Method for quantifying DNA in a biological sample
View Patent ↗Improved methods of quantifying nucleic acids that are unique to a transgenic corn event designated Bt11 in a biological sample and compositions thereof are disclosed. The invention further relates to primer pairs used in the method that are unique to event Bt11.
1. A method of quantifying event Bt11 DNA in a biological sample comprising corn nucleic acids, the method comprising:
(a) contacting said biological sample with a first pair of primers, comprising a first primer consisting of SEQ ID NO: 1 and a second primer consisting of SEQ ID NO: 2, and a fluorescent dye labeled probe comprising SEQ ID NO: 3, wherein said first pair of primers, when used in a nucleic-acid amplification reaction with genomic DNA from event Bt11 corn, produce a first amplicon comprising SEQ ID NO: 4, and wherein said first amplicon is diagnostic for event Bt11;
(b) contacting said biological sample with a second pair of primers, comprising a first primer consisting of SEQ ID NO: 5 and a second primer consisting of SEQ ID NO: 6, and a second fluorescent dye labeled probe comprising SEQ ID NO: 7, wherein said second pair of primers, when used in a nucleic-acid amplification reaction with corn genomic DNA, produce a second amplicon comprising SEQ ID NO: 8, and wherein said second amplicon is indicative of the presence of a maize adh1 gene;
(c) providing a nucleic acid amplification reaction condition and a PCR instrument capable of performing a quantitative real-time PCR;
(d) performing the quantitative real-time PCR using the primers and probes of (a) and (b), thereby producing said first amplicon and said second amplicon;
(e) detecting simultaneously said first amplicon and said second amplicon as they are produced by said PCR instrument; and
(f) calculating a relative amount of said first amplicon compared to said second amplicon, whereby the amount of said first amplicon is indicative of the quantity of Bt11 DNA in said biological sample.
2. The method of claim 1 , wherein said method has a limit of quantification (LOQ) of less than or equal to 0.08% Bt11 DNA concentration.
3. The method of claim 1 , wherein said method has a limit of detection (LOD) of less than or equal to 0.04% Bt11 DNA concentration.
4. The method of claim 1 , wherein said method has a mean coefficient of linearity (R 2 ) of at least 0.99.
5. The method of claim 1 , wherein said method has a relative reproducibility standard deviation (RSD R ) of 24% or less at a Bt11 DNA concentration of 0.090%.
6. The method of claim 1 , wherein said method has a relative repeatability standard deviation (RSD r ) of 17% or less at a Bt11 DNA concentration of 0.090%.
7. The method of claim 1 , wherein said method has a trueness value of ±5% or less across the entire dynamic range.