WO2017131463A1

WO2017131463A1 - Methods for providing signal for target nucleic acid sequence

Info

Publication number: WO2017131463A1
Application number: PCT/KR2017/000951
Authority: WO
Inventors: Jong Yoon Chun; Young Jo Lee; Han Bit LEE
Original assignee: Seegene, Inc. .
Priority date: 2016-01-26
Filing date: 2017-01-26
Publication date: 2017-08-03
Also published as: KR102110999B1; KR20180100446A

Abstract

The present invention is directed to a method for providing a signal for at least one target nucleic acid sequence in a sample from signals detected at different temperatures. The method of the present invention provides a signal for a target nucleic acid sequence with a signal extraction process only when particular criteria are satisfied, and provides a signal for a target nucleic acid sequence without a signal extraction process when a particular criterion is not satisfied. The method of the present invention can minimize the signal extraction process, thereby significantly reducing false positive results which may occur due to the signal extraction process. Accordingly, the method of the present invention can provide a signal for a target nucleic acid sequence using two detection temperatures in a more accurate and efficient manner, from which the presence or absence of a target nuclejc acid sequence can be determined.

Description

METHODS FOR PROVIDING SIGNAL FOR TARGET NUCLEIC ACID SEQUENCE

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from Korean Patent Application No. 2016- 0009509, filed on January 26, 2016, in the Korean Intellectual Property Office, the disclosure of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a method for providing a signal for at least one target nucleic acid sequence in a sample from signals detected at different temperatures.

BACKGROUND OF THE INVENTION

For detection of target nucleic acid sequences, real-time detection methods are widely used to detect target nucleic acid sequences with monitoring target amplification in a real-time manner. The real-time detection methods generally use t

labeled probes or primers specifically hybridized with target nucleic acid sequences. The exemplified methods by use of hybridization between labeled probes and target nucleic acid sequences include Molecular beacon method using dual-labeled probes with hairpin structure (Tyagi et al, Nature Biotechnology v.14 MARCH 1996),

HyBeacon method (French DJ et al., Mol. Cell Probes, 15(6): 363-374(2001)),

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Hybridization probe method using two probes labeled each of donor and acceptor

(Bernad et al, 147-148 Clin Chem 2000; 46) and Lux method using single-labeled oligonucleotides (U.S. Pat. No. 7,537,886). TaqMan method (U.S. Pat. Nos. 5,210,015 and 5,538,848) using dual-labeled probes and its cleavage by 5'-nuclease activity of

DNA polymerase is also widely employed in the art.

The exemplified methods using labeled primers include Sunrise primer method

(Nazarenko et al, 2516-2521 Nucleic Acids Research, 1997, v.25 no.12, and U.S. Pat.

No. 6,117,635), Scorpion primer method (Whitcombe et al, 804-807, Nature

l Biotechnology v.17 AUGUST 1999 and U.S. Pat. No. 6,326,145) and TSG primer method (WO 2011/078441).

As alternative approaches, real-time detection methods using duplexes formed depending on the presence of target nucleic acid sequences have been proposed: Invader assay (U.S. Pat. Nos. 5,691,142, 6,358,691 and 6,194,149), PTOCE (PTO cleavage AND extension) method (WO 2012/096523), PCE-SH (PTO Cleavage and Extension-Dependent Signaling Oligonucleotide Hybridization) method (WO 2013/115442), PCE-NH (PTO Cleavage and Extension-Dependent Non-Hybridization) method (WO 2014/104818).

The conventional real-time detection technologies described above detect signals generated from fluorescent labels at a selected detection temperature in signal amplification process associated with or with no target amplification. When a plurality of target nucleic acid sequences using a single type of label in a Single reaction tube are detected in accordance with the conventional real-time dete Iction technologies, generated signals for target nucleic acid sequences are not differentiated from each other. Therefore, the conventional real-time detection technologies generally employ different types of labels for detecting a plurality of target nucleic acid sequences. The melting analysis using Tm difference permits to detect a plurality of target nucleic acid sequences even using a single type of label. However, the melting analysis has serious shortcomings in that its performance time is longer than real-time technologies and design of probes with different Tm values becomes more difficult upon increasing target sequences.

Accordingly, where novel methods or approaches being not dependent on melting analysis are developed for detecting a plurality of target nucleic acid sequences using a single type of label in a single reaction vessel and a single type of detector, they enable to detect a plurality of target nucleic acid sequences with dramatically enhanced convenience, cost-effectiveness and efficiency. In addition, the combination of the novel methods with other detection methods {e.g., melting analysis) would result in detection of a plurality of target nucleic acid sequences using a single type of label in a. single reaction vessel with dramatically enhanced efficiency.

For this purpose, the present inventors have developed a novel method for detecting a plurality of target nucleic acid sequences using a single type of detector in

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a single reaction vessel (also referred to as 'MuDTl' technology; see WO 2015/147412). According to the method, the presence of the target nucleic acid i

sequence having a relatively low detection temperature can be determined by signals detected at a relatively high detection temperature and a relatively low detection temperature, and a reference value representing a relationship of change in signals at different detection temperatures. As a representative example, where the first target nucleic acid sequence is a target nucleic acid sequence having a relatively high detection temperature {i.e., capable of generating signals at both a relatively high detection temperature and a relatively low detection temperature) and the second target nucleic acid sequence is a target nucleic acid sequence haying a relatively low

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detection temperature {i.e., capable of generating a signal at only a relatively low detection temperature), the signal for the second target nucleic acid sequence can be extracted by eliminating the signal for the first target nucleic acid sequence from the signal detected at the relatively low detection temperature by using a reference value for the first target nucleic acid sequence. The extracted signal may be then used to determine the presence or absence of the second target nucleic acid sequence.

The reference value for the first target nucleic acid sequence used in the method may be obtained in a various manners. For instance, the reference value is determined by detecting two signals at a relatively low detection temperature and a relatively high detection temperature from a control sample containing only a first target nucleic acid sequence, followed by calculation of the relationship of change in the two signals, e.g., the ratio of the two signals.

The method as described above is surprisingly remarkable compared with the conventional methods in that the signal for the first target nucleic acid sequence and the signal for the second target nucleic acid sequence are easily distinguishable even by using a conventional single type of detector. In particular, the method as described above exhibits considerable accuracy in extracting a signal for a target nucleic acid sequence from a sample containing both a target nucleic acid sequence having a relatively high detection temperature and a target nucleic acid sequence having a relatively low detection temperature (a sample co-infected with two targets).

However, for a sample containing only one of two target nucleic acid sequences {e.g., a sample infected with a single target) or a sample containing no target nucleic acid sequence {e.g., non-infected sample), the method may lead to false positive results in extracting the signal for the target nucleic acid sequence having a relatively low detection temperature.

Accordingly, there remains a need in the art to develop a novel method for

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providing a signal for a target nucleic acid sequence in a more effective and accurate manner.

Throughout this application, various patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications in their entirety are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains. SUMMARY OF THE INVENTION

The present inventors have made intensive researches to improve the MuDTl technology for detecting a target nucleic acid sequence from signals detected at different temperatures (WO 2015/147412). As a result, the present inventors have developed a novel method for providing a signal for a target nucleic acid sequence in a more accurate and effective manner while significantly reducing false positive results by applying the signal extraction process only when the signals detected at two detection temperatures satisfy particular criteria.

Accordingly, it is an object of this invention to provide a method for providing a signal for at least one target nucleic acid sequence in a sample from signals detected at different temperatures.

It is another object of this invention to provide a computer readable storage medium containing instructions to configure a processor to perform a method for providing a signal for at least one target nucleic acid.:sequence in a sample from signals detected at different temperatures.

It is still another object of this invention to provide a devjce for providing a signal for at least one target nucleic acid sequence in a,_<sample from signals detected at different temperatures.

It is further object of this invention to provide a computer program to be stored on a computer readable storage medium to configure a processor to perform a method for providing a signal for at least one target^ nucleic acid sequence in a sample from signals detected at different temperatures.

Other objects and advantages of the present invention will become apparent from the detailed description to follow taken in conjugation with the appended claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 represents signals detected at two different detection temperatures (72°C and 60°C) in accordance with a PTOCE real-time PCR method (MuDTl Technology), for four (4) types of samples: (i) A-type: a sample containing only a first target nucleic acid sequence {Chlamydia trachomatis, CT); (ii) B-type: a sample containing only a second target nucleic acid sequence Neisseria gonorrhoeae, NG); (iii) C-type: a sample containing both the first target nucleic acid sequence (CT) and the second target nucleic acid sequence (NG); and (iv) D-type: a sample containing no target nucleic acid sequence.

Fig. 2A is a flow chart 100 representing an embodiment of this invention for providing signals for target nucleic acid sequences from signals detected at two different detection temperatures by applying two criteria. For a sample (B-type or D- type of sample) in which the signal detected at the relatively high detection temperature (RHT) does not satisfy a first criterion defined by a high-temperature threshold (HTT), a signal for a first target nucleic acid sequence and a signal for a second target nucleic acid sequence are provided without signal extraction process 125; whereas for a sample in which the signal detected at the RHT satisfies the first criterion, the next step 130 proceeds. Afterwards, for a sample (A-type of sample) in which a signal difference between the signal detected at the RHT and the signal detected at the relatively low detection temperature (RLT) does not satisfy a second criterion defined by a signal-difference threshold (SDT), a signal for a first target nucleic acid sequence and a signal for a second target nucleic acid sequence are

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provided without signal extraction process 135; whereas for a sample (C-type of sample) in which the signal difference between the signal detected at the RHT and the signal detected at the RLT satisfies the second criterion, a signal for a first target nucleic acid sequence is provided without signal extraction process and a signal for a second target nucleic acid sequence is provided with signal extraction process 140.

Fig. 2B represents signals for a first target nucleic acid sequence (CT) and a second target nucleic acid sequence (NG) provided according to an embodiment of this invention depicted in Fig. 2A, for four types of samples: A-type; B-type; C-type; and D-type.

Fig. 3 is a flow chart 200 representing another embodiment of this invention for providing a signal for a target nucleic acid sequence from signals detected at two different detection temperatures by applying three criteria. For a sample (D-type of sample) which does not satisfy a first criterion defined by a HTT and does not satisfy a third criterion defined by a low-temperature threshold (LIT), a signal for a first target nucleic acid sequence and a signal for a second target nucleic acid sequence are provided without signal extraction process 224; and for a sample (B-type of sample) which does not satisfy the first criterion but satisfies the third criterion, a signal for a first target nucleic acid sequence and a signal for a second target nucleic acid sequence are provided without signal extraction process 226. For a sample (A- type of sample) which satisfies the first criterion but does not satisfy a second criterion defined by a signal-difference threshold (SD^" ), a signal for a first target

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nucleic acid sequence and a signal for a second target nucleic acid sequence are

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provided without signal extraction process 232; whereas for a sample (C-type of sample) which satisfies both the first criterion and the second criterion, a signal for a first target nucleic acid sequence is provided without signal extraction process and a signal for a second target nucleic acid sequence is provided with signal extraction process 240.

Fig. 4 is a flow chart 300 representing another embodiment of this invention for providing a signal for a target nucleic acid sequence from signals detected at two different detection temperatures by applying three . criteria. For a sample (D-type of sample) which does not satisfy a third criterion - defined by a low-temperature threshold (ITT), a signal for a first target nucleic acid ^sequence and a signal for a second target nucleic acid sequence are provided without signal extraction - process

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322. For a sample (B-type of sample) which satisfies the third criterion but does not satisfies a first criterion defined by a high-temperature threshold (HTT), a signal for a first target nucleic acid sequence and a signal for a, second target nucleic acid sequence are provided without signal extraction process 332. For a sample (A-type of sample) which satisfies the third criterion and the first criterion but does not satisfies a second criterion defined by a signal-difference threshold (SDT), a signal for a first target nucleic acid sequence and a signal for a second target nucleic acid sequence are provided without signal extraction process 342. For a sample (B-type of sample) which satisfies all of the criteria described above, a signal for a first target nucleic acid sequence is provided without signal extraction process and a signal for a second target nucleic acid sequence is provided with signal extraction process 350.

Figs. 5A, 5B and 5C represent signals detected at three different detection temperatures (95°C, 72°C and 60°C) for eight (8) types of samples: (i) A-type: a sample containing only a first target nucleic acid sequence; (ii) B-type: a sample containing only a second target nucleic acid sequence; (Hi) C-type: a sample containing only a third target nucleic acid sequence; (iv) D-type: a sample containing both a first target nucleic acid sequence and a second target nucleic acid sequence; (v) E-type: a sample containing both a first target nucleic acid sequence and a third target nucleic acid sequence; (vi) F-type: a sample containing bQth a second target nucleic acid sequence and a third target nucleic acid sequence; (vii) G-type: a sample containing all of a first target nucleic acid sequence, a second target nucleic acid sequence and a third target nucleic acid sequence; and ^' (viii) H-type: a sample containing no target nucleic acid sequence.

