Detection of mycoplasma in cell cultures and cell culture derived biologicals

Abstract

Sequences having specificity for Mycoplasma and related Mollicutes genus strains and uses thereof. Methods of use include detection of samples contaminated with Mycoplasma. Kits are provided and comprise one or more oligonucleotides for the detection of Mycoplasma and related Mollicutes genus strains.

What is claimed is: 
     
         1 . An isolated or synthetic nucleic acid, wherein the isolated or synthetic nucleic acid hybridizes to at least 5 consecutive nucleic acids in a 5′ to 3′ or 3′ to 5′ direction of a 16S-23S ribosomal intergenic region nucleic acid of  Mycoplasma  and/or related  Mollicutes  genus strains. 
     
     
         2 . The isolated nucleic acid of  claim 1 , wherein the isolated nucleic acid comprises at least five nucleic acids. 
     
     
         3 . The isolated nucleic acid of  claim 1 , optionally comprising one or more mutations, variants, substitutions, analogs, deletions or combinations thereof. 
     
     
         4 . The isolated nucleic acid of  claim 3 , wherein the one or more mutations, variants, substitutions, analogs or combinations thereof, comprise: adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo-N 6 -methyladenine, 7-deazaxanthine, 7-deazaguanine, N 4 ,N 4 -ethanocytosin, N 6 ,N 6 -ethano-2,6-diaminopurine, 5-methylcytosine, 5-(C 3 -C 6 )-alkynylcytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin, isocytosine, isoguanin, inosine, non-naturally occurring nucleobases, locked nucleic acids (LNA), or peptide nucleic acids (PNA). 
     
     
         5 . The isolated nucleic acid of  claim 1 , wherein the  Mycoplasma  and/or related  Mollicutes  genus strains, comprising:  Acholeplasma laidlawii  ATCC23206 ; Spiroplasma citri  ATCC27556 ; M. arginini  ATCC23838 ; M. arthritidis  ATCC19611;  M. bovis  ATCC25025 ; M. capricolum capricolum  ATCC27343;  M. fermentans  ATCC49892 ; M. gallisepticum  ATCC19610 ; M. genitalium  ATCC33530;  M. hominis  ATCC27545 ; M. hyopneumoniae  ATCC25934 ; M. orale ATCC 23714 ; M. pirum  ATCC25960;  M. pneumoniae  ATCC15531 ; M. pulmonis  GenBank X58554 ; M. salivarium  ATCC23064 ; M. synoviae  ATCC25204 ; M. mirounga GenBank GU 905026 ; M. adleri strain G 145 ; M. agalactiae  strainGO-5 ; M. agassizii  strain PS6 ; M. alkalescens  ATCC29103 ; M. alligatoris  ATCC700619 ; M. alvi  strain Ilsley;  M. amphoriforme  strain A39 ; M. anseris  strain 1219 ; M. auris  strain UTA;  M. bovigenitalium  strain PG-11 ; M. bovirhinis  ATCC27748 ; M. bovoculi  ATCC29104 ; M. buccale  ATCC23636 ; M. buteonis  strain Bb/T2g; M.  californicum  strain ST6 ; M. canadense  strain 466 ; M. canimucosale  type strain 1642 ; M. canis  ATCC19525 ; M. caviae  strain ATCC27108 ; M. cavipharyngis  ATCC43016 ; M. citelli  strain RG2C;  M. cloacale  strain 383 ; M. collis  ATCC35278 ; M. columbinasale  ATCC33549 ; M. columbinum  strain FG295 ; M. columborale  ATCC29258 ; M. conjunctivae  Goat655 ; M. corogypsi  strain BV1-5 ; M. cottewii  ATCC43093 ; M. cricetuli  ATCC35279 ; M. crocodyli  ATCC51981 ; M. cynos  strain H381 ; M. dispar  ATCC27140 ; M. edwardii  strain 04-3440 ; M. elephantis  ATCC51980 ; M. equigenitalium  ATCC29869 ; M. equirhinis  ATCC29420 ; M. falconis  ATCC51372 ; M. fastidiosum  ATCC33229 ; M. faucium  ATCC25293 ; M. feliminutum  ATCC25749 ; M. felis GenBank AF 443608 ; M. flocculare  strain Ms42 ; M. gallinarum  strain B2Dinter;  M. gallisepticum  GenBank FJ468427 ; M. gallopavonis  ATCC33551 ; M. glycophilum  ATCC35277 ; M. gypis  ATCC51370 ; M. hyopharyngis  strain H36B-F;  M. hyorhinis  ATCC29052 ; M. iguanae  strain 2327 ; M. indiense  ATCC51125 ; M. finers  ATCC15969 ; M. iowae  ATCC33552 ; M. lagogenitalium  ATCC700289 ; M. leachii  strain PG50 ; M. leonicaptivi  ATCC49890 ; M. leopharyngis  ATCC49889 ; M. lipofaciens  ATCC35015 ; M. lipophilum  ATCC27104 ; M. maculosum  strain PG15 ; M. meleagridis  ATCC27764 ; M. microti  strain 11,371 ; M. moatsii  strain MK405 ; M. mobile  ATCC43663 ; M. molare  strain H542 ; M. muris  ATCC33757 ; M. mustelae  ATCC35214 ; M. neurolyticum  ATCC19988 ; M. opalescens  strain MHS408 ; M. ovipneumoniae  ATCC29419 ; M. oxoniensis  ATCC49694 ; M. phocicerebrale  strain CSL5195S2 ; M. phocirhinis  strain CSL7475-4 ; M. primatum  strain HRC292 ; M. pullorum  ATCC33553 ; M. putrefaciens  ATCC33756 ; M. simbae  ATCC49888 ; M. spermatophilum  strain AH159 ; M. sphenisci  strain UCMJ;  M. spumans  ATCC19526 ; M. sturni  ATCC51945 ; M. sualvi  ATCC33004 ; M. subdolum  ATCC29870 ; M. testudineum  ATCC700618 ; M. testudinis  ATCC43263 ; M. verecundum  ATCC27862 ; M. vulturii  strain Gb-V33 ; M. yeatsii  ATCC43094 ; M. zalophi  strain CSL5195; or  M. zalophidermidis  strain CSL4779. 
     
