Multiplex amplification and detection

Abstract

The invention relates to the field of multiplex amplification. In particular, the invention relates to methods for assaying a sample for one or more nucleic acid targets in a single reaction based on the distinct melting temperatures or melting profiles of primers and/or probes. The invention also provides probes and kits for use in such methods.

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, and 
 wherein 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 T m 1 in terms of its double-stranded portion, 
 wherein a second probe of said at least two of the probes has a melting temperature T m 2 in terms of its double-stranded portion, 
 wherein T m 1>T m 2, 
 wherein the same labels are independently attached to the first and second probes, 
 wherein a reduction of any melting peak at T m 1 and/or T m 2 provides indication of consumption of the first and/or second probe(s). 
   
     
     
         2 . A method according to  claim 1 , wherein said melting profile is measured before reaction/amplification takes place (pre-amplification melting profile), and/or is measured after completion of reaction/amplification (post-amplification melting profile), and/or is measured during reaction/amplification at each cycle or selected cycles (mid-amplification melting profile),
 wherein said method additionally comprises step (d)
 (i) comparing at least two melting profiles obtained in (c) 
 and/or 
 (ii) comparing a melting profile obtained in step (c)
 with a previously-obtained melting profile of the same probes or 
 with a melting profile of the same probes obtained in parallel at the same time in control reactions, or 
 with a theoretical melting profile of the same probes 
 
   
       wherein a change in the melting profile provides an indication of whether or not at least one target, nucleic acid is present in said sample/reaction mixture,
 wherein said the pre-amplification melting profile is measured in the same reaction vessel before the start of reaction/amplification, or is measured in a separate reaction vessel where no amplification takes place due to that reaction mixture lacking one or more ingredients necessary for the reaction/amplification, 
 wherein in step (d) the post-amplification or mid-amplification melting profile is compared with the pre-amplification melting profile of the duplex of probes to determine whether a particular probe is consumed, this being indicative of the presence of the corresponding target in the sample. 
 
     
     
         3 . A method according to any one of the preceding claims, wherein as least one detectable label is a fluorescent label, wherein step (b) further comprises the step (b1) obtaining cycle by cycle fluorescence emissions (FE) at various measuring temperatures (MT), wherein said fluorescence emissions (FE) is a baseline corrected fluorescence (dR). 
     
     
         4 . A method according to  claim 3 , wherein when said amplification reaction mixture comprises “n” probes for multiplex detection of “n” nucleic acid targets, wherein first probe has a melting temperature T m 1, second probe has a melting temperature T m 2, third probe has a melting temperature of T m 3, n-th probe has a melting temperature T m n, wherein T m 1>T m 2>T m 3 . . . >T m n, the percentages of the double-stranded form of each probe at a particular temperature or different temperatures are determined experimentally or are calculated in theory by a computer program, wherein a first fluorescence emission FEa is obtained at a measuring temperature MTa, at which more than 50% of first probe is in duplex form, second fluorescence emission FEb is obtained at a measuring temperature MTb, at which more than 50% of second probe is in duplex form, n−1 fluorescence emission FE(n−1) is obtained at a measuring temperature MT(n−1), at which more than 50% of (n−1) is duplex form, n-th fluorescence emission FEn is obtained at a measuring temperature MTn, at which more than 80% of n-th probe is in duplex form, and optionally a fluorescence emission FE0 is obtained at a measuring temperature MT0, at which no more than 10% of first probe is in duplex form, wherein n is a positive integer and n≧2. 
     
     
         5 . A method according to  claim 3 , wherein the step (b) further comprises the step (b2) determining cycle by cycle the Actual Consumed Amount (ACA) of fluorescence emission for each probe, wherein the Actual Consumed Amount of fluorescence emission of k-th probe is depicted as ACA k , wherein at a particular measuring temperature (MTa) the k-th probe has percentage (dska) % in ds (double-strand) form, the fluorescence emission FEa at this measuring temperature MTa contributed by the first probe will be (ds1a) %*(ACA 1 ), contributed by the second probe will be (ds2a) %*(ACA 2 ), contributed by the k-th probe will be (dska) %*(ACA k ), contributed by the n-th probe will be (dsna) %*(ACA n ), wherein the calculation of Actual Consumed Amount (ACA) uses the following formula: 
       at measuring temperature MTa, the total fluorescence emission will be
   FEa=(ACA1)*(ds1a) %+(ACA2)*(ds2a) %+(ACA3)*(ds3a) % . . . +(ACA n )*(ds n a) % 
 
       at measuring temperature MTb, the total fluorescence emission will be
   FEb=(ACA1)*(ds1b) %+(ACA2)*(ds2b) %+(ACA3)*(ds3b) % . . . +(ACA n )*(ds n a) % 
 