Figs. 5D and 5E are a flow chart 400 representing an embodiment of this

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invention for providing signals for target nucleic acid_, sequences from signals detected at three different detection temperatures by applying various criteria, for eight (8) types of samples containing up to three target nucleic acid sequences. In Figs. 5D and 5E, RHT represents a relatively high detection temperature; RMT represents a relatively middle detection temperature; RLT represents a relatively low detection temperature; HTT represents a threshold for determining significance of the signal at RHT; MTT represents a threshold for determining significance of the signal at RMT; LTT represents a threshold for determining significance of the signal at RLT; End- Ratio_M/H represents the ratio of End-RFU (RFU value at the end cycle) at RMT to End- RFU at RHT; End-Ratio _M represents the ratio of End-RFU at RLT to End-RFU at RMT; RT_{M H} represents a threshold for distinguishing between a sample containing 2^nd target and a sample containing no 2^nd target; and RT^M represents a threshold for distinguishing between a sample containing 3^rd target and a sample containing no 3^rd target.

DETAILED DESCRIPTION OF THIS INVENTION

I. Providing a Signal for a Target Nucleic Acid Sequence in a Sample

In one aspect of this invention, there is provided a method for providing a signal for at least one target nucleic acid sequence in a sample from signals detected at different temperatures, which comprises the steps of:

δ (a) incubating the sample with a first signal -generating means capable of generating a signal for a first target nucleic acid sequence and a second signal- generating means capable of generating a signal for a second target nucleic acid

■ ^~. ^{' '} ΐ ' sequence in a single reaction vessel, and detecting signals at a relatively high detection temperature and a relatively low detection temperature by a single type of detector; wherein the first signal-generating means generates signals at both the relatively high detection temperature and the relatively low detection temperature in the presence of the first target nucleic acid sequence ¾ the sample and the second

"s- signal-generating means generates a signal at the relatively low detection temperature in the presence of the second target nucleic acid sequence in the sample; wherein the two signals to be generated by the two signal-generating means are not differentiated by the single type of detector;

(b) identifying whether the signal detected at the relatively high detection temperature satisfies a first criterion defined by a high-temperature threshold (HTT);

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when the signal detected at the relatively high detection temperature satisfies the first criterion, the next step (c) proceeds;

(c) identifying whether a signal difference between the signal detected at the relatively low detection temperature and the signal detected at the relatively high detection temperature satisfies a second criterion defined by a signal-difference threshold (SDT); when the signal difference satisfies the second criterion, the next step (d) proceeds; and

(d) extracting the signal for the second target nucleic acid sequence by using the signal detected at the relatively high detection temperature in the step (a), the signal detected at the relatively low detection temperature in the step (a) and a reference value for the first target nucleic acid sequence, thereby providing the signal for the second target nucleic acid sequence; wherein the reference value for the first target nucleic acid sequence is a value representing a relationship of change in signals provided by the first signal-generating means at the relatively high detection temperature and the relatively low detection temperature; wherein the reference value for the first target nucleic acid sequence is determined from a control reaction using the first target nucleic acid sequence and the first signal-generating means; wherein the steps (b) and (c) may be performed in reverse order.

The present inventors have made intensive researches to improve the uDTl technology for detecting a target nucleic acid sequence from signals detected at different temperatures (WO 2015/147412). As a result, the present inventors have developed a novel method for providing a signal for a target nucleic acid sequence in

^;¾ - ί a more accurate and effective manner while significantly reducing false positive results by applying the signal extraction process only when the Signals detected at two detection temperatures satisfy particular criteria.

Although the inventive method is expressed as a method for providing a signal for a target nucleic acid sequence throughout the specification, it can be also referred

< ί to as "a method for determining the presence of a target nucleic acid sequence using a signal for the target nucleic acid sequence", since the signal provided is directly used to determine the presence of the target nucleic acid sequence.

One embodiment of the present invention 100 is illustrated in Fig. 2A. The present invention will be described in more detail with reference to Fig. 2A as follows:

Step (a): Detecting signals at two different temperatures 110

In step (a), the sample is incubated with a first signal-generating means capable of generating a signal for a first target nucleic acid sequence and a second signal-generating means capable of generating a signal for a second target nucleic acid sequence in a single reaction vessel. Afterwards, signals are detected at a relatively high detection temperature and a relatively low detection temperature by a single type of detector. The first signal-generating means generates signals at both the relatively high detection temperature and the relatively low detection temperature in the presence of the first target nucleic acid sequence in the sample and the second signal-generating means generates a signal at the relatively low detection temperature in the presence of the second target nucleic acid sequence in the sample. The two signals to be generated by the two signal-generating means are not differentiated by the single type of detector.

The term "sample" as used herein refers to any material undergoing the method of the present invention. Particularly, the term "sample" refers to any material containing or presumed to contain a nucleic acid of interest (one or both of a first target nucleic acid sequence and a second target nucleic acid sequence) or which is itself a nucleic acid containing or presumed to contain a target nucleic acid sequence of interest. More particularly, the term "sample" as used herein includes biological samples {e.g., cells, tissues, and fluid from a biological source) and non-biological samples {e.g., food, water and soil). The biological samples includes, but not limited to, virus, bacteria, tissue, cell, blood, serum, plasma, lymph, sputum, swab, aspirate, bronchoalveolar lavage fluid, milk, urine, feces, ocular fluid, saliva, semen, brain

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extracts, spinal cord fluid (SCF), appendix, spleen and tonsillar tissue extracts, amniotic fluid and ascitic fluid. The sample can be subjected to nucleic acid extraction process known in the art for efficient amplification reactions (see Sambrook, J. et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor (2001)). The nucleic acid extraction process may vary depending on the type of the sample. Moreover, if the extracted nucleic acid is RNA, a reverse transcription is further performed to synthesize cDNA therefrom (see supra).

The term used herein "target nucleic acid", "target nucleic acid sequence" or

"target sequence" refers to a nucleic acid sequence of interest for analysis, detection or quantification. The target nucleic acid sequence comprises a sequence in a single strand as well as in a double strand. The target nucleic acid sequence comprises a sequence newly generated in reactions as well as a sequence initially present in a sample.

The target nucleic acid sequence may include any DNA (gDNA and cDNA), RNA molecules and their hybrids (chimera nucleic acid). The sequence may be in either a double-stranded or single-stranded form. Where the nucleic acid as starting material is double-stranded, it is preferred to render the two strands into a single-stranded or partially single-stranded form. Methods known to separate strands includes, jbut not limited to, heating, alkali, formamide, urea and glycoxal treatment, enzymatic methods (e.g., helicase action), and binding proteins. For instance, strand separation can be achieved by heating at temperature ranging from 80°C to 105°C. General methods for accomplishing this treatment are provided by Joseph Sambrook, et al„ Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001).

The target nucleiq acid sequence includes any naturally occurring prokaryotic, eukaryotic (for example, protozoans and parasites, fungi, yeast, higher plants, lower and higher animals, including mammals and humans), viral (for example, Herpes viruses, HIV, influenza virus, Epstein-Barr virus, hepatitis virus, polio virus, ejtc), or viroid nucleic acid. The target nucleic acid sequence can also be any nucleic acid molecule which has been or can be recombinantly produced or chemically synthesized. Thus, the target nucleic acid sequence may or may not be found in nature. The target nucleic acid sequence may comprise a known or unknown sequence.

The target nucleic acid sequence should not be construed as limiting the sequence known at a given time or the sequence available as of a given time, but instead should be read to encompass the sequence that may be available or known now or at any time in the future. In other words, the target nucleic acid sequence may or may not be known at the time of practicing the present method. In case of unknown target nucleic acid, its sequence may be determined by one of conventional sequencing methods prior to performing the present method.

The target nucleic acid sequence used in the present invention comprises the first target nucleic acid sequence and the second target nucleic acid sequence. The terms "first target nucleic acid sequence" and "second target nucleic acid sequence" are used herein to distinguish two different target nucleic acid sequences. For instance, the first target nucleic acid sequence and the second target nucleic acid sequence may be two different genes, two different gene regions or two different DNA sequences of interest. Particularly, the first target nucleic acid sequence may be derived from one organism, whereas the second target nucleic acid sequence from another organism.

According to an embodiment of this invention, one of the two target nucleic acid sequences comprise a nucleotide variation, or one of the two target nucleic acid sequences comprises one type of the nucleotide variation and the other comprises the other type of the nucleotide variation.

The term "nucleotide variation" used herein refers to any single or multiple 4_f .._....

nucleotide substitutions, deletions or insertions in a DNA sequence at a particular

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location among contiguous DNA segments that are otherwise similar in sequence.

Such contiguous DNA segments include a gene or any other portion of a chromosome.

These nucleotide variations may be mutant or polymorphic allele variations. For example, the nucleotide variation detected in the present invention includes SNP

(single nucleotide polymorphism), mutation, deletion, insertion, substitution and translocation. Exemplified nucleotide variation includes numerous variations in a human genome (e.g., variations in the MTHFR (methylenetetrahydrofolate reductase) gene), variations involved in drug resistance of pathogens and tumorigenesis-causing variations. The term "nucleotide variation" used herein includes any variation at a particular location in a nucleic acid sequence. In other words, the term "nucleotide variation" includes a wild type and its any mutant type at a particular location in a nucleic acid sequence.

According to the present invention, the sample (or target nucleic acid sequences in the sample) is incubated with two signal-generating means in order to obtain signals for the target nucleic acid sequences.

The term "incubating," "incubate," or "incubation" as used herein refers to bring components together for their interaction or reaction. Particularly, the term refers to subjecting the components herein to a signal-generating process.

The term "signal" as used herein refers to a measurable output.

The signal change may serve as an indicator indicating qualitatively or quantitatively the presence or absence of an analyte (a target nucleic acid sequence).

Examples of useful indicators include, but not Jimited to, fluorescence intensity, luminescence intensity, chemiluminescence intensity,^' bioluminescence intensity, phosphorescence intensity, charge transfer, voltage, current, power, energy, temperature, viscosity, light scatter, radioactive intensity, reflectivity, transmittance and absorbance. The most widely used indicator is fluorescence intensity.

Signals include various signal characteristics from the signal detection, e.g., signal intensity [e.g., RFU (relative fluorescence unit) value or in the case of performing amplification, RFU values at a certain cycle, at selected cycles or at end- S. . ·'· .-Ί 1 point], signal change shape (or pattern) or Q value, or values obtained by

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mathematically processing the characteristics.

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According to an embodiment, the term "signal" with conjunction with reference value or sample analysis includes not only signals per se obtained at detection temperatures but also a modified signal provided by mathematically processing the signals.

According to an embodiment of this invention, w ien an amplification curve is obtained by real-time PCR, various signal values (or characteristics) from the amplification curve may be selected and used for determination of target presence (intensity, Q value or amplification curve data).

The terms "signal for a first target nucleic acid sequence" and "signal for a second target nucleic acid sequence" as used herein refer to signals representing the first target nucleic acid sequence and the second target nucleic acid sequence, respectively. In other words, the terms refer to signals which are generated and detected dependently on the presence of the first target nucleic acid sequence or the second target nucleic acid sequence, and thus a significant level of the signal for a first target nucleic acid sequence or second target nucleic acid sequence indicates the presence of the first target nucleic acid sequence or the second target nucleic acid sequence.

The signal (particularly, the signal intensity) may vary depending upon its detection temperature as well as a signahgenerating means employed.

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The incubation herein is performed by a signal-generating process using signal- generating means.

The term "signal-generating process" as used herein refers to any process capable of generating signals in a dependent manner on the presence of a target nucleic acid sequence in a sample.

The term "signal-generating means" as used herein refers to any material used in generation of signals indicating the presence of target nucleic acid sequences, for example including oligonucleotides, labels and enzymes! Alternatively, the term used herein "signal-generating means" can be used to refer to any methods using the materials for signal generation.

In particular, the terms "first signal-generating means" and "second signal- generating means" as used herein refer to means for generating signals for a first target nucleic acid sequence and a second target nucleic acid sequence, respectively. The first signal-generating means and the second signal-generating means may include common components (e.g., a single type of label and a single type of enzyme) or different components (e.g., different types of oligonucleotides) used in signal generation. In particular, the first signal-generating means and the second signal- generating means are characterized by having a single type of label (the same label). Thus, the signal derived from the label of the first signal-generating means is not differentiated from the signal derived from the label of the second signal-generating means by conventional methods.

A wide variety of the signal-generating means have been known to one of skill in the art. The signal-generating means include both labels per se and oligonucleotides with labels. The labels may include a fluorescent label, a luminescent label, a chemiluminescent label, an electrochemical label and a metal label. The label per se may serve as signal-generating means, for example, an intercalating dye. Alternatively, a single label or an interactive dual label containing a donor molecule and an acceptor molecule may be used as signal-generating means in the form of linkage to at least one oligonucleotide.

The signal-generating means may comprise 1 additional components for generating signals such as nucleolytic enzymes (e.g., 5'-nucleases and 3'-nucleases).

The signal-generating process is accompanied with signal change. The signal change may serve as an indicator indicating qualitatively or quantitatively the presence or absence of a target nucleic acid sequence.

The details of "signal-generating process" are disclosed in WO 2015/147412 filed by the present inventors, the teachings of which are incorporated herein by reference in its entirety.

According to an embodiment, the signal-generating process is a signal amplification process.

According to an embodiment of this invention, the signal-generating process is

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a process with amplification or with no amplification of a target nucleic acid sequence.

