RAPID DETECTION OF MYCOPLASMA CONTAMINATION IN CELL CULTURE SAMPLES
First Claim
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1. A method of detecting mycoplasma in a cell culture media, the method comprising,obtaining an aliquot of cell culture media;
- performing a real-time nucleic acid amplification reaction with a thermostable DNA polymerase to amplify a mycoplasma nucleic acid, if present, in the aliquot wherein;
nucleic acids in the aliquot are not further purified, andthe amplification reaction comprises an intercalating fluorescent dye that produces a fluorescent signal in the presence of double stranded DNA; and
detecting a melting temperature of an amplification product of the amplification reaction, wherein the presence of an amplification product indicates the presence of mycoplasma in the cell culture.
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Abstract
The present invention provides for methods of detecting mycoplasma, for example in cell culture media.
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Citations
33 Claims
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1. A method of detecting mycoplasma in a cell culture media, the method comprising,
obtaining an aliquot of cell culture media; -
performing a real-time nucleic acid amplification reaction with a thermostable DNA polymerase to amplify a mycoplasma nucleic acid, if present, in the aliquot wherein; nucleic acids in the aliquot are not further purified, and the amplification reaction comprises an intercalating fluorescent dye that produces a fluorescent signal in the presence of double stranded DNA; and detecting a melting temperature of an amplification product of the amplification reaction, wherein the presence of an amplification product indicates the presence of mycoplasma in the cell culture. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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14. A method of detecting mycoplasma in a cell culture media, the method comprising,
obtaining an aliquot of cell culture media; -
performing a nucleic acid amplification reaction with a DNA polymerase to amplify a mycoplasma nucleic acid, if present, in the aliquot wherein; (a) the performing step comprises amplifying the portion with a first and second primer, the first primer comprising the following degenerate sequence; GAAGAWATGCCWTATTTAGAAGATGG (SEQ ID NO;
1); andthe second primer comprising the following degenerate sequence; CCRTTTTGACTYTTWCCACCMAGTGGTTGTTG (SEQ ID NO;
2), wherein W represents A or T, Y represents C or T, R represents A or G, and M represents A or C; and(b) detecting the presence or absence of an amplification product of the amplification reaction, wherein the presence of an amplification product indicates the presence of mycoplasma in the cell culture. - View Dependent Claims (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)
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26. A kit for amplifying mycoplasma DNA, if present, from cell culture media, the kit comprising:
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a first degenerate primer comprising GAAGAWATGCCWTATTTAGAAGATGG (SEQ ID NO;
1); anda second degenerate primer comprising CCRTTTTGACTYTTWCCACCMAGTGGTTGTTG (SEQ ID NO;
2),wherein W represents A or T, Y represents C or T, R represents A or G, and M represents A or C. - View Dependent Claims (27, 28, 29, 30, 31, 32, 33)
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Specification