       At measuring temperature MTc, the total fluorescence emission will be
   FEc=(ACA1)*(ds1c) %+(ACA2)*(ds2c) %+(ACA3)*(ds3c) % . . . +(ACA n )*(ds n a) % 
 
       and so on, wherein the ACA for each probe can be calculated from the above formulas, wherein “*” denotes “multiply by”, “k” is positive integer, 1≦k≦n, and “n” is the number of probes. 
     
     
         6 . A method according to  claim 5 , wherein the Actual Consumed Amount of fluorescence emission of each probe is obtained through a computer program which performs the calculation at each measuring temperature at each cycle. 
     
     
         7 . 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 a reaction mixture comprising:
 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, and 
 wherein at least two of the probes comprise the same detectable label or different detectable labels with undistinguishable emission spectra and wherein the melting characteristics (melting temperature T m ) of the double-stranded portions between the first and second oligonucleotides of each of such probes are different, and are distinguishable in a melting profile analysis; 
   (b) performing the reaction on the sample/reaction mixture, wherein the reaction is a primer extension reaction under extension conditions, wherein, when a target nucleic acid is present, the first oligonucleotides of the corresponding probe which are extendable primers, are hybridised with target nucleic acid, therefore being consumed during the primer extension reaction, 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 the 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.   
     
     
         8 . 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 a hybridisation reaction mixture comprising:
 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, and 
 wherein 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 and are distinguishable in a melting profile analysis; 
   (b) perforating the hybridisation reaction on the sample/reaction mixture under hybridisation 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 target nucleic acid, therefore being consumed during the reaction, 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.   
     
     
         9 . A method according to  claim 1 , wherein said amplification is an isothermal amplification or a thermal cycling amplification reaction comprising two or more denaturing, annealing, and primer extension steps. 
     
     
         10 . A method for monitoring a PCR amplification of at least two nucleic acid targets, 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 a 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 fluorescent label or fluorescent label/quencher pair which is 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, and 
 wherein at least two of the probes comprise the same detectable label or different detectable labels with undistinguishable emissions 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 which comprises thermal cycling of the sample/amplification reaction mixture
 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 consumed during the amplification reaction; and 
 wherein the step (b) further comprises the step (b1) obtaining cycle by cycle fluorescence emissions (FE) at various measuring temperatures (MT), wherein said fluorescence emissions (FE) is baseline corrected fluorescence (dR), 
 wherein when said amplification reaction mixture comprises “n” probes for multiplex detection of “n” nucleic acid targets, wherein first probe has a melting temperature of T m 1, second probe has a melting temperature of T m 2, third probe has a melting temperature of T m 3, n-th probe has a melting temperature of T m n, wherein T m 1>T m 2>T m 3 . . . >T m n, wherein the percentages of the double-stranded form of each probe at a particular temperature or different temperatures are determined experimentally or are calculated in theory by a computer program, wherein a first fluorescence emission FEa is obtained at a measuring temperature MTa, at which more than 50% of first probe is in duplex form, second fluorescence emission FEb is obtained at a measuring temperature MTb, at which more than 50% of second probe is in duplex form, n−1 fluorescence emission FE(n−1) is obtained at a measuring temperature MT(n−1), at which more than 50% of (n−1)th probe is in duplex form, n-th fluorescence emission FEn is obtained at a measuring temperature MTn, at which more than 80% of n-th probe is in duplex form, and optionally a fluorescence emission FE0 is obtained at a measuring temperature MT0, at which no more than 10% of first probe is in duplex form, wherein n is a positive integer and n≧2. 
 wherein the step (b) further comprises the step (b2) determining cycle by cycle the Actual Consumed Amount of fluorescence emission for each probe, wherein the Actual Consumed Amount of fluorescence emission of k-th probe is depicted as ACA k , wherein at a particular measuring temperature (MTa) said k-th probe has percentage (dska) % in ds (double-strand) form, the fluorescence emission FEa at this measuring temperature MTa contributed by the k-th probe will be (dska) %*(ACA k ), contributed by the n-th probe will be (dsna) %*(ACA n ), wherein the calculation of Actual Consumed Amount (ACA) uses the following formula: 
   