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Particularly, the signal-generating process is a process with amplification of a

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target nucleic acid molecule. More particularly, the signal-generating process is a process with amplification of a target nucleic acid molecule and capable of increasing or decreasing signals (particularly, increasing signals) upon amplifying the target nucleic acid molecule.

The term used herein "signal generation" include appearance or disappearance of signals and increase or decrease in signals. Particularly, the term "signal generation" means increase in signals.

The signal-generating process may be performed in accordance with a multitude of methods known to one of skill in the art. The methods include TaqMan™ probe method (U.S. Pat. No. 5,210,015), Molecular Beacon method (Tyagi et al.,

Nature Biotechnology, 14(3):303(1996)), Scorpion method (Whitcombe et al., Nature

Biotechnology 17:804-807(1999)), Sunrise or Amplifluor method (Nazarenko et al.,

Nucleic Acids Research, 25(12):2516-2521(1997), and U.S. Pat. No. 6,117,635), Lux method (U.S. Pat. No. 7,537,886), CPT (Duck P, et al., Biotechniques, 9:142-

148(1990)), LNA method (U.S. Pat. No. 6,977,295), Plexor method (Sherrill CB, et al., Journal of the American Chemical Society, 126:4550-4556(2004)), Hybeacons¹" (D. J. French, et al., Molecular and Cellular Probes, 13:363-374(2001)) and U.S. Pat. No. 7,348,141), Dual-labeled, self-quenched probe (U.S. Pat. No. 5,876,930), Hybridization probe (Bernard PS, et al., Clin. Chem., 46: 147-148(2000)), PTOCE (PTO cleavage and extension) method (WO 2012/096523), PCE-SH (PTO Cleavage and Extension-Dependent Signaling Oligonucleotide Hybridization) method (WO 2013/115442) and PCE-NH (PTO Cleavage and Extension-Dependent Non- Hybridization) method (WO 2014/104818) and CER method (WO 2011/037306).

When the signal-generating process is performed in accordance with TaqMan™ probe method, the signal-generation means may comprise a primer pair, a probe with an interactive dual label and DNA polymerase having 5' to 3' nuclease activity. When the signal-generating process is performed in accordance with PTOCE method, the signal-generation means may comprise a primer pair, PTO (Probing and Tagging Oligonucleotide), CTO (Capturing and: Templating ^Oligonucleotide) and DNA

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polymerase having 5' to 3' nuclease activity. Either PTO or CTO may be labeled with suitable labels.

According to an embodiment, the signal-generating process is performed in a process involving signal amplification together with target amplification.

According to an embodiment, the amplification reaction as the signal- generating process is performed in such a manner , that signals are amplified simultaneously with amplification of the target nucleic acid sequence {e.g., real-time PCR). Alternatively, the amplification reaction is performed in such a manner that signals are amplified with no amplification of the target nucleic acid molecule [e.g., CPT method (Duck P, et al., Biotechniques, 9: 142-148 (1990)), Invader assay (U.S. Pat. Nos. 6,358,691 and 6,194,149)].

A multitude of methods have been known for amplification of a target nucleic acid molecule, including, but not limited to, PCR (polymerase chain reaction), LCR (ligase chain reaction, see Wiedmann M, et al., "Ligase chain reaction (LCR)- overview and applications." PCR Methods and Applications, 3(4):S51-64(1994)), GLCR (gap filling LCR, see WO 90/01069, EP 0439182 and WOs 93/00447), Q-beta (Q-beta replicase amplification, see Cahill P, et al., Clin Chem., 37(9): 1482-5(1991), U.S. Pat.

No. 5,556,751), SDA (strand displacement amplification, see G T Walker et al.,

Nucleic Acids Res. 20(7): 16911696(1992), EP 0497272), NASBA (nucleic acid sequence-based amplification, see Compton, J. Nature^ 350(6313):912(1991)), TMA

(Transcription-Mediated Amplification, see Hofmann WP et al., J CNn Virol. 32(4): 289?

93(2005); U.S. Pat. No. 5,888,779) or RCA (Rolling Circle Amplification, see Hutchison

C.A. et al., Proc. Natl Acad. Sci. USA. 102:1733217336(2005)).

The detection of signals from the two signal-generating means at a relatively high detection temperature and a relatively low detection, temperature is performed at

: ? ^{' :'} '■-'· !i

one or more cycles of the signal-generating process to obtain a signal value at each of the one or more cycles.

The term used herein "cycle" refers to a unit of changes of conditions in a plurality of measurements accompanied with changes of conditions. For example, the changes of conditions include changes in temperature, reaction time, reaction number, concentration, pH and/or replication number of a target nucleic acid molecule sequence. Therefore, the cycle may include time or process cycle, unit operation cycle and reproductive cycle. For example, an isothermal amplification allows for a plurality of measurements for a sample in the course of reaction time under isothermal conditions and the reaction time may correspond to the changes of conditions and a unit of the reaction time may correspond to a cycle.

Particularly, when repeating a series of reactions or repeating a reaction with a time interval, the term "cycle" refers to a unit of the repetition.

For example, in a polymerase chain reaction (PCR), a cycle refers to a reaction unit comprising denaturation of a target molecule, annealing (hybridization) between the target molecule and primers and primer extension. The increases in the repetition of reactions may correspond to the changes of conditions and a unit of the repetition may correspond to a cycle.

The signals detected at the relatively high detection temperature and the relatively low detection temperature may be a signal value at a particular cyde, a set h ^{' "'■} i

of signal values at particular cycles, a set of signal values within a particular cycle interval, or a set of signal values at all cycles. The detection of signals provides

interchangeably.

The signals generated by the two signal-generating means are not differentiated by a single type of detector. The term "signals not differentiated by a single type of detector" means that signals are not differentiated from each other at a certain detection temperature by a single type of detector due to their identical or substantially identical signal properties (e.g., optical properties, emission wavelength and electrical signal).

The term used herein "a single type of signal" means signals providing identical or substantially identical signal properties (e.g., optical properties, emission wavelength and electrical signal). For example, FAM and CAL Fluor 610 provide different types of signals from one another.

According to an embodiment of the present invention, the two signal- generating means comprise an identical label and the signals from the label is not differentiated from each other by a single type of detector.

The first signal-generating means generates signals at both the relatively high detection temperature and the relatively low detection temperature in a dependent manner on the presence of the first target nucleic acid sequence; whereas the second signal-generating means generates a signal at the relatively low detection k I

temperature in a dependent manner on the presence of the second target nucleic acid

i ^' x - ^■ .

sequence. According to an embodiment of the present invention, the detection temperatures for target nucleic acid sequences are determined in considering a temperature range to allow signal generation by the signal-generating means.

The term "target nucleic acid sequence having a relatively high detection temperature" as used herein refers to a target nucleic acid sequence which is capable of generating a signal at the relatively high detection temperature of the two detection temperatures, and thus generating a signal at the relatively low detection temperature as well. A target nucleic acid sequence having a relatively high detection

? " ^"3 ί ^;

temperature herein may be used interchangeably with a first target nucleic acid sequence. In contrast, the term "target nucleic acid sequence haying a relatively low detection temperature" refers to a target nucleic.acid sequence which is capable of generating a signal at the relatively low detection temperature of the two detection temperature, but not generating a signal at the relatively high detection temperature. A target nucleic acid sequence having a relatively low detection temperature herein may be used interchangeably with the second target nucleic acid sequence.

According to an embodiment of the present invention, a detection temperature determined by a corresponding signal-generation is assigned to one target nucleic acid sequence.

The relatively high detection temperature is a temperature capable of generating only a signal for the target nucleic acid sequence having the relatively high detection temperature (first target nucleic acid sequence), and the relatively low detection temperature is a temperature capable of generating both a signal for the target nucleic acid sequence having the relatively low detection temperature (second target nucleic acid sequence) and a signal for the target nucleic acid sequence having the relatively high detection temperature (first target nucleic acid sequence). The relatively high detection temperature may be referred to a first detection temperature, and the relatively low detection temperature may be referred to a second detection temperature. According to MuDTl technology, the presence o the two target nucleic acid sequences may be differentially determined by detecting the signals indicative of the presence of the two target nucleic acid sequences at_¾different temperatures. The technology employs that there is a certain temperature range to allow signal generation by signal-generating means.

For example, when a signal-generating means generates a signal upon hybridization (or association) between two nucleic acid molecules and does not generate a signal upon non-hybridization (or dissociation) between them, a signal is generated at temperatures allowing hybridization between two nucleic acid molecules,

¾ ^' 'i ζ however, no signal is generated at temperatures failing to hybridize between two nucleic acid molecules. As such, there is a certain temperature range to allow signal generation {i.e., signal detection) and other temperature range not to allow signal generation. The temperature ranges are affected by the T_m value of the hybrid of the

.

two nucleic acid molecules employed in the signal- generation means.

Where the signal generation method using a released fragment with a label after cleavage is employed, the signal may be theoretically detected at any temperature {e.g., 30-99°C).

A detection temperature is selected from the temperature range to allow signal generation by the signal generation means.

The term "detection temperature range" is used herein to particularly describe the temperature range to allow signal generation {i.e., signal detection).

Where there are different detection temperature ranges depending on signal- generating means for two target nucleic acid sequences, a relatively high detection temperature may be selected from a non-overlapped detection temperature range. A target nucleic acid sequence providing a signal at the relatively high detection temperature is determined as the target nucleic acid sequence having the relatively high detection temperature. A relatively low detection temperature may be selected from an overlapped detection temperature range. A target nucleic acid sequence providing a signal at the relatively low detection temperature and not providing a signal at the relatively high detection temperature is determined as the target nucleic add sequence having the relatively low detection temperature.

ί ^'

According to an embodiment, the detection temperature may be determined in considering the overlapped detection temperature range and the non-ovedapped detection temperature range.

According to an embodiment, the detection temperatures allocated for the target nucleic acid sequences are different from each other by at least 2°C, 3°C, 4°C , 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 15°C or 20°C

According to the present invention, a temperature for detecting the presence of each of target nucleic acid sequences may be allocated in considering signal- generating means.

According to an embodiment of this invention, one of the two target nucleic

* i ■ ^■· ' <

acid sequences is assigned with a relatively high detection temperature and the other is assigned with a relatively low detection temperature, and then signal-generating

. ^■ j? ^■ ,- :^{■ ■}

means suitable for the detection temperatures are constructed.

According to an embodiment, the relatively high detection temperature and the relatively low detection temperature at which the detection is carried out may be determined. For example, the relatively high detection temperature and the relatively low detection temperature are determined as 72°C and 60°C, respectively, and then signal-generating means suitable for the detection temperatures are constructed.

According to an embodiment of this invention, when the signal- generating means generates a signal in a dependent manner on the formation of a duplex, the detection temperature is selected based on a T_m value of the duplex.

According to an embodiment of this invention, when the signal-generating means generates a signal in a dependent manner on the formation of a duplex, the detection temperature is controllable by adjusting a T_m value of the duplex.

For example, where the signal is generated by a detection oligonucleotide specifically hybridized with the target nucleic acid sequence, the detection of the signal is successfully done at the determined temperature by adjusting the T_m value of the oligonucleotide. Where Scorpion primer is used, the detection of the signal is successfully done at the determined temperature by jadjusting the T_m value of a portion to be hybridized with extended strand.

X.

Where the signal is generated by the duplex, formed dependent on the presence of the target nucleic acid sequence, the detection of the signal is successfully done at the determined temperature by adjusting the T_m value of the duplex. For example, where the signal is generated by the PTOCE method, the

fragment on the CTO.

The PTOCE-based methods have advantages to readily adjust T_m values of the duplex or a third hybrid whose hybridization is affected y the duplex.

According to an embodiment of this inventions when the signal-generating means generates a signal in a dependent manner on cleavage of a detection oligonucleotide, the detection temperature is arbitrarily .selected. As described above, where the signal is generated being dependent manner on cleavage of the detection oligonucleotide, the label released by the cleavage may be detected at various temperatures (e.g., 30°C to 130°C).

The signals are generated by the signal-generating process, thereby providing a data set. The term used herein "data set" refers to a set of data points. The term used herein "data point" means a coordinate value comprising a cycle and a signal value at the cycle. Data points obtained by the signal-generating process may be plotted with coordinate values in a rectangular coordinate system, giving a curve {e.g., amplification curve). The curve may be a fitted or normalized {e.g., baseline- subtracted) curve. In particular, the signals detected are plotted against cycles.

In a particular embodiment, the signals, and the data sets, the data points and the curve thereof are baseline-subtracted.

According to the present invention, the signals detected at the relatively high detection temperature and at the relatively low detection temperature exhibit distinct pattern depending upon the types of samples.

For instance, the samples containing up to two target nucleic acid sequences may be divided into four (4) types:

the signal at the relatively low detection temperature and the signal at the relatively high detection temperature is higher than that for A-type; and (iv) for a D-type sample, no significant signals are detected both at the relatively high detection temperature and the relatively low detection temperature.

Such differences in signal patterns allow the samples to be distinguished into the four types using appropriate criteria as described below.

Step (b): Evaluating the signal at the relatively high detection temperature by a high-temperature threshold 120

In step (b), it is identified whether the signal detected at the relatively high detection temperature satisfies a first criterion defined by a high-temperature threshold (HTT) 120. When the signal detected at the relatively high detection temperature satisfies the first criterion, the next step (c) 130 proceeds.

