Variability and Biological Properties of Rice Orange Leaf Phytoplasma in Southeast Asia

et al. 2021

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Rice orange leaf phytoplasma (ROLP) causes clear orange to yellowish leaf discoloration and severe stunting in rice seedlings. The ecological and biological characteristics of ROLP are largely unknown because the disease has not widely caused serious problems in rice cultivated areas, thereby leading to the low accumulation of research data. However, in the past decade, the disease became a threat to rice production, particularly in South China and India; it has also been recognised in other Asian countries, such as Vietnam, Thailand and the Philippines. Here, we observed the occurrence of ROLP in paddies of the Southeast Asian counties (Cambodia, Vietnam and the Philippines) and found that the isolates in the Philippines and Vietnam were monophyletic, while those in India, Thailand and Cambodia were more diverse, suggesting their potential origins. In Cambodia, it was revealed that following polymerase chain reaction (PCR) detection, the known ROLP-insect vectors, N. virescens Distant and Recilia dorsalis Motchulsky, were ROLP-positive, indicating their roles in pathogen dispersal. Moreover, fluorescent and scanning electron microscopy revealed the intensive accumulation of the phytoplasma in phloem tissues and massive accumulation of storage starch in vascular bundle sheath and parenchyma. Altogether, this study illustrated the genetic variability of global ROLP isolates and the pathogen’s biological impact on rice tissue.

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DAPI Staining and Fluorescence Microscopy Techniques for Phytoplasmas

Cited by 10 publications

References 17 publications

Real-time PCR assay is superior to other methods for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran

Real-time PCR assay is superior to other methods for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran

<i>In vitro</i> oral epithelium cytotoxicity and <i>in vivo</i> inflammatory inducing effects of anesthetic rice gel

Geographic Distribution, Genetic Variability and Biological Properties of Rice Orange Leaf Phytoplasma in Southeast Asia

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