       at measuring temperature MTa, the total fluorescence emission will be
   FEa=(ACA1)*(ds1a) %+(ACA2)*(ds2a) %+(ACA3)*(ds3a) % . . . +(ACA n )*(ds n a) % 
 
       at measuring temperature MTb, the total fluorescence emission will be
   FEb=(ACA1)*(ds1b) %+(ACA2)*(ds2b) %+(ACA3)*(ds3b) % . . . +(ACA n )*(ds n a) % 
 
       At measuring temperature MTc, the total fluorescence emission will be
   FEc=(ACA1)*(ds1c) %+(ACA2)*(ds2c) %+(ACA3)*(ds3c) % . . . +(ACA n )*(ds n a) % 
 
       and so on, wherein the individual ACA can be calculated from the above formulas, wherein “*” denotes “multiply by”, “k” is positive integer, 1≦k≦n, and “n” is the number of probes
 wherein the Actual Consumed Amount of fluorescence emission of each probe is obtained though a computer program which performs the calculation at each measuring temperature at each cycle 
 and wherein the Actual Consumed Amount of fluorescence emission of each probe is related to the degree of amplification of the target nucleic acid to which the first oligonucleotide of that probe binds. 
 
     
     
         11 . A method according to any one of the preceding claims, wherein said consumption of probes is achieved through hybridisation of the first oligonucleotide of the probe to the target sequence, which is followed by the incorporation of the first oligonucleotide of the probe into the amplified product, wherein when the first oligonucleotide of the probe can be incorporated into the amplified product, the first oligonucleotide is extendable primer or is one of the pair of forward/reverse oligonucleotide primers. 
     
     
         12 . A method according to any one of the preceding claims, wherein said consumption of probes is achieved through hybridisation of the first oligonucleotide of the probe to the target sequence, which is followed by degradation of the first and/or second oligonucleotide of the probe, wherein when the first oligonucleotide of the probe is degraded during the reaction, the reaction mixture comprises double-strand dependent nuclease activity. 
     
     
         13 . A method according to any one of the preceding claims, wherein the probe comprises a first label and a second label, wherein the first label is a fluorophore and the second label is a quencher, or vice versa. 
     
     
         14 . A method according to  claim 13 , wherein the first label is attached to the first oligonucleotide and the second label is attached to the second oligonucleotide such that said first label and second label are in close proximity when the probe's internal duplex is formed. 
     
     
         15 . A method according to  claim 13 , wherein labels are on one oligonucleotide of the probe, either the first oligonucleotide or the second oligonucleotide. 
     
     
         16 . A method according to  claim 15 , wherein the first oligonucleotide of the probe does not comprise a label, and the second oligonucleotide of the probe comprises at least one, preferably two, labels, wherein the second oligonucleotide comprises a first label and a second label, the first label being attached at or near one end of second oligonucleotide and the second label is attached at or near the other end of the second oligonucleotide, whereby when the second oligonucleotide is not hybridised to the first oligonucleotide, the second oligonucleotide is in a random-coiled or a stem-loop structure which brings the first label and second label into close proximity. 
     
     
         17 . A method according to  claim 15 , wherein the first oligonucleotide does not comprise a label and the second oligonucleotide comprises a label wherein, when the second oligonucleotide hybridises to the first oligonucleotide to form the double-stranded portion of the probe, the label is capable of changing the signal emission relative to the emission of the single-stranded form of the second oligonucleotide. 
     
     
         18 . A method according to  claim 15 , wherein the first oligonucleotide of the probe does not comprises a label and wherein the probe comprises two second oligonucleotides which are capable of hybridising adjacently or substantially adjacently to different parts of the second region of the first oligonucleotide, wherein one of the second oligonucleotides is attached with a first label and the other second oligonucleotide is attached with a second label, such that when the two second oligonucleotides are hybridised to the first oligonucleotide, the two labels are brought into close proximity and one label affects the signal from the other. 
     
     
         19 . A method according to  claim 13 , wherein the first and second oligonucleotides of a probe are joined by a linker moiety which comprises nucleotides or a non-nucleotide chemical linker, allowing the first oligonucleotide and second oligonucleotide to form a stem-loop structure, wherein the first and second oligonucleotides are labelled with a first and second label, respectively, such that, when the probe forms an internal stem-loop structure, the labels are brought into close proximity and one label affects the signal from the other. 
     