The term "high-temperature threshold (HTT)" as used herein refers to a threshold used for determining the significance of the signal detected at the relatively high detection temperature. Determining the significance of the signal means determining whether the signal is generated at a meaningful level at a certain temperature relative to a specific threshold. The significance of the signal can be determined by a suitable threshold (e.g., HTT) for distinguishing a signal generated by a specific signal-generating means from other signals. Specifically, the threshold (e.g., HTT) used to determine the significance of the signal is set to any value that can exclude a signal not derived from a nucleic acid sequence of interest, for example, a background signal or a noise signal. As an example, if the signal is amplified as the amplification reaction proceeds, the signal above the threshold may be determined to be significant.

The significance of the signal can be applied to the "low-temperature threshold (LTT)" or the "verification threshold (VT)" in a similar manner as will be described below. The HTT used herein may also be expressed, as the "first signal threshold" in order to distinguish it from other thresholds. The value of the HTT used to determine the significance of the signal may differ from that of the threshold used to finally determine the presence of the target nucleic acid sequence.

The HTT may be determined by a conventional method for setting thresholds.

For example, the HTT may be determined by considering the background signal, sensitivity, label characteristics, signal variation of the detector, or error range. The HTT may be set to any value within the exponential region of the signal-generating reaction. The HTT may be set to any value which is greater than the signal values in the baseline region and which is lower than the signal values in the plateau region of the signal-generating reaction. Alternatively, the HTT may be set to a value obtained by multiplying by a certain value (e.g., 10) the standard deviation of the signal values in the baseline region in a baseline-subtracted amplification curve.

The HTT may be set automatically by the detector or directly by the user. As used herein, the expression "first criterion defined by a high-temperature threshold" refers to a criterion which is defined in consideration of the HTT in o ΐrder to s ϊ . - determine the significance of the signal detected at the relatively high detection temperature. In other words, the signal satisfying the first criterion indicates that a significant signal is generated at the relatively high detection temperature, whereas the signal not satisfying the first criterion indicates that no significant signal is generated at the relatively high detection temperature.

According to an embodiment of the present invention, the first criterion for the 4 . ^■' ^■ '? , » signal detected at the relatively high detection temperature is the magnitude relation between the signal detected at the relatively high detection temperature and t e HTT.

For example, the first criterion for the signal detected at the relatively high detection temperature is that the signal is not less than, above, not more than, or below the HTT. Specifically, when the signal is amplified as amplification products accumulate, the first criterion is that the signal is not less than or above the HTT. On the other

? hand, when the signal is decreased as amplification products accumulate, the first criterion is that the signal is not more than or below the HTT.

More specifically, the first criterion for the signal detected at the relatively high detection temperature is that the signal is not less than the HTT.

For example, when the signal amplification reaction is performed according to real-time PCR and the HTT is RFU (relative fluorescence unit) 100, "the signal value not less than RFU 100" may be the first criterion.

According to the method of the present invention, the signal detected at the relatively high detection temperature will have only the signal derived from the first signal-generating means, i.e., the signal for the first target nucleic acid sequence, because the second signal-generating means is designed not to generate a signal at the relatively high detection temperature. Thus, if the signal detected at the relatively high detection temperature satisfies the first criterion, i.e., if the signal detected at the relatively high detection temperature is identified as being significant, it indicates directly that the first target nucleic acid sequence is present in a sample. Such significant signal for the first target nucleic acid sequence may be provided as the signal for the first target nucleic acid sequence. In this case, the presence or absence of the first target nucleic acid sequence may be identified by use of an additional threshold, or the presence of the first target nucleic acid sequence may be determined directly based on the result of satisfying the first criterion without using the additional threshold. Further, such significant signal for the first target nucleic acid sequence is then used to calculate the signal difference between the signal detected at the relatively low detection temperature and the signal detected at the relatively high detection temperature in the next step (c).

Conversely, if the signal detected at the relatively high detection temperature does not satisfy the first criterion, i.e., if the signal detected at the relatively high detection temperature is identified as being insignificant, it indicates directly that the first target nucleic acid sequence is absent in a sample. Such insignificant signal for the first target nucleic acid sequence may be provided as the signal for the first target nucleic acid sequence. In this case, the presence or absence of the first target nucleic acid sequence may be identified by use of an additional threshold, or the absence of the first target nucleic acid sequence may be determined directly based on the result of not satisfying the first criterion without using the additional threshold. Further, it may also indicate that the signal detected at the relatively low detection temperature includes only the signal provided by the second target nucleic acid sequence.

The first criterion defined by the HTT as used in this step allows distinguishing A-type or C-type from B-type or D-type among the typical four (4) types of samples (see Fig. 1). Typically, an A-type or C-type of samples generates significant signals at the relatively high detection temperatures, whereas a B-type or D-type of samples generates no significant signals at the relatively high detection temperatures. Therefore, if the signal detected at the relatively high detection temperature satisfies the first criterion defined by the HTT, the sample is determined to be either an A-type or C-type sample, whereas if the signal detected at the relatively high detection temperature does not satisfy the first criterion defined by the HTT, the sample is determined to be either a B-type or D-type sample.

According to an embodiment of the present invention, when the signal detected at the relatively high detection temperature does not satisfy the first criterion defined by the HTT, the signal detected at the relatively low detection temperature in the step (a) is provided as the signal for the second target nucleic acid sequence, without the signal extraction process of the step (d) 125. This embodiment may provide the signal detected at the relatively low detection temperature in the step (a) as the signal for the second target nucleic acid sequence, without using the third criterion defined by the low-temperature thresholds will be described below. For example, when a signal is detected at the relatively high detection temperature in a real-time PCR reaction but the signal values of the signal are all below the HTT, the signal detected at the relatively low detection temperature in the step (a) may be provided as the signal for the second target nucleic acid sequence, and the signal provided may be used to determine the presence of the second target nucleic acid sequence. The provided signal for the second target nucleic acid sequence is analyzed using an additional threshold for determining the presence of the second target nucleic acid sequence.

According to one embodiment of the present invention, the signal detected at the relatively high detection temperature in the step (a) is provided as the signal for the first target nucleic acid sequence.

According to an alternative embodiment of the present invention, when the signal detected at the relatively high detection temperature does not satisfy the first criterion defined by the HTT, any signal indicative of the absence of a target nucleic acid sequence is provided as the signal for the absence of the first target nucleic acid sequence.

As used herein, the expression "signal indicative of the absence of a target nucleic acid sequence" is used to encompass a signal having signal values by which the absence of the target nucleic acid sequence is determined. For example, the expression is used to encompass a signal indicating that there is no or substantially no target nucleip acid present in a sample {e.g., a signaj_. having signal values of RFU zero or signal values close to RFU zero), or a signal having such low signal values that the target nucleic acid sequence is determined not to be significantly present by a predetermined threshold {e.g., a signal having signal values lower than the threshold).

According to one embodiment of the present invention, when the signal detected at the relatively high detection temperature does not satisfy the first criterion defined by the HTT, any of the following signals is provided as the signal for the first target nucleic acid sequence: (i) the signal detected at the relatively high detection temperature in_;the step (a); (ii) the signal obtained from a negative control that does not contain the target nucleic acid sequence; or (iii) any signal indicative of the absence of a target nucleic acid sequence. These signals indicate that the sample does not contain the first target nucleic acid sequence.

According to one embodiment of the present invention, when the signal detected at the relatively high detection temperature^ does not satisfy tlie first criterion defined by the HTT 120, any of the signals (i)-(iii) is provided as the signal for the first target nucleic acid sequence, and the signal detected at the relatively low detection temperature in the step (a) is provided as the signal for the second target nucleic acid sequence 125.

Therefore, according to the embodiment of the present invention, in the case of a sample containing only the second target nucleic acid sequence (B-type), any of the signals (i)-(iii) is provided as the signal for the first target nucleic acid sequence, and the significant signal detected at the relatively low detection temperature in the step (a) is provided as the signal for the second target nucleic acid sequence. Also, in the case of a sample not containing the first target nucleic acid sequence and the second target nucleic acid sequence (D-type), any of the signals (i)-(iii) is provided as the signal for the first target nucleic acid sequence, and the insignificant signal detected at the relatively low detection temperature in the step (a) is provided as the signal for the second target nucleic acid sequence.

The provided signals may be used to determine the presence or absence of each target nucleic acid sequence by applying new additional thresholds.

When the signal detected at the relatively high detection temperature satisfies the first criterion, the next step (c) 130 proceeds. Step (c): Evaluating the signals detected at the relatively low detection temperature and the relatively high detection temperature bv a signal- difference threshold 130

When the first criterion is satisfied in the step (b), it is then performed to identify whether the signal difference between the signal detected at the relatively low detection temperature and the signal detected at «the relatively high detection

' ^■ 4, ; . .^{■ "}-^■·^■

temperature satisfies a second criterion defined by a signal-difference threshold (SDT) 130. When the signal difference satisfies the second criterion, the next step 140 proceeds.

The signal difference and SDT may be used to determine the presence or absence of a second target nucleic acid sequence.

The "signal difference" between the signal detected at the relatively low detection temperature and the signal detected at the relatively high detection temperature as used in this step is a value representing a change, a signal change or a signal difference between the signals detected at the relatively low detection temperature and the relatively high detection temperature from the sample (both signals obtained in the step (a)). That is, the "signal difference" includes any value reflecting the change of signals at the different detection temperatures from the sample. The signal difference may be expressed in various manners. For example, a signal difference may be expressed by a numerical value, the presence/absence of a signal, or a plot of signal characteristics.

The signal difference is a value representing a change of the signals detected at the relatively high detection temperature and the relatively low detection temperature from the sample of interest.

According to one embodiment of the present invention, the signal difference may be obtained by mathematically processing the signals detected at the relatively high detection temperature and the relatively low detection temperature from the sample.

According to one embodiment of the present invention, the mathematical processing includes calculation (e.g., addition, subtraction, multiplication and division) using signals or other values derived from the signals.

Particularly, the mathematical processing is a subtraction or ratio of the signal detected at the relatively low detection temperature and the signal detected at the relatively high detection temperature, more particularly a ratio, and most particularly a ratio of the signal detected at the relatively low detection temperature to the signal detected at the relatively high detection temperature.

In a particular embodiment of the invention, the signal difference is a ratio of

I

signal values at one cycle, i.e., a ratio of a signal value at one cycle in the signal detected at the relatively high detection temperature and a signal value at the same cycle in the signal detected at the relatively low detection temperature. The cycle selected for the calculation of the ratio is any cycle following the baseline region, particularly any cycle within the plateau region, and more particularly the end cycle in the amplification curve. According to one embodiment of the present invention, the ratio may be an average value of the ratios in several cycles, for example, two, three, four or five consecutive cycles. According to an embodiment of the present invention, the signal difference is a ratio of the signal value at the end cycle in the signal detected at the relatively low detection temperature versus the signal value at the end cycle in the signal detected at the relatively high detection temperature.

The mathematical processing for obtaining the signal difference may be performed in various ways. Mathematical processing may be performed using a machine. For example, the signals may be mathematically processed by a processor in a detector or real-time PCR instrument. Alternatively, the signals may be mathematically processed manually, in particular according to a certain algorithm.

The term "signal-difference threshold (SDT)" as used herein refers to a determined value for distinguishing a sample containing only the first target nucleic acid sequence (A-type) from a sample containing both the first target nucleic acid sequence and the second target nucleic add sequence (C-type). The SDT is compared with the signal difference as described above and the comparison result allows for distinguishing between the sample containing only the first target nucleic acid sequence and the sample containing both the first target nucleic acid sequence and the second target nucleic acid sequence. The SDT may be referred to as a second criterion.

The SDT may be set in various manners and applied to the present invention. According to one embodiment, the SDT is determined by considering: (i) the signal difference between the signals at the relatively low detection temperature and the

^{■ ■} . · ^' ' ^' "i

relatively high detection temperature for a control sample containing only the first target nucleic acid sequence; and (ii) the signal difference between the signals at the relatively low detection temperature and the relatively high detection temperature for the control sample containing both the first target nucleic acid sequence and the second target nucleic acid sequence. The signal differences which are obtained from the control samples and used to set the SDT and which are obtained from the control samples may be calculated in the same manner as the signal difference which is obtained from the test sample as described above. However, it is noted that the signal difference is obtained by analyzing the signals from a test sample of interest (i.e., unknown sample), while the SDT is obtained by analyzing the signals from two control samples to acquire two signal differences and then selecting a value in considering the two signal differences.

Specifically, the SDT may be determined as follows:

First, a control sample (a first control sample) containing only the first target nucleic acid sequence is incubated with the first signal generating means and two signals are detected at the relatively high detection temperature and the relatively low detection temperature. Afterwards, a signal difference is calculated from the signals at the relatively high detection temperature and the relatively low detection temperature for the first control sample;

Second, a control sample (a second control sample) containing both the first target nucleic acid sequence and the second target nucleic acid sequence is also incubated with the first signal generating means and , the second signal generating means, and two signals are detected at the relatively high detection temperature and the relatively low detection temperature. Afterwards, the signal difference is calculated from the signals at the relatively high detection temperature and the relatively low detection temperature for the second control sample.

