| 主权项 |
1. A method for assaying a sample for one or more target nucleic acids, said method comprising:
(a) contacting a sample comprising one or more target nucleic acids with an amplification reaction mixture comprising:
(i) one or more pairs of forward/reverse oligonucleotide primers, wherein the primer pairs are capable of amplifying one or more target nucleic acids, if present in the sample,(ii) two or more probes, wherein each probe comprises
a first oligonucleotide which comprises a first region which is substantially complementary to part of one target nucleic acid and a second region, and at least one second oligonucleotide which comprises a region which is substantially complementary to the second region of the first oligonucleotide, such that the first and second oligonucleotides are capable of forming a double-stranded portion,wherein each probe comprises a detectable label or detectable combination of labels which is/are capable of producing a changeable signal which is characteristic of the presence or absence of a double-stranded portion between the first and second oligonucleotides of that probe, andwherein at least two of the probes comprise the same detectable label or different detectable labels with undistinguishable emission spectra and wherein the melting characteristics of the double-stranded portions between the first and second oligonucleotides of each of such probes are different; (b) performing an amplification reaction on the sample/reaction mixture under amplification conditions, wherein, when a target nucleic acid is present, the first oligonucleotides of probes which are substantially complementary to part of that target nucleic acid are hybridised with the target nucleic acid, therefore being consumed, wherein the consumed oligonucleotides of probes are no longer able to participate in forming double stranded portion (the duplex) of the probe, and (c) measuring, at least, once, the melting profile of the double-stranded portions between the first and second oligonucleotides of unconsumed probes in the reaction mixture by detecting the signal(s) from the labels in those probes as a function of temperature, wherein the melting profile provides an indication of whether or not at least one target nucleic acid is present in said sample,
wherein a first probe of said at least two of the probes has a melting temperature Tm1 in terms of its double-stranded portion,wherein a second probe of said at least two of the probes has a melting temperature Tm2 in terms of its double-stranded portion,wherein Tm1>Tm2,wherein the same labels are independently attached to the first and second probes,wherein a reduction of any melting peak at Tm1 and/or Tm2 provides indication of consumption of the first and/or second probe(s). |
