Methods for detecting contamination in molecular diagnostics using PCR
View Patent ↗The invention provides methods for detecting contamination in a PCR reaction. Methods of the invention are especially useful for detection of contamination in heterogeneous samples containing a rare nucleic acid to be detected.
1. A method for detecting cross-sample contamination in an amplification reaction, said method comprising the steps of:
conducting an amplification reaction in a first nucleic acid sample, using at least one chimeric primer comprising a first portion that hybridizes with at least a portion of a target nucleic acid, the amplification of which is desired, and a second, contamination detection portion that does not hybridize with said target nucleic acid;
conducting a subsequent control amplification reaction in a second nucleic acid sample, using at least one primer to amplify specifically said contamination detection portion of said chimeric primer; and
determining whether said second sample has been contaminated by an amplicon from said first sample by determining whether said control reaction produces an amplicon.
2. The method of claim 1 , wherein said second portion is 5′ to said first portion in each of said at least one chimeric primers.
3. The method of claim 1 , wherein said at least one primer in said control reaction is not complementary to any contiguous nucleic acid sequence in any target nucleic acid in said second sample.
4. The method of claim 1 , wherein said at least one primer used in said control reaction is substantially complementary to said contamination detection portion.
5. The method of claim 1 , wherein said at least one primer used in said control reaction is substantially identical to said contamination detection portion.
6. The method of claim 1 , wherein at least one of said amplification reactions is selected from the group consisting of PCR, quantitative PCR, and reverse-transcriptase PCR.
7. The method of claim 1 , wherein said samples comprise a heterogeneous population of nucleic acids.
8. The method of claim 7 , wherein said samples comprise a stool sample.
9. The method of claim 7 , wherein said samples comprise a blood sample.
10. The method of claim 1 , wherein said contamination detection portion is about 20 nucleotides.
11. The method of claim 1 , wherein said nucleic acid comprises DNA.
12. The method of claim 1 , wherein said determination step comprises using a sequence-specific nucleic acid probe to capture said amplicon from said first sample.