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 bacterial contaminant within a sample, the method comprising the steps of:
 purifying nucleic acid comprising a sample;   preparing a reaction mixture including a first primer and a second primer, the first and second primers being specific for a target sequence of nucleic acid comprising a bacterial contaminant within the sample;   combining the reaction mixture with the nucleic acid purified in said step of purifying;   combining the reaction mixture with a predetermined amount of a second purified nucleic acid from a second sample, the second purified nucleic acid being free of the bacterial contaminant;   performing polymerase chain reaction on the reaction mixture combined with the nucleic acid purified in said step of purifying and the second purified nucleic acid, said step of performing amplifying the target sequence comprising the bacterial contaminant; and   detecting presence of the target sequence.   
     
     
         20 . The method according to  claim 19 , wherein the 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 bacterial 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 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 . The method according to  claim 19 , wherein the first primer comprises a sequence at least 80% identical to SEQ ID NO:1 and the second primer comprises a sequence at least 80% identical to SEQ ID NO:2. 
     
     
         24 . The method according to  claim 23 , wherein the target sequence comprises a 16S-rRNA complement of prokaryotic DNA. 
     
     
         25 . The method according to  claim 19 , wherein the second sample comprises Chinese hamster ovary cells. 
     
     
         26 . The method according to  claim 19 , wherein the predetermined amount is at least based in part on the volume of the reaction mixture having the nucleic acid purified in said step of purifying combined therewith. 
     
     
         27 . The method according to  claim 26 , wherein the predetermined amount is in the range of 10 μg to 250 μg of the second purified nucleic acid per 1 ml of the reaction mixture having the nucleic acid purified in said step of purifying combined therewith. 
     
     
         28 . The method according to  claim 19 , wherein said step of detecting comprises real-time polymerase chain reaction. 
     
     
         29 . A composition comprising,
 nucleic acid comprising a sample; and   DNA comprising Chinese hamster ovary cells, the DNA comprising the Chinese hamster ovary cells being free of prokaryotic DNA, wherein the sample is selected from the group consisting of amniotic fluid and eukaryotic cells.   
     
     
         30 . The composition according to  claim 29 , further comprising:
 a first primer having a sequence at least 80% identical to SEQ ID NO:1;   a second primer having a sequence at least 80% identical to SEQ ID NO:2; and a thermostable DNA polymerase.   
     
     
         31 . The composition according to  claim 30 , further comprising an intercalating dye. 
     
     
         32 . The composition according to  claim 29 , wherein the sample comprises a suspension of eukaryotic cells. 
     
     
         33 . A kit comprising:
 a thermostable DNA polymerase;   purified DNA from a eukaryotic source;   a first primer; and   a second primer, the first and second primers being specific for amplifying a target sequence of nucleic acid of a prokaryotic source, the purified DNA from the eukaryotic source being free of the target sequence of the prokaryotic source.   
     
     
         34 . The kit of  claim 33 , wherein the first primer comprises a sequence at least 80% identical to SEQ ID NO: 1 and the second primer comprises a sequence at least 80% identical to SEQ ID NO: 2. 
     
     
         35 . The kit of  claim 33 , wherein the eukaryotic source comprises Chinese hamster ovary cells. 
     
     
         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 . The kit of  claim 33 , wherein the target sequence of the nucleic acid of the prokaryotic source comprises a 16S-rRNA complement of prokaryotic DNA.