Multi-primer assay for mycoplasma detection

Abstract

Disclosed is a multi-primer amplification assay, method and kits for detecting Mycoplasma species and closely related species utilizing a plurality of oligonucleotide primers in contact with a sample in a single vessel and detecting the amplification product, wherein the presence of an amplification product indicates Mycoplasma in the sample.

1 . A multi-primer assay for detecting  Mycoplasma  comprising:
 a.) contacting a sample with the plurality of oligonucleotide primers selected from the group consisting of SEQ ID NOs:1-144 in a single vessel;   b.) performing a multi-primer amplification reaction in the vessel, wherein each of the plurality of oligonucleotide primers is present for participation in amplifying a target nucleic acid in the sample to produce an amplification product; and   c.) detecting the amplification product, wherein the presence of an amplification product indicates  Mycoplasma  in the sample.   
     
     
         2 . The assay according to  claim 1 , wherein at least one of the oligonucleotide primers comprises a modified nucleobase at the third nucleobase from the 3′ end of the primer. 
     
     
         3 . The assay according to  claim 1 , wherein at least one of the oligonucleotide primers comprises a modified nucleobase at the second nucleobase from the 3′ end of the primer. 
     
     
         4 . The assay of  claim 1  wherein the plurality of primers comprises at least 15 forward primers and at least 16 reverse primers. 
     
     
         5 . The assay according to  claim 1 , wherein at least twenty oligonucleotide primers are present in the multi-primer amplification reaction. 
     
     
         6 - 8 . (canceled) 
     
     
         9 . The assay according to  claim 1 , wherein the sample is selected from a cell culture, a tissue culture, a food sample, an environmental sample, an agricultural sample, a biopharmaceutical sample, a pharmaceutical sample, or water. 
     
     
         10 - 11 . (canceled) 
     
     
         12 . The assay according to  claim 2 , wherein the modification averts primer-dimer formation in the amplification reaction. 
     
     
         13 . The assay according to  claim 3 , wherein the modification averts primer-dimer formation in the amplification reaction. 
     
     
         14 . The assay according to  claim 1 , wherein the contacting comprises hybridizing one or more of the oligonucleotide primers to a target nucleic acid in the sample. 
     
     
         15 . The assay according to  claim 14 , wherein the target nucleic acid is extracted from the sample prior to hybridizing with the one or more oligonucleotide primers. 
     
     
         16 . The assay according to  claim 1 , wherein the detecting comprises a detector selected from the group consisting of a nucleic acid dye, a reporter probe, or a reporter probe and a nucleic acid dye. 
     
     
         17 . The assay according to  claim 1 , wherein the assay comprises a discriminatory positive control (DPC) nucleic acid. 
     
     
         18 . The assay according to  claim 17 , wherein the DPC nucleic acid is added to the sample prior to extraction of the target nucleic acid of the sample. 
     
     
         19 . The assay according to  claim 18 , wherein the DPC nucleic acid is extracted simultaneously with the target nucleic acid of the sample. 
     
     
         20 . The assay according to  claim 19 , wherein the DPC nucleic acid is amplified in the multi-primer amplification reaction. 
     
     
         21 . The assay according to  claim 20 , wherein the DPC amplification product is detected. 
     
     
         22 . The assay according to  claim 21 , wherein detection of the DPC amplification product and no detection of the  Mycoplasma  amplification product is a confirmed negative assay for  Mycoplasma.    
     
     
         23 . The assay according to  claim 21 , wherein detection of the DPC amplification product and detection of the  Mycoplasma  amplification product is a confirmed positive assay for  Mycoplasma.    
     
     
         24 - 25 . (canceled) 
     
     
         26 . The assay according to  claim 21 , wherein the detecting comprises a melting temperature for the discriminatory positive control distinguishable from a melting temperature for the  Mycoplasma.    
     
     
         27 . The assay according to  claim 1 , wherein the assay detects more than 90  Mycoplasma  species as well as related microorganisms  Acholeplama laidlwaii  and  Spiroplasma citri.    
     
     
         28 . A method for detecting  Mycoplasma  comprising:
 a.) extracting nucleic acid from a sample to be tested for  Mycoplasma;      b.) combining in a single vessel a plurality of oligonucleotide primers and the nucleic acid from the sample;   c.) contacting the nucleic acid from the sample with the plurality of oligonucleotide primers in the vessel;   d.) performing a multi-primer amplification reaction in the vessel, wherein each of the plurality of oligonucleotide primers is present for participation in amplifying the sample to produce an amplification product; and   e.) detecting the amplification product, wherein the presence of the amplification product indicates  Mycoplasma  in the sample.   
     
     
         29 . The method according to  claim 28 , wherein the plurality of oligonucleotide primers is selected from the group consisting of SEQ ID NOS:1-144. 
     
     
         30 - 33 . (canceled) 
     
     
         34 . A kit for detecting  Mycoplasma  comprising a container having disposed therein a plurality of oligonucleotide primers selected from the group consisting of SEQ ID NOs: 1-144. 
     
     
         35 . The kit of  claim 34 , further comprising one or more of a polymerase, a detectable reporter, a protocol and a discriminating positive control nucleic acid. 
     
     
         36 . The kit of  claim 35 , wherein the detectable reporter is selected from the group consisting of a nucleic acid dye, a reporter probe or a reporter probe and a nucleic acid dye.