PCR and qPCR method for detection of a Bt11 nucleic acid and a maize adh1 gene in a transgenic corn sample

摘要

Discloses a PCR based method for detection and quantification of Bt11 event DNA in a biological sample of corn nucleic acids, the method comprising: (a) contacting the biological sample with a first primer pair (SEQ ID NO: 1 and SEQ ID NO: 2) and a fluorescent dye labelled probe (SEQ ID NO: 3), wherein the primers when used in a nucleic acid amplification reaction with corn genomic DNA, generate a first amplicon (SEQ ID NO: 4) indicative of the presence of Bt11 event DNA; (b) contacting said biological sample with a second primer pair (SEQ ID NO: 5 and SEQ ID NO: 6), and a second fluorescent dye labelled probe (SEQ ID NO: 7), wherein the primers when used in nucleic acid amplification reaction with corn genomic DNA, generate a second amplicon (SEQ ID NO: 8) indicative of the presence of a maize adh1 gene; (c) providing a nucleic acid amplification reaction condition and a PCR machine capable of performing 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 (d) calculating a relative amount of said first amplicon compared to said second amplicon, where the amount of said first amplicon is indicative of the quantity of Bt11 DNA in said biological sample. Further discloses statistical requirements of the method.