     
         6 . The isolated nucleic acid of  claim 1 , wherein the nucleic acid hybridizes to the 16S-23 S ribosomal intergenic region of  Mycoplasma  and related  Mollicutes  genus strains, under low-, medium- or high stringency conditions. 
     
     
         7 . (canceled) 
     
     
         8 . (canceled) 
     
     
         9 . A nucleic acid sequence, wherein the nucleic acid sequence is complementary to at least five consecutive nucleic acids in a 5′ to 3′ or 3′ to 5′ direction, of any one or more sequences set forth as SEQ ID NOS: 1 to 15. 
     
     
         10 . The nucleic acid sequence of  claim 9 , wherein the nucleic acid comprises at least five nucleic acids. 
     
     
         11 . The nucleic acid of  claim 10 , optionally comprising one or more mutations, variants, substitutions, analogs, deletions or combinations thereof. 
     
     
         12 . The nucleic acid of  claim 11 , wherein the nucleic acid comprises one, or combinations thereof, of: adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo-N 6 -methyladenine, 7-deazaxanthine, 7-deazaguanine, N 4 ,N 4 -ethanocytosin, N 6 ,N 6 -ethano-2,6-diaminopurine, 5-methylcytosine, 5-(C 3 -C 6 )-alkynylcytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin, isocytosine, isoguanin, inosine, non-naturally occurring nucleobases, locked nucleic acids (LNA), or peptide nucleic acids (PNA). 
     
     
         13 . (canceled) 
     
     
         14 . (canceled) 
     
     
         15 . (canceled) 
     
     
         16 . (canceled) 
     
     
         17 . (canceled) 
     
     
         18 . (canceled) 
     
     
         19 . A nucleic acid sequence set forth as SEQ ID NOS: 1 to 15 or fragments thereof, comprising one or more nucleic acid base mutant, variants, analogs, deletions, substitutions or combinations thereof. 
     
     
         20 . The nucleic acid sequence of  claim 19 , wherein the nucleic acid base mutant, variants, analogs, or substitutions comprise: adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo-N 6 -methyladenine, 7-deazaxanthine, 7-deazaguanine, N 4 ,N 4 -ethanocytosin, N 6 ,N 6 -ethano-2,6-diaminopurine, 5-methylcytosine, 5-(C 3 -C 6 )-alkynylcytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin, isocytosine, isoguanin, inosine, non-naturally occurring nucleobases, locked nucleic acids (LNA), peptide nucleic acids (PNA), or combinations thereof. 
     
     
         21 . A method of detecting whether a sample is contaminated with  Mycoplasma  and/or related  Mollicutes  genus strains comprising:
 incubating a sample with one or more primers or oligomers comprising nucleic acid sequences set forth as SEQ ID NOS: 1 to 15;   assaying the samples; thereby,   detecting whether the sample is contaminated with  Mycoplasma  and/or related  Mollicutes  genus strains.   
     
     
         22 . The method of  claim 21 , wherein incubating the samples further comprises incubating the samples with at least one discriminatory positive control set forth as SEQ ID NOS: 1 to 9. 
     
     
         23 . The method of  claim 21 , wherein methods of assaying the samples comprise, polymerase chain reaction (PCR), immunoassays, blots, gels, microarrays, high-throughput screening assays, or combinations thereof. 
     
     
         24 . The method of  claim 23 , wherein the PCR is a real-time polymerase chain reaction (RT-PCR). 
     
     
         25 - 39 . (canceled)