Detection of bacterial (mollicutes) contamination
AssigneeRoche Diagnostics Operations, Inc.
InventorChristian Birkner
Life of this patent
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Abstract
The present disclosure provides a method and system for the PCR amplification of a target sequence which suppresses non-specific amplification products. The disclosure concerns the use of a primer pair optimized to amplify a nucleic acid of a contaminant in the background of genomic DNA of a first organism. When DNA from a second organism suspected for comprising the contaminant is subjected to the same PCR-based amplification reaction, detection sensitivity and specificity of the contaminant is enhanced when an amount of genomic DNA of the first organism is present in the amplification reaction.
Claims19
A method of detecting a Mollicute contaminant within a eukaryotic sample, the method comprising the steps of:
- extracting nucleic acid from the eukaryotic sample in a liquid form, the extracted nucleic acid comprising extracted nucleic acid from the Mollicute contaminate;
- preparing a reaction mixture including a thermostable polymerase, a first synthetic nucleotide primer having a nucleic acid sequence with less than or equal to three nucleic acid variations from the nucleic acid sequence of SEQ ID NO:1 and a second synthetic nucleotide primer having a nucleic acid sequence with less than or equal to three nucleic acid variations from the nucleic acid sequence of SEQ ID NO:2;
- combining the reaction mixture prepared in said step of preparing with the nucleic acid extracted in said step of extracting;
- combining the reaction mixture with a predetermined amount of a purified DNA obtained from a second eukaryotic sample, the purified DNA being free of prokaryotic DNA;
- performing polymerase chain reaction on the reaction mixture combined with the nucleic acid extracted in said step of extracting and the purified DNA obtained from a second eukaryotic sample, wherein said step of performing comprises the thermostable polymerase coupling to a 3′ end of each of the first and second synthetic nucleotide primers and amplification of extracted nucleic acid from Mollicute contaminant;
- and
- detecting presence of amplified extracted nucleic acid from the Mollicute contaminate.
A kit for detecting Mollicute nucleic acid in a sample of one of amniotic fluid of embryonated cells, a eukaryotic cell culture supernatant, and an eukaryotic cell culture suspension, the kit comprising:
- a thermostable DNA polymerase;
- purified DNA obtained from Chinese hamster ovary cells and being free of prokaryotic DNA;
- a first synthetic nucleotide primer having a nucleic acid sequence with less than or equal to three nucleic acid variations from the nucleic acid sequence of SEQ ID NO:1;
- and
- a second synthetic nucleotide primer having a nucleic acid sequence with less than or equal to three nucleic acid variations from the nucleic acid sequence of SEQ ID NO:2.
A composition for detecting contaminating Mollicute nucleic acid within a eukaryotic sample, the composition comprising:
- nucleic acid extracted from the eukaryotic sample, said nucleic acid present in liquid form and comprising extracted nucleic acid from a Mollicute contaminate within the eukaryotic sample;
- a detection reagent;
- a thermostable DNA polymerase;
- a first synthetic nucleotide primer having a 5′ and a 3′ end and having a nucleic acid sequence with less than or equal to three nucleic acid variations from the nucleic acid sequence of SEQ ID NO:1, said first synthetic nucleotide primer having thermostable DNA polymerase coupled to the 3′ end;
- a second synthetic nucleotide primer having a 5′ and a 3′ end and having a nucleic acid sequence with less than or equal to three nucleic acid variations from the nucleic acid sequence of SEQ ID NO:2, said second synthetic nucleotide primer having a thermostable DNA polymerase coupled at the 3′ end;
- and
- purified DNA obtained from a eukaryote source, said purified DNA being free of prokaryotic DNA, the eukaryote source being the same eukaryote source as the eukaryotic sample.
Dependencies are inferred from the claim text.