Method for detecting nucleic acid sequences using competitive amplification

A method for detecting the amount of at least one target nucleic acid sequence which may be present in a test sample, comprising the steps of:

• (a) performing a nucleic acid amplication reaction with said test sample by contacting said test sample with:
• (i) two target primer sets, wherein a first primer set represents a first target sequence and a second primer set represents a second target sequence within the genome of the target nucleic acid sequence, and wherein each primer set comprises four oligonucleotide primers of which two are sense primers and the other two are antisense primers,
• (ii) an internal standard (IS) sequence, wherein the IS sequence comprises a chimeric molecule wherein said chimeric molecule comprises a sequence from either a first or second half of one of the target nucleic acid sequences and wherein the sequence of said chimeric molecule is colinear with a sequence from either the first or second half of the other target nucleic acid sequence such that a combination of the first target nucleic acid sequence and the second target nucleic acid sequence is formed,
• (iii) an IS primer set, wherein the IS primer set comprises four oligonucleotide primers of which two are derived from the first target nucleic acid sequence and the other two are derived from the second target nucleic acid sequence, and
• (iv) a set of dNTPs, under hybridizing conditions such that, for each pair of target oligonucleotide primer sequences or IS oligonucleotide primer sequences which hybridizes to each strand of said target nucleic acid sequences or said IS sequence or its complementary strand, a ligase chain reaction takes place whereby a nucleotide gap between the oligonucleotide primer sequences is filled by DNA polymerase in the presence of said dNTPs and in the presence of the target nucleic acid sequence, wherein each of the polymerase reactions require the same set of dNTPs, or a subset thereof, to fill the nucleotide gap resulting in juxtaposed 3' and 5' ends which are joined by DNA ligase, and
• (b) determining the ratio of target amplification products to IS amplification products present in the test sample as a measure of the amount of target nucleic acid sequence present in the test sample.