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Quantification of a cell culture contaminant using 16S rDNA
Author(s) -
Xu Xuebo,
Lai Yueyang,
Zhou Wenzhao,
Wu Leyang,
Hua Zichun
Publication year - 2019
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1002/bab.1792
Subject(s) - 16s ribosomal rna , propidium iodide , biology , contamination , bacteria , microbiology and biotechnology , staining , gentamicin , chromatography , a549 cell , cell culture , chemistry , antibiotics , biochemistry , genetics , programmed cell death , ecology , apoptosis
In this study, we identified a “black dot”‐like cell culture contaminant as a species belonging to the genus of Pusillimonas using 16S rDNA sequencing. Among all antibiotics tested, a combinatorial treatment of ampicillin and gentamicin both at 100 µg/mL was able to eliminate this contaminant. The contaminant was then visualized by fluorescence microscopy using propidium iodide staining and was found inside the cytosol of contaminated A549 cells. To characterize the efficacy of antibiotics for contaminant removal, we devised a quantitative method to determine the average number of 16S rDNA copies associated with a single A549 cell, which is directly proportional to the average number of contaminant per A549 cell. By using primers specific to the 16S rDNA sequence of the contaminant, we were able to estimate contaminants per single contaminated cell using both qPCR‐based relative and absolute quantification.