Kit for together detecting multiple target nucleic acids differing from each other and detection method using the same
Provided is a kit for detecting multiple target nucleic acids capable of simultaneously amplifying and detecting multiple genes by means of one reaction vessel containing one kind of reaction solution and one kind of labels. Solution may contain first target nucleic acid ( 10 ) and second target nucleic acid ( 20 ) each of which dissociates at denaturation temperature T 0 . The solution further contains: DNA polymerase ( 30 ); a first target's primer ( 13 ) at annealing temperature T 1 bonding with first single strands derived from the first target nucleic acid; a second target's primer ( 23 ) at annealing temperature T 1 bonding with second single strands derived from the second target nucleic acid; a first target's probe ( 15 ) at annealing temperature T 1 bonding with the first single strands derived from the first target nucleic acid; and a second target's probe ( 25 ) at second target detection temperature T 3 which is lower than the annealing temperature T 1 and elongation temperature T 2 bonding with the second single strands derived from the second target nucleic acid. A condition that: T 0 is higher than T 2 ; T 2 is not lower than T 1 ; and T 1 is higher than T 3 is satisfied.
1 . A detection method for detecting a first double stranded target nucleic acid and a second double stranded target nucleic acid differing from each other, comprising the steps of:
(a) subjecting a composition to an initial denaturation temperature, T 0 , wherein the composition comprises:
(i) a first target's primer for specifically hybridizing a first target nucleic acid and a second target's primer for specifically hybridizing a second target nucleic acid;
(ii) a first labeled probe for specifically detecting a first target nucleic acid and a second labeled probe for specifically detecting a second target nucleic acid, wherein the label of the first and second probe are identical; and
(iii) double stranded first target nucleic acid and double stranded second nucleic acid and
(iv) DNA polymerase and deoxyribonucleoside triphosphate;
(b) subjecting the composition to amplification of n cycles, wherein each cycle comprises the steps of:
(b1) dissociating the first double stranded target nucleic acid into single strands and denaturing the second double stranded target nucleic acid into single strands by subjecting the composition to the denaturation temperature, T 0 , and measuring the fluorescence signal from the first probe and second probe;
(b2) decreasing the temperature to an annealing temperature, T 1 , annealing the first target primer to each single strand of the first target nucleic acid and annealing the second target primer to each single strand of the second target nucleic acid and measuring the fluorescence signal of the first probe;
(b3) elongating the first target primer and the second target primer at an elongation temperature, T 2 , and measuring the fluorescence signal of the first probe;
(c) subjecting the composition to a step to detect the second target after the amplification cycles are complete, comprising the steps of:
(c1) increasing the temperature of the composition to denaturing temperature T 0 and measuring the fluorescence intensity of the second probe; and
(c2) decreasing the temperature of the composition and measuring the fluorescence signal from the second probe at a second target detection temperature, T 3 ;
(d) comparing the fluorescence of the first probe to a first threshold value and the fluorescence of the second probe to a second threshold value comprising:
(d1) a first comparing step of comparing a first value of a measurement sample with a first threshold so as to judge that the first target nucleic acid is positive or negative,
wherein said first threshold is a fluorescence intensity at the final amplification cycle for a negative reference sample;
(d2) a second comparing step of comparing a second value of the measurement sample with a second threshold so as to judge that the second target nucleic acid is positive or negative,
wherein said second threshold is a fluorescence intensity for a reference sample negative for the second target nucleic acid;
and
a step of capturing a difference caused by changes of the first and second fluorescent signals according to the second labeling substance so as to judge existence or non-existence of the second target nucleic acid;
wherein T 0 >T 2 ≥T 1 >T 3 .
2 . The detection method of claim 1 , wherein for every cycle of amplification reaction and the second target detecting step, calculation is carried out according to formulae, including:
fn=fhyb.n/fden.n (Formula 1); and
fe=fhyb.e/fden.e (Formula 1′),
wherein:
fn: a fluorescence intensity value in an n-th cycle calculated according to Formula 1;
fhyb.n: a fluorescence intensity value at an elongation step in the n-th cycle;
fden.n: a fluorescence intensity value at a denaturation step in the n-th cycle;
fe: a fluorescence intensity value at the second target detecting step calculated according to Formula 1′;
fhyb.e: a fluorescence intensity value (55 Centigrade) at the second target detecting step; and
fden.e: a fluorescence intensity value (95 Centigrade) at the second target detecting step.
3 . The detection method of claim 2 , wherein the formulae further include:
Fn=fn/f 10 (Formula 2); and
Fe=fe/f 10 (Formula 2′),
wherein:
Fn: a relative value in the n-th cycle assuming that a fluorescence intensity value in a tenth cycle obtained according to Formula 1 is equal to a value of “1”; and
Fe: a relative value at the second target detecting step assuming that the fluorescence intensity value in the tenth cycle obtained according to Formula 1′ is equal to a value of “1”.
4 . The detection method of claim 1 , wherein the fluorescent signals are shown by quenching light when the annealing occurs.
5 . The detection method of claim 1 , wherein the fluorescent signals are shown by emitting light when the annealing occurs.
6 . The detection method of claim 1 , wherein the first target nucleic acid is a Mycoplasma pneumoniae P1 genes and the second target nucleic acid is internal control composition.
7 . The detection method of claim 1 , wherein the first target nucleic acid is a Chlamydia trachomatis endogenous plasmid gene and the second target nucleic acid is a Neisseria gonorrhoeae CMT gene.