Patent ID: 7709232
Filing Date: 2010-05-04
Classification: A61P,C12N,C12Q,Y02A

Abstract:
1. An in vitro quantitative detection method for determining the content ratio of a selected genetic recombinant line of a species in a sample which may contain the selected genetic recombinant line comprising: (i) preparing a first DNA by extraction of the DNA from the sample; (ii) preparing a standard molecule, which is a recombinant DNA molecule comprising a recombinant DNA sequence present only in said selected genetic recombinant line, and an endogenous DNA sequence of a non-recombinant line of the species, the endogenous DNA sequence being common to the selected recombinant line of the species and the non-recombinant line of the species, when the selected recombinant line exists in the sample; (iii) selecting the selected genetic recombinant line of the species from the sample and selecting a single DNA sequence specific for the selected genetic recombinant line when the selected genetic recombinant line is in the sample; (iv) preparing a second DNA, by extraction of the DNA from the selected genetic recombinant line of step (iii) when the selected genetic recombinant line is in the sample; (v) performing a quantitative polymerase chain reaction (PCR) using the second DNA as a template and a probe specifically hybridized to DNA sequence specific for the selected genetic recombinant line of step (iii), performing a quantitative PCR and preparing a first standard curve of the DNA sequence specific for the selected genetic recombinant line using sets of defined quantities of the standard molecule and the probe specifically hybridized to the DNA sequence specific for the selected genetic recombinant line, and determining the number of molecules of the DNA sequence specific for the selected genetic recombinant line in the second DNA using the first standard curve; (vi) performing a quantitative PCR using the second DNA as a template and a probe of the endogenous DNA sequence, performing a quantitative PCR and preparing a second standard curve using sets of defined quantities of the standard molecule and the probe of the endogenous DNA sequence, and determining the number of molecules of the endogenous DNA sequence in the second DNA using the second standard curve; (vii) performing a quantitative PCR using the first DNA as a template and the probe in step (v), and determining the number of molecules of the DNA sequence specific for the selected genetic recombinant line in the first DNA of step (i) using the first standard curve; (viii) performing a quantitative PCR using the first DNA as a template and the probe in step (vi), and determining the number of molecules of the endogenous DNA sequence in the first DNA extract of step (i) using the second standard curve; and (ix) determining the content ratio of the selected genetic recombinant line of the species in the sample according to formula (I):