Finally, a SDT is set in consideratipn of both the ^signal difference for the first control sample and the signal difference for the second control sample.

For example, when the signal difference (e.g., a ratio) for the first control sample is 1.15 and the signal difference (e.g., a ratio) for the second control sample is 2.09, the SDT may be set to any appropriate value between 1.15 and 2.09 (e.g., 1.50).

According to one embodiment, the SDT is any value between (i) the signal difference between the signals at the relatively high detection temperature and the relatively low detection temperature for the control sample containing only the first target nucleic acid sequence and (ii) the signal difference between the signals at the relatively high detection temperature and the relatively low detection temperature for the control sample containing both the first target nucleic acid sequence and the second target nucleic acid sequence.

The signal difference for the control samples may be both acquired in a specific range via repeated experiments. For example, when the signal difference for the control sample containing only the first target nucleic acid sequence is acquired in the range of 1.05 to 1.30 via repeated experiments, and the signal difference for the control sample containing both the first target nucleic acid sequence and the second target nucleic acid sequence is acquired in the range of 1.80 to 2.30 via repeated experiments, the SDT may be set to a value which is between the two ranges and does not overlap the two range, for example a value higher than 1.30 and lower than 1.80.

According to one embodiment, the SDT is set to any value which is higher than the ratio of the signal values at end cycles in the signals detected at the relatively low detection temperature and the relatively high detection temperature from a control containing only the first target nucleic acid sequence and which is lower than the ratio of the signal values at end cycles in the signals detected at the relatively low detection temperature and the relatively high detection temperature from a control containing both the first-target nucleic acid sequence and the second target nucleic acid sequence.

When the SDT is expressed by a ratio, the SDT value may or may not be identical to the reference value as will be described below.

Although the SDT is explained mainly by the ratio of the signal values in the specification, it will be appreciated by those skilled in the art that various other modified signal-difference thresholds can be devised by considering the calculating method for a signal difference or considering the signal pattern difference between a control sample containing only the first target nucleic acid sequence and a control sample containing both the first target nucleic acid sequence and the second target nucleic acid sequence.

The expression "second criterion defined by a signal-difference threshold (SDT)" as used herein refers to a criterion which is defined in consideration of the SDT. For example, the second criterion defined by the signal-difference threshold is that the signal difference is more than, not less than, not more than, or less than the signal-difference threshold. For example, if the SDT is set to 1.5 based on the ratio of the signal values at the end cycle, the second criterion defined by the SDT may be that the signal difference is 1.5 or more.

In the method of the present invention, the SDT and the signal difference should be calculated by the same mathematical processing. For example, if the SDT is calculated by subtraction of the signals, the signal difference should be also calculated by subtraction of the signals. Alternatively, if the SDT is calculated by the ratio of the signals, the signal difference should also be calculated by^the ratio of the signals.

According to an embodiment, when the signal difference calculated in the step

(c) does not satisfy the second criterion defined by the SDT, the method of the present invention provides any signal indicative of the absence of a target nucleic acid sequence as the signal for the absence of the second target nucleic acid sequence, without signal extraction process 135. Such a signaj indicates that the sample contains the first target nucleic acid sequence but does not contain the second target nucleic acid sequence. In this case, the signal detected at the relatively high detection temperature or at the relatively low detection temperature in the step (a) may be provided as the signal for the first target nucleic acid sequence without signal extraction.

For example, if the signal difference is calculated as the ratio of the signal value at the end cycle in the signal detected at the relatively low detection temperature to the signal value at the corresponding cycle in the signal detected at the relatively high detection temperature, the signal difference of less than the SDT indicates that the sample contains only the first target nucleic acid sequence _>(A-type of sample).

The signals provided may be used to determine the presence or absence of each target nucleic acid sequence by applying a new additional threshold.

When the first criterion defined by the HTT is satisfied in the step (b), the sample is determined to be either A-type or C-type. For the A-type or C-type sample, signals are generated at both the relatively high detection temperature and the relatively low detection temperature. However, the signal difference between the signal at the relatively high detection temperature and the signal at the relatively low detection temperature is much higher in C-type compared to A-type. Therefore, in this step, it is possible to distinguish A-type and C-type samples by analyzing the signal differences by the second criterion.

When the signal difference satisfies the second criterion, the next step (c) 140 proceeds. It will be appreciated by those skilled in the art that the steps (b) and (c) may be performed in reverse order.

According to an embodiment, the method may be performed as follows:

(a) incubating the sample with a first signal-generating means capable of generating a signal for a first target nucleic acid sequence and a second signal- generating means capable of generating a signal for a second target nucleic acid sequence in a single reaction vessel, and detecting signals at a relatively high detection temperature and a relatively low detection temperature by a single Jtype of detector; wherein the first signal-generating means generates signals at both the relatively high detection temperature and the relatively low detection temperature in the presence of the first target nucleic acid sequence in the sample and the second signal-generating means generates a signal at the relatively low detection temperature in the presence of the second target nucleic acid sequence in the sample; wherein the two signals to be generated by the two signal-generating means are not differentiated by the single type of detector;

(c) identifying whether a signal difference between the signal detected; at the relatively low detection temperature and the signal detected at the relatively high detection temperature satisfies a second criterion defined by a signal-difference threshold (SDT); when the signal difference satisfies the second criterion, the next step (b) proceeds;

(b) identifying whether the signal detected at the relatively high detection temperature satisfies a first criterion defined by a high-temperature threshold (HTT); when the signal detected at the relatively high detection temperature satisfies the first criterion, the next step (d) proceeds; and

(d) extracting the signal for the second target nucleic acid sequence by using the signal detected at the relatively high detection temperature in the step (a), the signal detected at the relatively low detection temperature in the step (a) and a reference value for the first target nucleic acid sequence, thereby providing the signal for the second target nucleic acid sequence; wherein the reference value for the first target nucleic acid sequence is a value representing a relationship of change in signals provided by the first signal-generating means at the relatively high detection temperature and the relatively low detection temperature; wherein the reference value for the first target nucleic acid sequence is determined from a control reaction using the first target nucleic acid sequence and the first signal-generating means.

According to the embodiment, when the signal difference does not satisfy the second criterion, any signal indicative of the absence f a target nucleic acid sequence is provided as the signal for the absence of the second target nucleic acid sequence without signal extraction process, and the signal detected at the relatively high detection temperature in the step (a) is provided as the signal for the first target nucleic acid sequence without signal extraction.

According to the embodiment, when the signal detected at the relatively high detection temperature does not satisfy the first criterion, the signal detected at the relatively low detection temperature in the step (a) is provided as the signal for the second target nucleic acid sequence, and any signal indicative of the absence of a target nucleic acid sequence or the signal detected at the relatively high detection temperature in the step (a) is provided as the signal for the first target nucleic acid sequence.

According to the embodiment, when the second criterion and the first criterion are all satisfied, the step (d) is performed to extract the signal for the second target nucleic acid sequence.

Step (d): Providing a signal for a second target nucleic acid sequence using a reference value ( 140) - signal extraction process

When the first criterion in the step (b) and the second criterion in the step (c) are all satisfied, a signal for the second target nucleic: acid sequence is extracted using the signal detected at the relatively high detection temperature in the step (a), the signal detected at the relatively low detection temperature in the step (b and a reference value for the first target nucleic acid sequence,, thereby providing the signal for the second target nucleic acid sequence. In the step (d), the reference value for the first target nucleic acid sequence is a value representing a relationship of change in signals provided by the first signal-generating means at the relatively high f i

detection temperature and the relatively low detection, temperature. The reference value for the first target nucleic acid sequence is determined from a control reaction using the first target nucleic acid sequence and the first signal-generating means.

:^{■ ■}* ^! I

As described above, a process of extracting a signal for a specific target nucleic acid sequence by using signals detected at two different detection temperatures and

-. '

a reference value is referred herein to as a "signal extraction process". Specifically,

^■ ¾ ^r (,

the signal extracting process refers to a process of extracting only signal derived from the target nucleic acid sequence of interest by eliminating other signals f om the signal detected at a particular detection temperature.

The term "reference value" as used herein describes a relationship or degree of change in signals, a signal change or a signal difference, in particular numerically, when two signals are generated at different detection temperatures (i.e., relatively high detection temperature and relatively low detection temperature). Stated otherwise, the "reference value" includes any value reflecting a pattern (rule) of a signal change at different detection temperatures. Also, the term "reference value" indicates a value representing the degree of change between a signal detected at relatively high detection temperature and a signal detected at relatively low detection temperature for a particular target nucleic acid sequence. The reference value may indicate a value used herein to transform, convert, adjust or modify a signal detected at one temperature into a signal at another temperature. The reference value may vary depending upon the types of the target nucleic acid sequences, the types of the signal-generating means and the conditions of incubation and detection. Thus, a variety of reference values may be determined for different or same target nucleic acid sequences.

The reference value may be expressed in various aspects. For example, the reference value may be expressed as numerical values, the presence/absence of signal or plot with signal characteristics.

Particularly, the term "reference value" as used herein refers to a reference value which is determined for the first target nucleic acid sequence and which is used in the signal extraction of the second target nucleic acid sequence.

In an embodiment, the reference value may be determined considering a signal value at a selected cycle. In other words, the reference value may be determined considering a signal value at a selected cycle among signals detected at a

¾ .7 relatively high detection temperature and at a relatively low detectipn temperature. In such case, the selected cycle may be one of cycles following a baseline region of an amplification curve, particularly one of cycles in a plateau region, more particularly an end cycle.

In an alternative embodiment, the reference value may be dete/mined considering a plurality of signal values at different selected cycles. For instance, the reference value may be determined considering a mean of a plurality of signal values at different selected cycles.

The reference values will be described in detail below.

The reference value for the first target nucleic acid sequence is a value representing a relationship of change in signals provided by the first signal-generating means at the relatively high detection temperature and the relatively low detection temperature.

According to an embodiment, the reference value for the first target nucleic acid sequence may be determined by incubating a first target nucleic acid sequence with a signal-generating means, detecting signals at a relatively high detection temperature and a relatively low detection temperature, and then obtaining a relationship of change in signals detected at the relatively high detection temperature and the relatively low detection temperature.

According to an embodiment, the reference value for the first target nucleic f

acid sequence is determined by using a standard material corresponding to a first target nucleic acid sequence.

determined by acquiring a certain range of values via iterative reactions for the control reaction (or control sample) under various conditions (e.g., concentrations of a target nucleic acid sequence and types of primers) and selecting a suitable one among the acquired values.

In an embodiment, the reference value for the first target nucleic acid sequence may be selected such that the signal for the first target nucleic acid sequence not to be extracted is eliminated. In other embodiment, the reference value for the first target nucleic acid sequence may be selected such that the signal for the second target nucleic acid sequence to be extracted is not eliminated or eliminated as little as possible. In another embodiment, the reference value for the first target nucleic acid sequence may be selected such that the signal for the first target nucleic acid sequence not to be extracted is eliminated and the signal for the second target nucleic acid sequence to be extracted is not eliminated or eliminated as little as possible.

For example, when the target nucleic acid sequences comprise ""Chlamydia trachomatis (CT)" and ""Neisseria gonorrhoeae (NG)" and a signal for "NG" is intended to be extracted, the reference value for CT may be selected such that the signal for i

CI is completely eliminated and the signal for NG is not eliminated or eliminated as little as possible.

The reference value for the first target nucleic acid sequence may be empirically obtained by repetitive experiments.

The reference value for the first target nucleic acid sequence may be selected among a certain range empirically obtained by repetitive experiments. In this regard,

ί

it is advantageous that a relatively high value within the range is selected as a reference value, because the relatively high reference value has a higher propensity

.

to eliminate a signal intended not to be extracted, compared to relatively low reference values. For example, when a range from 1.1 to 1.50 is obtained, a value i ί around 1.50 may be more suitable as a reference value. Alternatively, a value exceeding the range may be selected as a reference value. For example, a. value of

5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% greater than the t

upper limit of the range may be selected a reference value. However, it is noted that too high reference value is not desirable, since it may rather eliminate a signal intended to be extracted.

According to an embodiment, the reference value for the first target nucleic acid sequence can be calculated by the difference between signals provided by the first signal-generating means at the relatively high detection temperature and the relatively low detection temperature.

According to an embodiment, the reference value for the first target nucleic acid sequence can be calculated by mathematically processing the signals provided by the first signal-generating means at the relatively high detection temperature and the relatively low detection temperature.

In certain embodiment, the mathematical processing includes calculation (e.g., addition, multiplication, subtraction and division) using signals or other values derived from signals.

According to an embodiment of this invention, the mathematical processing of the signals to obtain the reference value for the first target nucleic acid sequence is a calculation of a ratio of the signal provided by the first signal-generating means at the relatively low detection temperature to the signal provided by the first signal- generating means at the relatively high detection temperature" According to an embodiment of this invention, the mathematical processing of the signals to obtain the reference value for the first target nucleic acid sequence is a calculation of a ratio of the signal provided by the first signal-generating means at the relatively high detection temperature to the signal provided by the first signal-generating means at the relatively low detection temperature.

