Detection of bacterial (mollicuties) contamination

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.

What is claimed is: 
     
         1 . 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.   
     
     
         2 . The kit of  claim 1  further comprising a lysis reagent. 
     
     
         3 . The kit of  claim 1  further comprising a detection reagent selected from the group consisting of an intercalating dye and an oligonucleotide probe. 
     
     
         4 . The kit of  claim 1 , wherein the purified DNA is total DNA obtained from Chinese hamster ovary cells. 
     
     
         5 . 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.   
     
     
         6 . The composition of  claim 5 , wherein the eukaryote source is Chinese hamster ovary cells. 
     
     
         7 . The composition of  claim 6 , wherein the purified DNA is total DNA obtained from Chinese hamster ovary cells. 
     
     
         8 . The composition of  claim 7 , wherein said purified DNA obtained from Chinese hamster ovary cells is present in the composition at a concentration of between about 10 μg to about 250 μg per 1 ml of volume of said nucleic acid. 
     
     
         9 . The composition of  claim 5 , wherein the eukaryotic sample is one of amniotic fluid of embryonated cells, a eukaryotic cell culture supernatant, and an eukaryotic cell culture suspension. 
     
     
         10 . The composition of  claim 5  further comprising a plurality of deoxyribose nucleotides. 
     
     
         11 . The composition of  claim 10  further comprising uracil-N-glycosylase enzyme, wherein the plurality of deoxyribose nucleotides includes a uracil deoxyribose nucleotide. 
     
     
         12 . The composition of  claim 5 , wherein the detection reagent is selected from the group consisting of an intercalating dye and an oligonucleotide probe having a detectable label. 
     
     
         13 . The composition of  claim 5  further comprising a lysis reagent comprising a chaotropic reagent and proteinase K.