Species specific DNA detection
View Patent ↗The present invention relates to methods and compositions for the identification of species-specific material in pharmaceutical products. In particular, the present invention relates to methods for the identification of species-specific DNA in a population of a different species or following contact (e.g., growth) with cells of a different species.
1. A method for detecting species-specific nucleic acid, comprising:
a) providing
i) a nucleic acid sample from a culture of cells of a first species, wherein said culture has had previous exposure to a second culture of cells from a second species or a cell product derived from said cells from a second species;
ii) first nucleic acid probes specific for nucleic acid from said second species; and
(iii) second nucleic acid probes specific for said first species;
b) exposing said sample to said first nucleic acid probes under conditions such that said first nucleic acid probes hybridize to nucleic acid from said second species and do not hybridize to nucleic acid from said first species to detect nucleic acid from said second species; and
c) exposing said sample to said second nucleic acid probes, wherein said second nucleic acid probes are selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:4, under conditions such that said second nucleic acid probes hybridize to nucleic acid from said first species and do not hybridize to nucleic acid from said second species to detect nucleic acid from said first species.
2. The method of claim 1 , wherein said first nucleic acid probes are specific for a repetitive element of nucleic acid in the genome of said second species.
3. The method of claim 2 , wherein at least 20 copies of said repetitive element are present in the genome of said second species.
4. The method of claim 1 , wherein said cells from a second species are selected from the group consisting of rat cells, mouse cells, and porcine cells.
5. The method of claim 1 , wherein said exposing of b) and/or c) comprises PCR.
6. The method of claim 5 , wherein said first nucleic acid probes and/or said second nucleic acid probes are PCR primers.
7. The method of claim 1 , wherein said first nucleic acid probes and said second nucleic acid probes are PCR primers, and wherein said exposing of b) and c) comprises PCR, and wherein said PCR is a multiplex PCR reaction.
8. The method of claim 1 , wherein said first nucleic acid probes are selected from the group consisting of SEQ ID NOs: 1, 2, and 5-26.
9. The method of claim 1 , wherein said culture of cells of a first species is a cultured human skin tissue.
10. The method of claim 9 , wherein said cultured human skin tissue comprises keratinocytes selected from the group consisting of primary keratinocytes and immortalized keratinocytes.
11. The method of claim 10 , wherein said immortalized keratinocytes are NIKS cells.
12. The method of claim 1 , wherein said culture of cells of a first species comprises stem cells.
13. The method of claim 1 , wherein said cells from a second species comprise feeder cells.
14. The method of claim 13 , wherein said feeder cells are mouse fibroblast cells.
15. A method for detecting species-specific nucleic acid, comprising:
a) providing
i) a nucleic acid sample from a culture of cells from a first species, wherein said culture has had previous exposure to a feeder layer from a second species;
ii) first nucleic acid probes specific for nucleic acid derived from said feeder layer from said second species, wherein said first nucleic acid probes are selected from the group consisting of SEQ ID NOs: 1, 2, and 5-26; and
iii) second nucleic acid probes specific for said first species selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:4;
b) exposing said sample to said first nucleic acid probes under conditions such that said first nucleic acid probes hybridize to nucleic acid derived from said feeder layer and do not hybridize to nucleic acid from said first species to detect nucleic acid from said second species; and
c) exposing said sample to said second nucleic acid probes under conditions such that said second nucleic acid probes hybridize to nucleic acid from said first species and do not hybridize to nucleic acid from said second species to detect nucleic acid from said first species.
16. The method of claim 15 , wherein said first nucleic acid probes are specific for a repetitive element of nucleic acid in the genome of said second species.
17. The method of claim 16 , wherein at least 20 copies of said repetitive element are present in the genome of said second species.
18. The method of claim 15 , wherein said feeder layer is a mouse cell feeder layer.
19. The method of claim 15 , wherein said exposing of b) and/or c) comprises PCR.
20. The method of claim 19 , wherein said first nucleic acid probes and/or said second nucleic acid probes are PCR primers.
21. The method of claim 15 , wherein said first nucleic acid probes and said second nucleic acid probes are PCR primers, and wherein said exposing of b) and c) comprises PCR, and wherein said PCR is a multiplex PCR reaction.
22. The method of claim 15 , wherein said culture of cells from a first species is a cultured human skin tissue.
23. The method of claim 22 , wherein said cultured human skin tissue comprises NIKS cells.