Detection of bacterial (mollicutes) 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.

1 - 18 . (canceled) 
     
     
         19 . 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 the  Mollicute  contaminant; and   detecting presence of amplified extracted nucleic acid from the  Mollicute  contaminate.   
     
     
         20 . The method according to  claim 19 , wherein the eukaryotic sample is selected from the group consisting of amniotic fluid and a suspension of eukaryotic cells. 
     
     
         21 . The method according to  claim 19 , wherein the  Mollicute  contaminant comprises a genus selected from the group consisting of  Acholeplasma, Bacillus, Clostridium, Corynebacterium, Micrococcus, Mycoplasma, Spiroplasma, Staphylococcus , and  Streptococcus.    
     
     
         22 . The method according to  claim 21 , wherein the bacterial  Mollicute  contaminant comprises a species selected from the group consisting of  M. hyorhinis, M. arginini, M. pneumoniae, M. fermentans, M. orale , and  M. pirium , or the bacterial contaminant is  Acholeplasma laidlawii , or the bacterial contaminant is  Spiroplasma mirium.    
     
     
         23 - 24 . (canceled) 
     
     
         25 . The method according to  claim 19 , wherein the second eukaryotic sample comprises Chinese hamster ovary cells. 
     
     
         26 . The method according to  claim 19 , wherein the predetermined amount of the purified DNA obtained from the second eukaryotic sample is at least based in part on a total volume of the reaction mixture prepared in said step of preparing combined with the nucleic acid extracted in said step of extracting. 
     
     
         27 . The method according to  claim 26 , wherein the predetermined amount is a concentration of between about 10 μg to 250 μg of the purified DNA obtained from a second eukaryotic sample per 1 ml of the reaction mixture prepared in said step of preparing combined with the nucleic acid extracted in said step of extracting. 
     
     
         28 . The method according to  claim 19 , wherein said step of detecting comprises real-time polymerase chain reaction. 
     
     
         29 - 32 . (canceled) 
     
     
         33 . 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.   
     
     
         34 - 35 . (canceled) 
     
     
         36 . The kit of  claim 33  further comprising a lysis reagent. 
     
     
         37 . The kit of  claim 33  further comprising a detection reagent selected from the group consisting of an intercalating dye and an oligonucleotide probe. 
     
     
         38 . (canceled) 
     
     
         39 . 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.   
     
     
         40 . The composition of  claim 39 , wherein the eukaryote source is Chinese hamster ovary cells. 
     
     
         41 . The composition of  claim 40 , 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. 
     
     
         42 . The composition of  claim 39 , wherein the eukaryotic sample is one of amniotic fluid of embryonated cells, a eukaryotic cell culture supernatant, and an eukaryotic cell culture suspension. 
     
     
         43 . The composition of  claim 39  further comprising a plurality of deoxyribose nucleotides. 
     
     
         44 . The composition of  claim 43  further comprising uracil-N-glycosylase enzyme, wherein the plurality of deoxyribose nucleotides includes a uracil deoxyribose nucleotide. 
     
     
         45 . The composition of  claim 39 , wherein the detection reagent is selected from the group consisting of an intercalating dye and an oligonucleotide probe having a detectable label. 
     
     
         46 . The composition of  claim 39  further comprising a lysis reagent comprising a chaotropic reagent and proteinase K.