The ratio may be a ratio of a signal value at a cycle of signals detected at the relatively low detection temperature to a signal value at the cycle of signals detected at the relatively high detection temperature. Alternatively^", the ratio may be a ratio of a signal value at a cycle of signals detected at the relatively high detection temperature to a signal value at the cycle of signals detected at the relatively low detection temperature. The cycle selected for ratio calculation may be one of cycles following a baseline region of an amplification curve. Particularly, the cycle for ratio calculation may be one of cycles in a plateau region. More particularly, the cycle for ratio calculation may be the end cycle.

In an embodiment, the reference value for the first target nucleic acid sequence can be obtained by a mean of ratios at several cycles, e.g., consecutive two cycles, three cycles, four cycles, five cycles, etc. Further, the reference value for the first target nucleic acid sequence can be suitably selected in consideration of ratios at several cycles. For example, the reference value for the first target nucleic acid sequence can be selected as being a slightly higher than a ratio of a signal value at a cycle or ratios of signal values at several cycles.

In a particular embodiment, the reference value for the first target nucleic acid sequence may be calculated in accordance with the mathematical equation III:

< Equation III>

Reference value for the first target nucleic acid sequence = [signal at the relatively low temperature for a sample containing only the first target nucleic acid sequence] ÷ [signal at the relatively high temperature for a sample containing only the first target nucleic acid sequence]

The mathematical processing for obtaining the reference value may be carried out in various fashions. The mathematical processing may be carried out by use of a machine. For example, the signals may undergo a mathematical processing by a processor in a detector or real-time PCR device. Alternatively, the signals may manually undergo a mathematical processing particularly according to a determined algorithm.

According to an embodiment of this invention, signal-generating means for the reference value for the first target nucleic acid sequence, may be the same as that for the step (a).

According to an embodiment, the signal for the second target nucleic acid sequence is provided either (i) by using the signal detected at the relatively high detection temperature in the step (a) and the reference value for the first target nucleic acid sequence to eliminate the signal for the first target nucleic acid sequence {i.e., the signal generated by the first signal generating means) at the relatively low detection temperature from the signal detected at the relatively low detection temperature in the step (a), or (ii) by using the signal detected at the relatively low detection temperature in the step (a) and the reference value for the first target nucleic acid sequence to eliminate the signal for the first target nucleic acid sequence {i.e., the signal generated by the first signal generating means) at the relatively high detection temperature from the signal detected at the relatively high detection temperature in the step (a).

More particularly, the elimination of the signal generated by the first signal generating means at the relatively low detection temperature is to mathematically eliminate the signal generated by the first signal generating means at the relatively low detection temperature from the signal detected at the relatively low detection temperature in the step (a), and the elimination of the signal generated by the first signal generating means at the relatively high detection temperature is to mathematically eliminate the signal generated by the first signal generating means at the relatively high detection temperature from the signal detected at the relatively high detection temperature in the step (a).

i

Still more particularly, the signal for the second target nucleic acid sequence

v, - - may be provided by the following mathematical equation I:

< Equation I>

Signal for the second target nucleic acid sequence = [signal at the relatively low detection temperature in the step (a)] - [(signal at the relatively high detection temperature in the step (a)) x (the reference value for the first target nucleic acid sequence)];

i . ^' >..

wherein the reference value for the first target nucleic acid sequence is a ratio of a signal provided by the first signal-generating rpeans at the relatively low detection temperature to a signal provided by the first signal-generating means at the relatively high detection temperature.

In the mathematical equations described herein, the symbol "-" represents minus, particularly signal subtraction. For example, the signal subtraction may be performed at each cycle by subtracting a signal value at a cycle in one signal from a signal value at a corresponding cycle in another signal.

In the mathematical equations described herein, the symbol "x" represents multiplication.

In the mathematical equations described herein, the symbol represents division.

According to the extraction of the signal for the second target nucleic acid sequence by the mathematical equation I, the signal detected at the relatively low detection temperature reflects a combination of the signal for the first target nucleic acid sequence and the signal for the second target nucleic acid sequence, whereas the signal detected at the relatively high detection temperature only reflects the signal for the first target nucleic acid sequence. Thus, the signal for the second target nucleic acid sequence may be obtained by subtracting the signal detected at the relatively high detection temperature frqm the signal detected at the relatively low detection temperature, with a proviso that the signals for the first target nucleic acid sequence at the relatively high detection temperature and the relatively low detection temperature are not changed. However, considering that the signals vary depending upon the detection temperatures, it is necessary to adjust (transform) the signal detected at the relatively high detection temperature into a signal to be expected at the relatively low detection temperature, prior to the signal subtraction. For this purpose, the reference value for the first target nucleic acid sequence is used.

Still more particularly, the signal fpr the second target nucleic acid sequence may be provided by the following mathematical equation II:

< Equation II>

Signal for the second target nucleic acid sequence = [signal at the relatively high detection temperature in the step (a)] - [(signal at the relatively low detection temperature in the step (a)) ÷ (the reference value for the first target nucleic add sequence)];

wherein the reference value for the first target nucleic acid sequence is a ratio of a signal provided by the first signal-generating means at the relatively low detection temperature to a signal provided by the first signal-generating means at the relatively high detection temperature.

Unlike the signal extraction using the mathematical equation I, the signal extraction using the mathematical equation II is intended to convert the signal generated from the first signal-generating means at the relatively low detection temperature in the step (a) into a signal expected to be detected at the relatively high detection temperature and then subtract it from the signal generated from the first signal-generating means at the relatively high detection temperature in the step (a). Although the signal extracted by using the mathematical equation II is slightly lower in its intensity compared to that of the signal extracted by using the mathematical equation I, the former can be used for the qualitative determination of the target nucleic acid sequence in a similar manner to the latter. In the mathematical equations I and II, the reference value for the first target nucleic acid sequence is represented by the ratio of the signal at the relatively low detection temperature to the signal at the relatively high detection temperature. However, it would be understood by one of skill in the art that the signal extraction for the second target nucleic acid sequence may be accomplished with a modification using a reference value represented by the ratio of the signal at the relatively high detection temperature to the signal at the relatively low detection temperature. Those of skill in the art would understand that such modification falls within the spirit and scope of the invention.

presence or absence of the second target nucleic acid sequence. In other words, the presence of the second target nucleic acid sequence (or the significance of the second target nucleic acid sequence) may be verified by applying an additional threshold to the signal.

According to one embodiment, the method further comprises the following step (e): (e) identifying whether the signal for the second target nucleic acid sequence provided in the step (d) satisfies a fourth criterion defined by a verification threshold (vT); when the signal for the second target nucleic acid sequence does not satisfy the fourth criterion, any signal indicative of the absence of a target nucleic acid sequence is provided as the signal for the absence of the second target nucleic acid sequence. In this case, the signal detected at the relatively high detection temperature of the step (a) or the signal detected at the relatively low detection temperature of the step (a) may be provided as the signal for the first target nucleic acid sequence. ^■. ·:^■■ -' f

The "verification threshold (VT)" as used herein refers to a threshold used for

. ^h , *

verifying the significance of the signal provided by the signal extraction process. The

VT is similar to the HTT and the LTT in the sense that it is used to check the significance of the signal. The details of the VT can be referred to the disclosures of the HTT and the LTT. The VT may also be expressed as a fourth threshold.

<^■ ,

The VT used to determine the significance of the signal may or may not differ

. 5; /

from a threshold used to finally determine the presence of a target nucleic acid sequence.

The expression "fourth criterion defined by the verification threshold (VT)" as

^{■ ■} <f ^'■*- -^{■ ■} - · - b

used herein means a criterion which is defined in consideration of the VT. For example, if the VT value is RFU 300, the fourth criterion defined by the VT may be that the signal is not less than RFU 300.

If the signal for the second target nucleic acid sequence provided using the reference value satisfies the fourth criterion defined by the VT, it can be again verified that the signal provided using the reference value per se is a signal for the second target nucleic acid sequence. If not, the signal provided using the reference value is used to indicate that the second target nucleic acid sequence is absent in a sample, or any signal indicative of the absence of a target nucleic acid sequence is provided as the signal for the absence of the second target nucleic acid sequence instead of the signal provided using the reference value.

According to one embodiment, when the signal for the second target nucleic acid sequence is provided by the mathematical equation I, the fourth criterion defined by the VT is that the signal is not less than the VT. In other words, when the signal for the second target nucleic acid sequence is not less than the VT, the significance of the signal and thus the presence of the second target nucleic acid sequence are determined.

According to one embodiment, when the signal for the second target nucleic acid sequence is provided by the mathematical equation II, the fourth criterion defined by the VT value is that the signal is not more than the verification threshold. The VT for verifying the signal for- the second target nucleic acid sequence provided using the reference value can be set by referring to the disclosure of the above-mentioned HTT. For example, if the signal for the second target nucleic acid sequence is provided by the mathematical equation I, the VT of RFU 300 and the fourth criterion of not less than RFU 300 may be determined by taking into account background signal, sensitivity, characteristics of label or error range.

The provided signals may be used to determine the presence or absence of each target nucleic acid sequence by applying an additional threshold. According to the embodiments of the present invention, a signal for each target nucleic acid sequence can be provided for a sample containing one or more target nucleic acid sequences. The embodiments has technical features: (i) a signal for a target nucleic acid sequence, particularly the second target nucleic acid sequence is provided with "signal extraction process" only when the criteria of the steps (b) and (c) are satisfied, and (ii) any appropriate signal selected from the signal detected in the step (a) and the signal indicative of the absence of a target nucleic acid sequence is provided as the signal for the absence of the second target nucleic acid sequence without "signal extraction process" when any one of the criteria of the steps (b) and (c) is not satisfied.

The method of the present invention may be performed with a little modification. Hereinafter, two different embodiments with a little modification will be described in detail. These two different embodiments are shown in Figures 3 and 4, respectively.

The method according to another embodiment 200 will now be described with reference to Fig. 3.

Step (a): Detecting signals at two different temperatures 210

Since this step (a) 210 is the same as the step (a) 110 in the embodiment of the present invention 100, a detailed description thereof is omitted. Step (b): Evaluating the signal at the relatively high detection temperature by a high-temperature threshold 220

This step (b) 220 will be referred to the step (b) 120 in the embodiment of the present invention 100.

According to the embodiment of the present invention 100, when the signal detected at the relatively high detection temperature does not satisfy the first criterion defined by the HTT, the signal detected at the relatively low detection temperature in the step (a) is provided as the signal for the second target nucleic acid sequence 125.

In contrast, according to the another embodiment of the present invention 200, when the signal detected at the relatively high detection temperature does not satisfy the first criterion defined by the HTT, the method further comprises the step of identifying whether the signal detected at the relatively low detection temperature satisfies a third criterion defined by a low-temperature threshold (LTT) 222.

The signal detected at the relatively low detection temperature satisfies the third criterion, the next step 230 proceeds.

Hereinafter, the step 222 will be described. Step {by. Evaluating the signal at the relatively low detection temperature by a low-temperature threshold 222

In the step (b¹), the signal detected at the relatively low detection temperature is evaluated in order to determine whether a second target nucleic acid sequence is present in the sample which had been determined to contain no first target nucleic acid sequence 222.

The description of the LTT for evaluating the signal detected at the relatively low detection temperature can be referred to the disclosure to the HTT as described above.

The expression "low-temperature threshold (LTT)" as used in this step (b⁷) means a threshold for determining the significance of the signal detected at the relatively low detection temperature. The LTT may be expressed as a third threshold.

As used herein, the expression "third criterion determined by the low- temperature threshold" means a criterion which is defined by considering the LTT for determining the significance of the signal detected at the relatively low detection temperature. For example, if the LTT is RFU 500, the third criterion defined by the LTT may be RFU 500 or higher.

·^■ i

When the signal detected at the relatively low detection temperature satisfies . ., ^'" ■ · ·- the third criterion defined by the LTT, it indicates that the second target nucjeic acid sequence is present in the sample. If it is determined in the prior step (b) that the first target nucleic acid sequence is not present, the signal detected at the relatively low detection temperature will have only. the signal provided by the second target nucleic acid sequence. Thus, when the signal detected at the relatively low detection temperature satisfies the third criterion defined by the LTT, it indicates that the second target nucleic acid sequence is likely to be present in the sa.mple.

In other words, when the signal detected at the relatively high detection temperature does not satisfy the first criterion defined by the HTT, but satisfies the third criterion defined by the LTT, the method provides the signal detected at the relatively low detection temperature as the signal for the second target nucleic acid sequence without signal extraction process 226. This signal provided indicates that the sample does not contain the first target nucleic acid sequence but contains the second target nucleic acid sequence (B-type of sample). In this case, the signal detected at the relatively high detection temperature in the step (a) or any signal indicative of the absence of a target nucleic acid sequence may be provided as the signal for the absence of the first target nucleic acid sequence.

When the signal detected at the relatively low detection temperature in the step (b does not satisfy the third criterion defined by the LTT, the method provides any signal indicative of the absence of a target nucleic acid sequence as the signal for the absence of the second target nucleic acid sequence 224. The signal indicative of the absence of a target nucleic acid sequence may be any one selected from the group consisting of (i) the signal detected at the relatively low detection temperature in the step (a), (ii) a signal obtained from a negative control containing no target nucleic acid sequence, and (iii) any signal in which the target nucleic acid sequence is absent. This signal indicates that the sample does not contain both the first target nucleic acid sequence and the second target nucleic acid sequence. In this case, any signal indicative of the absence of a target nucleic acid sequence may be provided as the signal for the absence of the first target nucleic acid sequence.

