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Rapid Detection and Differentiation of the Major Mycoplasma Contaminants in Cell Cultures Using Real‐Time PCR with SYBR Green I and Melting Curve Analysis
Author(s) -
Harasawa Ryô,
Mizusawa Hiroshi,
Fujii Masashi,
Yamamoto Junko,
Mukai Hiroyuki,
Uemori Takashi,
Asada Kiyozo,
Kato Ikunoshin
Publication year - 2005
Publication title -
microbiology and immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.664
H-Index - 70
eISSN - 1348-0421
pISSN - 0385-5600
DOI - 10.1111/j.1348-0421.2005.tb03675.x
Subject(s) - melting curve analysis , sybr green i , biology , mycoplasma , real time polymerase chain reaction , polymerase chain reaction , contamination , microbiology and biotechnology , standard curve , biochemistry , gene , ecology
A quantitative real‐time polymerase chain reaction (PCR) procedure followed by melting curve analysis, using the green fluorescence dye SYBR Green I, was developed for rapid detection and differentiation of mycoplasma contaminants in cell cultures. This method showed that the detection of the target sequence was linear over a range from 10 4 to 10 colony‐forming units (CFU) of the mycoplasma cells. Analysis of the melting temperature of the PCR products allowed differentiation of the major mycoplasma contaminants. These results demonstrate that the protocol described in the present study can decrease the time to obtain reproducible results by simultaneous detection and differentiation of the Mycoplasma species contaminating cell cultures.