Wide dynamic range nucleic acid detection using an aggregate primer series
First Claim
1. A method for determining an approximate amount of a target nucleic acid sequence in a test sample comprising the steps of:
- (a) contacting said test sample with a nucleic acid amplification reaction mixture comprising a first primer set capable of hybridizing with a first sub-target region of said target nucleic acid sequence, and a second primer set capable of hybridizing with a second sub-target region of said target nucleic acid sequence, wherein said first and said second sub-target regions are different and wherein said first and said second primer sets are selected such that(i) said first primer set is capable of producing a detectable amplicon at or above a first pre-selected threshold concentration of said target nucleic acid sequence,(ii) said second primer set is capable of producing a detectable amplicon at or above a second pre-selected threshold concentration of said target nucleic acid sequence, and(iii) said first and second threshold concentrations are different from one another;
(b) subjecting the reaction mixture to amplification conditions sufficient to produce a detectable amplicon from said primer sets when said test sample contains a concentration of target nucleic acid sequence which is at or above the different threshold concentrations at which the primer sets are capable of producing a detectable amplicon; and
(c) detecting whether amplicons are produced in said reaction mixture from said primer sets so as to determine whether the test sample contains a concentration of target nucleic acid sequence at or above said threshold concentrations corresponding to said primer sets.
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Abstract
The present invention provides a method of detecting the amount of a target sequence which may be present in a test sample. The method uses an aggregate primer series, which comprises at least two primer sets, in an amplification reaction to detect the relative concentration of a target sequence which may be present in a test sample. The primer sets have different sensitivities and hybridize with sub-target sequences which are different regions of the target sequence. The method generally comprises cycling a test sample suspected of containing a target sequence, an aggregate primer series, and means necessary for performing an amplification reaction; and detecting any amplified sub-target sequences. Based on a qualitative detection of the amplified sub-target sequences generated by individual primer sets, the relative quantity of the target sequence can be determined.
131 Citations
13 Claims
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1. A method for determining an approximate amount of a target nucleic acid sequence in a test sample comprising the steps of:
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(a) contacting said test sample with a nucleic acid amplification reaction mixture comprising a first primer set capable of hybridizing with a first sub-target region of said target nucleic acid sequence, and a second primer set capable of hybridizing with a second sub-target region of said target nucleic acid sequence, wherein said first and said second sub-target regions are different and wherein said first and said second primer sets are selected such that (i) said first primer set is capable of producing a detectable amplicon at or above a first pre-selected threshold concentration of said target nucleic acid sequence, (ii) said second primer set is capable of producing a detectable amplicon at or above a second pre-selected threshold concentration of said target nucleic acid sequence, and (iii) said first and second threshold concentrations are different from one another; (b) subjecting the reaction mixture to amplification conditions sufficient to produce a detectable amplicon from said primer sets when said test sample contains a concentration of target nucleic acid sequence which is at or above the different threshold concentrations at which the primer sets are capable of producing a detectable amplicon; and (c) detecting whether amplicons are produced in said reaction mixture from said primer sets so as to determine whether the test sample contains a concentration of target nucleic acid sequence at or above said threshold concentrations corresponding to said primer sets. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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Specification