According to an embodiment, the signal detected at: the relatively high detection temperature in the step (a) is provided as the signal for the first target nucleic acid sequence.

Step (c): Evaluating the signals detected at the relatively low detection temperature and the relatively high detection temperature by a signal-difference threshold 230

Since this step (c) 230 is the same as the step (c) 130 in the embodiment of the present invention 100, a detailed description thereof is omitted.

Step (d): Providing the signal for the second target nucleic acid sequence using the reference value 240

Since this step (d) 240 is the same as the step (d) 140 in the embodiment of the present invention 100, a detailed description thereof is omitted.

Next, the method according to still another embodiment 300 will now be described with reference to Fig. 4.

Step (a): Detecting signals at two different temperatures 310

Since this step (a) 310 is the same as the step (a) 110 in the embodiment of the present invention 100, a detailed description thereof is omitted.

Step (b"): Evaluating the signal at the relatively low detection temperature by a low-temperature threshold 320

In the step (b"), the signal detected at the relatively low detection temperature

- f 7 I is evaluated to determine whether the signal at the relatively low detection temperature is significant 320.

The another embodiment of the present invention 300 is characterized by further comprising the step of identifying whether the signakdetected at the relatively low detection temperature satisfies a third criterion defined by a LTT 320 prior to the step (b), compared with the embodiment of the present invention 100.

In the step (b"), the description of the LTT for eva uating the signal detected at the relatively low detection temperature is as described above. However, satisfying the third criterion defined by the LTT as used in the step (b means that the second target nucleic acid sequence is likely to be present in the sample; whereas satisfying the third criterion defined by the LTT as used in the step (b") means that either or both of the first target nucleic acid sequence and the second target nucleic acid sequence are likely to be present in the sample.

When the signal detected at the relatively low detection temperature does not satisfy the third criterion defined by the LTT, the method provides any signal indicative of the absence of a target nucleic acid sequence as the signal for the absence of the second target nucleic acid sequence 322. The signal indicative of the absence of a target nucleic acid sequence may be any one selected from the group consisting of (i) the signal detected at the relatively low detection temperature in the step (a), (ii) a signal obtained from a negative control containing no target nucleic acid sequence, and (iii) any signal in which a target nucleic acid sequence is absent. This signal indicates that the sample does not contain both the first target nucleic acid sequence and the second target nucleic acid sequence. In this case, any signal indicative of the absence of a target nucleic acid sequence may be provided as the signal for the absence of the first target nucleic acid sequence.

Meanwhile, when the signal detected at the relatively low detection temperature satisfy the third criterion defined by the LTT, the next step (b) 330 proceeds. Step (b): Evaluating the signal at the relatively hiah_detection temperature by a high-temperature threshold 330

The step (b) 330 is referred to the step (b) 110 in the embodiment of the present invention 100.

According to the embodiment of the present invention 100, when the signal detected at the relatively high detection temperature does not satisfy the first criterion defined by the HTT, it indicates that the sample contains only the .second target nucleic acid sequence or the sample contains no target nucleic acid sequence;

^' " k - *^' i} ^' ~^~~ whereas according to another embodiment of the present invention 300, it indicates that the sample contains only the second target nucleic afcid sequence.

Step (c): Evaluating the signals detected at the relatively low detection temperature and the relatively high detection temperature by a signal-difference threshold 340

Since this step (c) 340 is the same as the step (c) 130 in the embodiment of the present invention 100, a detailed description thereof is omitted.

Step (d): Providing the signal for the second target nucleic acid sequence using a reference value 350

Since this step (d) 350 is the same as the step (c) 140 in the embodiment of the present invention 100, a detailed description thereof is omitted.

Although the invention has been described in terms of three embodiments providing a signal for each target nucleic acid sequence for a sample containing up to two target nucleic acid sequences, it will be appreciated that those skilled in the art will be able to devise various modifications, which are within the scope of the present invention.

II. Storage medium, Computer program and Device for Signal Extraction In another aspect of this invention, there is provided a computer readable storage medium containing instructions to configure a processor to perform a.rnethod for providing a signal for at least one target nucleic acid sequence in a sample from signals detected at different temperatures, comprising trie steps of:

(a) incubating the sample with a first signal-generating means capable of generating a signal for a first target nucleic acid sequence and a second signal- generating means capable of generating a signal for a second target nucleic acid sequence in a single reaction vessel, and receiving signals detected at a rejatively high detection temperature and a relatively low detection temperature by a single

"ϊ

type of detector; wherein the first signal-generating means generates signals at both the relatively high detection temperature and the relatively low detection temperature in the presence of the first target nucleic acid sequence in the sample and the second signal-generating means generates a signal at the relatively low detection temperature in the presence of the second target nucleic acid sequence in the sample; wherein the two signals to be generated by the two signal-generating means are not differentiated by the single type of detector;

(b) identifying whether the signal detected at the relatively high detection temperature satisfies a first criterion defined by a high-temperature threshold (HTT); when the signal detected at the relatively high detection temperature satisfies the first criterion, the next step (c) proceeds;

(c) identifying whether a signal difference between the signal detected at the relatively low detection temperature and the signal detected at the relatively high detection temperature satisfies a second criterion defined by a signal-difference threshold (SDT); when the signal difference satisfies the second criterion, the next step (d) proceeds; and (d) extracting the signal for the second target nucleic acid sequence by using the signal detected at the relatively high detection temperature in the step (a), the signal detected at the relatively low detection temperature in the step (a) and a reference value for the first target nucleic acid sequence, thereby providing the signal for the second target nucleic acid sequence; wherein the reference value for the first target nucleic acid sequence is a value representing a relationship of change in signals provided by the first signal-generating means at the relatively high detection temperature arid the relatively low detection temperature; wherein the reference value for the first target nucleic acid sequence is determined from a control reaction using the first target nucleic acid sequence and the first signal-generating means; wherein the steps (b) and (c) may be performed in reverse order.

In still another aspect of this invention, there is provided a computer program to be stored on a computer readable storage medium to configure a processor to perform a method for providing a signal for at least one target nucleic acid sequence in a sample from signals detected at different temperatures, comprising the steps of:

(a) incubating the sample with a first signal-generating means capable of generating a signal for a first target nucleic acid sequence and a second signal- generating means capable of generating a signal for a second target nucleic acid sequence in a single reaction vessel, and receiving signals detected at a relatively high detection temperature and a relatively low detection temperature by a single type of detector; wherein the first signal-generating means generates signals at both the relatively high detection temperature and the relatively low detection temperature in the presence of the first target nucleic acid sequence in the sample and the second signal-generating means generates a signal at the relatively low detection temperature in the presence of the second target nucleic acid sequence in the sample; wherein the two signals to be generated by the two signal-generating means are not differentiated by the single type of detector;

(b) identifying whether the signal detected at the relatively high detection temperature satisfies a first criterion defined by a high-temperature threshold (HIT); when the signal detected at the relatively high detection temperature satisfies the first criterion, the next step (c) proceeds;

Since the storage medium, the device and the computer program of the prevent invention are intended to perform the present methods in a computer, the common descriptions between them are omitted in order to avoid undue redundancy leading to the complexity of this specification.

The program instructions are operative, when performed by the processor, to cause the processor to perform the present method described above. The program instructions for performing the present method may comprise: (i) an instruction to identify whether the signal detected at the relatively high detection temperature satisfies the first criterion defined by the HTT; (ii) an instruction to identify whether the signal difference between the signal detected at the relatively low detection temperature and the signal detected at the relatively high detection temperature satisfies the second criterion defined by the SDT; and (iii) an instruction to provide a signal for the second target nucleic acid sequence by using the signal detected at the relatively high detection temperature in the step (a), the signal detected at the relatively low detection temperature in the step (a) and the reference value for the first target nucleic acid sequence.

The present method described above is implemented in a processor, such as a processor in a stand-alone computer, a network attached computer or a data acquisition device such as a real-time PCR machine.

The types of the computer readable storage medium include various storage medium such as CD-R, CD-ROM, DVD, flash memory, floppy disk, hard drive, portable HDD, USB, magnetic tape, MINIDISC, nonvolatile memory ca/d, EEPROM, optical disk, optical storage medium, RAM, ROM, system memory and web server.

The signals from the signal-generating process may be received through several mechanisms. For example, the signals may be acquired by a processor resident in a PCR data acquiring device. The signals may be provided to the processor in real time as the signals are being collected, or it may be stored in a memory unit or buffer and provided to the processor after the experiment has been completed. Similarly, the signals may be provided to a separate system such as a desktop computer system via a network connection (e.g., LAN, VPN, intranet and Internet) or direct connection (e.g., USB or other direct wired or wireless connection) to the acquiring device, or provided on a portable medium such as a CD, DVD, floppy disk, portable HDD or the like to a stand-alone computer system. Similarly, the signals may be provided to a server system via a network connection (e.g., LAN, VPN, intranet, Internet and wireless communication network) to a client such as a notebook or a desktop computer system.

The instructions to configure the processor to perform the present invention may be included in a logic system. The instructions may be downloaded and stored in a memory module (e.g., hard drive or other memory such as a local or attached RAM or ROM), although the instructions can be provided on any software storage medium such as a portable HDD, USB, floppy disk, CD and _KDVD. A computer code for

.· . ■ ^{' ■}· ·^■ - k ¹ .

implementing the present invention may be implemented in a variety of coding languages such as C, C++, Java, Visual Basic, VBScript, JavaScript, Perl and XML. In

- ^■ A ^'* ■ A

addition, a variety of languages and protocols may be used in external and internal storage and transmission of data and commands according to the present invention.

In a further aspect of this invention, there is provided a device for providing a signal for at least one target nucleic acid sequence in a sample from signals detected at different temperatures, comprising (a) a computer processor any (b) the computer readable storage medium described above coupled to the computer processor.

According to an embodiment, the device further comprises a reaction vessel to

,..(..,.■,_* . ■^'.,, ■ - , - accommodate the sample and signal-generating means, a temperature controlling

. ^' . .■ ^"S ^; ■ - i : ^' ; ^{' ?}

means to control temperatures of the reaction vessel and/or a detector to detect signals at cycles.

The computer processor may be prepared in such a manner that a single processor can do several performances; Alternatively, the processor unit may be prepared in such a manner that several processors do the several performances, respectively.

According to an embodiment, the processor may be embodied by installing software into conventional devices for detection of target nucleic acid sequences (e.g. real-time PCR device).

For example, the present device may be embodied into a real-time PCR system. The system comprises a real-time PCR device for performing a real-time PCR amplification, and a computer system as a logic system connected to the real-time PCR device via a cable for providing and displaying signals. The computer system may display the resultants in various forms such as graphs, tables and words according to demands of users. The computer system may comprise instructions contained in a computer readable storage medium for performing the present method. The real-time PCR device and the computer system may be integrated into a system. The signals may be received with amplification curves in various fashions. For example, the signals may be received and collected by a processor in a data collector of the real-time PCR device. Upon collecting the signals, they may be provided to a processor in a real-time manner, or stored in a memory unit or buffer and then provided to a processor after experiments.

Likely, the signals may be provided from the real-time PCR device to the computer system such as a desktop computer system via network connection {e.g., LAN, VPN, intranet and internet) or direct connection {e.g., USB and wired or wireless direct connections), or via portable media such as CD, DVD, floppy disk and portable HDD. Alternatively, the signals may be provided to a server system via network connections {e.g., LAN, VPN, intranet, internet and wireless communication network) connected to a client such as notebook and desktop computer systems.

After the signals are received or obtained, a signal processor proceeds to evaluate the signals based on the determined HTT (optionally, the LTT and/or the VT) and SDT, and to provide a signal for a desired target nucleic acid sequence by using a determined reference value.

The HTT (optionally, the LTT and/or the VT), the SDT and the reference value may be determined by the product supplier or the user. Alternatively, such values may be determined by the apparatus of the present invention.

The process of the present invention may be undertaken by an application {i.e., program) installed into the computer system. Alternatively, it may be made by an application directly installed into the computer system through application store server or application provider servers in which the application is operable in an operating system of the computer system. The operating system includes Window, Macintosh and mobile operating systems such as iOS and Android that are installed into mobile terminals such as Smartphones and Tablet PC.

As described above, the present method may be embodied by an application {i.e., program) supplier-installed or user-direct installed into the computer system, and recorded in a computer readable storage medium. A computer program embodying the present method may implement aji functions. The computer program may a program comprising program instructions stored on a computer readable storage medium to configure a processor to perform the present method.

The computer program may be coded by using suitable computer languages such as C, C++, JAVA, Visual basic, VBScript, JavaScript, Perl, XML and machine languages. The program codes may include function codes for mathematical functions described above and control codes for implementing process in order by a processor of the computer system.

The codes may further comprise memory reference codes by which additional information or media required in implementing the above-described functions by the processor is referred at location (address) of internal or external memory of the computer system.

When the computer system requires communication with another computer or server in remote for implementing functions of the processor, the codes may further comprise communication-relating codes encoding how the processor is communicated with another computer or server in remote by using communication module {e.g., wired and/or wireless communication module) or what information or media is transmitted.