     
         20 . A method according to any one of the preceding claims, wherein said first region of said first oligonucleotide is not substantially overlapping with second region of said first oligonucleotide. 
     
     
         21 . A method according to any one of the preceding claims, wherein said first region of said first oligonucleotide is substantially overlapping with the second region of said first oligonucleotide or the second region is embedded in the first region, wherein the T m  of the duplex of said first oligonucleotide hybridised with the target sequence is higher than the T m  of the duplex of said first oligonucleotide hybridised with the second oligonucleotide such that if a target is present, the first oligonucleotide forms stronger hybrids with the target and consequently melts at a higher temperature than the first/second oligonucleotide duplex. 
     
     
         22 . A method according to any one of the preceding claims, wherein said first oligonucleotide comprises a third region which is identical or substantially identical to the sequence of a primer which is used in the amplification reaction. 
     
     
         23 . A method according to any one of the preceding claims, wherein both the first and second oligonucleotides of probes are capable of being consumed during amplification. 
     
     
         24 . A method according to any one of the preceding claims, wherein said first oligonucleotide is blocked at the 3′ end, and wherein said second oligonucleotide is blocked at the 3′ end. 
     
     
         25 . A method for assaying a sample for one or more variant nucleotides on the target nucleic acids, said method comprising:
 (a) contacting a sample comprising target nucleic acids with 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) at least one pair of probes, wherein first probe in the pair comprises sequence complementary to wild-type target nucleic acid sequence, second probe in the pair comprises sequence complementary to the target nucleic acid sequence containing variant nucleotides, wherein each probe in the pair 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 in the pair comprise the same second oligonucleotide, 
 
 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, and 
 wherein 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/amplification reaction mixture 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 consumed during the amplification reaction; and   (c) measuring, at least once, the melting profile of the double-stranded portions between the first and second oligonucleotides of unconsumed probes 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 has been amplified in said sample/amplification reaction mixture.   
     
     
         26 . A method according to  claim 25 , wherein the same second oligonucleotide in the pair of the probes comprise universal base or Inosine which corresponds to the variant nucleotide in the target nucleic acid sequence, wherein said universal base is 3-nitropyrrole 2′-deoxynucleoside, 5-nitroindole, pyrimidine analog or purine analog. 
     
     
         27 . A method according to any one of the preceding claims, wherein multiple first oligonucleotides of different probes hybridise to different sites of the same strand of a target sequence. 
     
     
         28 . A computer software product for use with a method according to any one of the preceding claims, when run on suitable data processing means, for comparing melting profiles of probes and/or quantifying a real time PCR amplification of multiple targets which, when executed by a computer processor, performs the calculation of Actual Consumed Amount (ACA) using the following formula: 
       at measuring temperature MTa, the total fluorescence emission will be
   FEa=(ACA1)*(ds1a) %+(ACA2)*(ds2a) %+(ACA3)*(ds3a) % . . . +(ACA n )*(ds n a) % 
 
       at measuring temperature MTb, the total fluorescence emission will be
   FEb=(ACA1)*(ds1b) %+(ACA2)*(ds2b) %+(ACA3)*(ds3b) % . . . +(ACA n )*(ds n a) % 
 
       At measuring temperature MTc, the total fluorescence emission will be
   FEc=(ACA1)*(ds1c) %+(ACA2)*(ds2c) %+(ACA3)*(ds3c) % . . . +(ACA n )*(ds n a) % 
 
       and so on, wherein the individual ACA can be calculated from the above formulas, wherein “*” denotes “multiply by”, “k” is positive integer, 1≦k≦n, and “n” is the number of probes 
     
     
         29 . A computer system comprising a computer memory having a computer software program stored therein, wherein the computer software program, when executed by a processor or in a computer, performs a method according to any one of  claims 1  to  27 . 
     
     
         30 . A computer system according to  claim 29 , wherein the computer software program performs a method comprising the step of calculating the Actual Consumed Amount of fluorescence emission t of each probe and/or determination of features of melting profiles during amplification or at the end amplification. 
     
     
         31 . A kit for assaying for one or more nucleic acid targets, which kit comprises a probe comprising:
 a first oligonucleotide of 15-150 nucleotides 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 of 4-150 nucleotides 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 composes 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,   
       and wherein
 (a) the first oligonucleotide of the probe does not comprise a label, the second oligonucleotide comprises a first label and a second label wherein the first label is attached at or near one end of second oligonucleotide and the second label is attached at or near the other end of the second oligonucleotide, whereby when the second oligonucleotide is not hybridised with the first oligonucleotide, the second oligonucleotide is in a random-coiled or a stem-loop structure which brings the first label and second label in close proximity and wherein when the second oligonucleotide is hybridised with the first oligonucleotide, the two labels are held away from each other; or 
 (b) the first oligonucleotide does not comprise a label and the second oligonucleotide comprises a label, wherein when the second oligonucleotide hybridises in the first oligonucleotide to form the double-stranded portion of the probe, the label is capable of changing its detectable signal emission relative to the emission of the label when in the single-stranded form of the second oligonucleotide; or 
 (c) the first oligonucleotide of the probe does not comprises a label, the probe comprises two second oligonucleotides which are capable of hybridising adjacently or substantially adjacently to different parts of the second region of the first oligonucleotide, wherein one of the second oligonucleotides is attached with a first label, and the other second oligonucleotide is attached with a second label, such that when the two second oligonucleotides are hybridised to the first oligonucleotide, the two labels are brought in close proximity and one label effects the signal from the other. 
 
     
     
         32 . A kit for assaying for one or more nucleic acid targets, which kit comprises a probe mixture containing two or more probes, wherein each probes comprises:
 a first oligonucleotide of 15-150 nucleotides 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 of 4-150 nucleotides 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,   
       and wherein
 (a) the first label is attached to the second region of the first oligonucleotide and the second label is attached to the region of the second oligonucleotide which is complementary to the second region of the first oligonucleotide such that the first and second labels are brought into close proximity upon formation of the probe's internal duplex, or 
 (b) the first oligonucleotide of the probe does not comprise a label, the second oligonucleotide comprises a first label and a second label, wherein the first label is attached at or near one end of second oligonucleotide and the second label is attached at or near the other end of the second oligonucleotide, whereby when the second oligonucleotide is not hybridised with the first oligonucleotide, the second oligonucleotide is in a random-coiled or a stem-loop structure which brings the first label and second label in close proximity and wherein when the second oligonucleotide is hybridised with the first oligonucleotide, the two labels are held away from each other; or 
 (c) the first oligonucleotide does not comprise a label and the second oligonucleotide comprises a label, wherein when the second oligonucleotide hybridises to the first oligonucleotide to form the double-stranded portion of the probe, the label is capable of changing its detectable signal emission relative to the emission of the label when in the single-stranded form of the second oligonucleotide; or 
 (d) the first oligonucleotide of the probe does not comprises a label, the probe comprises two second oligonucleotides which are capable of hybridising adjacently or substantially adjacently to different parts of the second region of the first oligonucleotide, wherein one of the second oligonucleotides is attached with a first label, and the other second oligonucleotide is attached with a second label such that when the two second oligonucleotides are hybridised to the first oligonucleotide, the two labels are brought in close proximity and one label affects the signal from the other; or 
 (e) the first and second oligonucleotides of a probe are joined by a linker moiety which comprises nucleotides or a non-nucleotide chemical linker, allowing the first oligonucleotide and second oligonucleotide to form a stem-loop structure, wherein the first and second oligonucleotides are labelled with a first and second label, respectively, such that, when the probe forms an internal stem-loop structure, the labels are brought into close proximity and one label affects the signal from the other;
 and wherein 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. 
 
 
     
     
         33 . A kit according to  claim 31  or  claim 32 , wherein at least one label is a fluorescent label. 
     
     
         34 . A kit according to  claim 31 , parts (a) or (c) or  claim 32  parts (a), (b), (d) or (e), wherein the probe comprises a first label and a second label. 
     
     
         35 . A kit according to  claim 34 , wherein the first label is a fluorophore and the second label is a quencher, or vice versa. 
     
     
         36 . Use of a probe as defined in any one of parts (a)-(c) of  claim 31 , a probe mixture of  claim 32 , in a method as defined in any one of  claims 1 - 27 . 
     
     
         37 . A method according to any one of  claims 1  to  27 , substantially as described herein and/or with reference to the Examples.