Functional programs and codes (code segments) for embodying the present invention may be easily inferred or modified by programmers in the art in considering system environments of computers reading storage media and executing programs.

The storage medium network-connected to the computer system may be distributed and computer-readable codes may be stored and executed in a distribution manner. In such case, at least one computer among a plurality of distributed computers may implement a portion of the functions and transmit results of the implementation to at least one computer that may also implement a portion of the functions and transmit results of the implementation to at least one computer.

The storage medium in which application {i.e., program) is recorded for executing the present invention includes a storage medium {e.g., hard disk) contained in application store servers or application provider servers, application provider servers perse, another computer having the program and its storage medium.

The computer system capable of reading the storage medium may include general PC such as desk top or notebook computers, mobile terminals such as

i

Smartphone, Tablet PC, PDA (Personal Digital Assistants) and mobile communication terminals as well as all computing-executable devices. i

III. Providing a Signal for a Target Nucleic Acid Sequence in a Sample

Containing Three or More Target Nucleic Acid Sequences

The method for providing a signal for a target nucleic acid sequence described above can be applied in the similar manner to a sample containing three or more target nucleic acid sequences.

A method for providing a signal for each target nucleic acid sequence from a sample containing three or more target nucleic acid sequences is characterized in that the step (a) further comprises incubating the sample with at least one additional signal-generating means capable of generating a signal for at least one additional target nucleic acid sequence in a single reaction vessel, and detecting signals at at least one additional detection temperature by a single type of detector; wherein the at least one additional detection temperature is lower than the relatively high detection temperature and the relatively low detection temperature.

In addition, a method for providing a signal for each target nucleic acid sequence from a sample containing three or more target nucleic acid sequences is characterized by comprising the method for providing a signal for each target nucleic acid sequence from a sample containing one or two target nucleic acid sequences described above. In other words, even if three or more detection temperatures are used, the signal provision using signals detected at at least two temperatures, i.e., the highest detection temperature and the second highest detection temperature, is performed according to the method described above.

Hereinafter, one embodiment for providing a signal for each target nucleic acid sequence from a sample containing up to three target nucleic acid sequences is illustrated.

The signals detected at three different temperatures for typical samples

^■ k

containing up to three target nucleic acid sequences are shown in Figs. 5A, 5B and 5C.

As shown in Figs. 5A, 5B and 5C, samples containing Up to three target nucleic acid sequences can be divided into a total of eight (8) types: (i) A-type: a sample containing only a first target nucleic acid sequence; (ii) B-type: a sample containing only a second target nucleic acid sequence; (iii) C-type: a sample containing only a third target nucleic acid sequence; (iv) D-type: a sample containing both a first target nucleic acid sequence and a second target nucleic acid sequence; (v) E-type: a sample containing both a first target nucleic acid sequence and a third target nucleic acid sequence; (vi) F-type: a sample containing both a second target nucleic acid sequence and a third target nucleic acid sequence; (vii) G-type: a sample containing all of a first target nucleic acid sequence, a second target nucleic acid sequence and a third target nucleic acid sequence; and (viii) H-type: a sample containing no target nucleic acid sequence.

The procedures for providing signals for a first target nucleic acid sequence, a second target nucleic acid sequence and a third target nucleic acid sequence for the eight samples are shown in Figs. 5C and 5D.

First, an unknown sample is incubated with a first signal-generating means capable of generating a signal for a first target nucleic acid sequence, a second signal-generating means capable of generating a signal for a second target nucleic acid sequence, and a third signal-generating means capable of generating a signal for a third target nucleic acid sequence in a single reaction vessel, and signals are detected at a relatively high detection temperature (RHT), a relatively middle detection temperature (RMT) and a relatively low detection temperature (RLT) by a

Afterwards, it is identified whether the signal detected at the relatively high detection temperature is not less than a high-temperature threshold (HTT) 420. When the signal detected at the relatively high detection temperature is not less than the HTT, the step 430 proceeds; while when the signal detected at the relatively high detection temperature is less than the HTT, the step 450 proceeds. The step 420 corresponds to the step 120 in the embodiment of the present invention 100 of identifying whether the first criterion defined by the HTT is satisfied. The HTT herein is a threshold for determining the significance of the signal detected at the relatively high detection temperature. The HTT in this step is referred to the description of the HTT in the embodiment of the present invention 100. When the signal detected at the relatively high detection temperature is not less than the HTT, it can be determined that the first target nucleic acid sequence is present in a sample; while when the signal detected at the relatively high detection temperature is less than the HTT, it can be determined that the first target nucleic acid sequence is absent in a sample.

When the signal detected at the relatively high detection temperature is not less than the HTT, it is then performed to identify whether End-Ratio_M/H is not less than a RT_M/H 430. When the End-Ratio_M H is not less than the RT_M/H, the step 440 proceeds; while when the End-Ratio_M/H is less than the RT_M H_/ the step 432 proceeds. The End-Ratio_M/H represents a difference, particularly a ratio, between the signal detected at the relatively middle detection temperature and the signal detected at the relatively high detection temperature, which corresponds^ to one example of the signal difference described above. Since the Enc_-Ratio_M/H is used in a similar manner to the signal difference as described above, its detailed description will be referred to the explanation of the signal difference described above. Although the End-Ratio_{M H} is described as an example of the signal difference in step 430 of this embodiment 400, it will be appreciated by those skilled in the art that various other signal differences may be used as well by reference to the embodiment of the present invention 100. For example, the signal difference may be calculated by mathematically processing the signal detected at the relatively middle detection temperature and the signal detected at the relatively high detection temperature. More specifically, the mathematical processing may be a subtraction or a ratio of the signal detected at the relatively middle detection temperature and the signal detected at the relatively high detection temperature. Still more specifically, the signal difference may be an End- Ratio_M H, and still more specifically, the End-Ratio_M/H is a ratio of the signal value at an end cycle in the signal detected at the middle detection temperature to the signal value at the end cycle in the signal detected at the relatively high detection temperature. The RT_M/H is a threshold for distinguishing a sample containing a second target nucleic acid sequence from a sample containing no second target nucleic acid sequence, which corresponds to one example of the aforementioned signal-difference threshold (SDT). Since the RT_M/H is used in a similar manner to the above-described SDT, the RT_M/H will be referred to the above description of the SDT. With respect to this embodiment 400, while the RT_M/H is mentioned as an example of the SDT in step

430, it will be appreciated by those skilled in the art: that various other signal- difference thresholds may be used as well by reference to the embodiment of the present invention 100. The SDT may be any value between a difference in signals

relatively high detection temperature for a control sample containing only the first target nucleic acid sequence and the ratio of the signal at the relatively middle detection temperature to the signal at the relatively high detection temperature for a control sample containing both the target nucleic acid sequence and the second target nucleic acid sequence.

When the End-Ratio_M/H is not less than the RT_{M H}, it can be determined that the second target nucleic acid sequence is present in a sample; while when the End- Ratio_M/H is less than the RT_M/H, it can be determined that the second target nucleic acid sequence is absent in a sample.

When the End-Ratio_M/_H is not less than the RT_{M H}, it is identified whether End- RatiO_L/M is not less than a RT_yM 440. The End-Ratio^ represents a difference, particularly a ratio, between the signal detected at the relatively low detection temperature and the signal detected at the relatively middle detection temperature, which corresponds to one example of the signal difference described above. Since the End-RatiO_L is used in a similar manner to the above-described signal difference, the detailed description thereof will be referred to the description of the signal difference as described above. Although the End-Ratio^ is described as an example of the signal difference in the step 440 of this embodiment 400, it will be appreciated by those skilled in the art that various other signal differences may be used as well

as an example of the SDT in step 440 of this embodiment 400, it will be appreciated by those skilled in the art that various other signal-difference thresholds may be used as well by reference to the embodiment of the present invention 100. Specifically, the SDT may be RTL/_M, and the

may be any value between the ratio of the signal at the relatively low detection temperature to the signal at the relatively middle detection temperature for a control sample containing both a first target nucleic acid sequence and a second target nucleic acid sequence and the ratio of the signal at the relatively low detection temperature to the signal at the relatively middle detection temperature for a control sample containing a first target nucleic acid sequence, a second target nucleic acid sequence and a third target nucleic acid sequence. When the End-Ratio^ is not less than the RTL M, it can be determined that the third target nucleic acid sequence is present in a sample 442 (G-type of sample); while when the

it can be determined that the third target nucleic acid sequence is absent in a sample 444 (D-type of sample).

When the steps 420, 430 and 440 are all satisfied, the signal detected at the relatively high detection temperature is provided without signal extraction as the signal for the first target nucleic acid sequence, and a signal for the second target nucleic acid sequence is provided by signal extraction- process of eliminating the signal for the first target nucleic acid sequence from the signal detected at the relatively middle detection temperature 442. In this case, the signal for the second target nucleic acid sequence may be extracted by using a reference value calculated from the signal detected at the relatively high detection temperature and the signal detected at the relatively middle detection temperature for a control sample containing only the first target nucleic acid sequence. Meanwhile, a signal for the third target nucleic acid sequence may be provided by signal extraction process of eliminating the signals for the first target nucleic acid sequence and the second target nucleic acid sequence from the signal detected at the relatively low detection temperature 442. In this case, the signal for the third target nucleic acid sequence may be extracted by using a reference value calculated from the signal detected at the relatively middle detection temperature and the signal detected at the relatively

i low detection temperature for a control sample containing only the first target nucleic acid sequence and the second target nucleic acid sequence.

When the steps 420 and 430 are satisfied but the step 440 is not satisfied, the signal detected at the relatively high detection temperature is provided without signal extraction as the signal for the first target nucleic acid sequence, a signal for the second target nucleic acid sequence is provided by signal extraction process of eliminating the signal for the first target nucleic acid sequence from the signal detected at the relatively middle detection temperature, and any signal indicative of the absence of a target nucleic acid sequence is provided without signal extraction as the signal for the absence of the third target nucleic acid sequence 444.

Further, when the step 420 is satisfied but the step 430 is not satisfied, it is identified whether the End-Ratio _M is not less than RTL/M, in order to determine whether the third target nucleic acid sequence is present in a sample 432. The End- RatiO_L M represents a difference, particularly a ratio, between the signal detected at the relatively low detection temperature and the signal detected at the relatively middle detection temperature, which corresponds to , one example of the signal

^{■ ■ ·}. ^. ... i; ^r ' - i

difference described above. Since the End-RatiO_L/M is used in a similar manner to the above-described signal difference, the detailed description thereof will be referred to the description of the signal difference as described above. Although the End-RatiOL/M is described as an example of the signal difference in step -432 of this embodiment

400, it will be appreciated by those skilled in the art that various other signal differences may be used as well by reference to the embodiment of the _¾present invention 100. Further, the P _UM is a threshold for distinguishing a sample containing a third target nucleic acid sequence from a sample containing no third target nucleic i

acid sequence, which corresponds to one example of the aforementioned signal- difference threshold (SDT). Since the RT_L/M is used in a similar manner to the above- described SDT, a detailed description thereof will be referred to the description of the SDT value. Although the RT_{L M} is described as an example of the SDT in step 432 of this embodiment 400, it will be appreciated by those skilled in the art that various other signal-difference thresholds may be used as _f well by reference to the embodiment of the present invention 100. Specifically, . the SDT may be RT_L/_M, and the TL M may be any value between the ratio of the signal at a relatively low detection temperature to the signal at the relatively middle detection temperature for a control sample containing only a first target nucleic acid sequence and the ratio of the signal at the relatively low detection temperature to the signal at the relatively middle detection temperature for a control sample containing a first target nucleic acid sequence and a third target nucleic acid sequence. In an embodiment of the present invention, when the second target nucleic acid sequence is determined to be absent in a sample, End-RatiOL/_H is used instead of End-Ratio^, and RT^H is used instead of RTL _M- In this case, RT_UH may be any value between the ratio of the signal at a relatively low detection temperature to the signal at the relatively high detection temperature for a control sample containing only a first target nucleic acid sequence and the ratio of the signal at the relatively low detection temperature to the signal at the relatively high detection temperature for a control sample containing a first target nucleic acid sequence and a third target nucleic acid sequence. The RTL _M used in the step 432 may or may not be identical to the

used in the step 440.

When the End-Ratio^ is not less than the RT _M, it can be determined that the third target nucleic acid sequence is present in a sample; whereas when the End- Ration is less than the RTL/M, it can be determined that the third target nucleic acid

sample containing only a first target nucleic acid sequence.

When the step 420 is satisfied, the step 430 is not satisfied, and the step 432 is not satisfied, the signal detected at the relatively high detection temperature, the signal detected at the relatively middle detection temperature, or the signal detected at the relatively low detection temperature is provided without signal extraction as the signal for the first target nucleic acid sequence, and any signal indicative of the absence of target nucleic acid sequence is provided as the signal for the second target nucleic acid sequence or a signal for the absence of the third target nucleic acid sequence 436 (A-type of sample). Meanwhile, when the step 420 is not satisfied,^* it is performed to Identify whether the signal detected at the relatively middle detection temperature is not less than a middle-temperature threshold (MTT) in order to determine whether the second target nucleic acid sequence is present in a sample 450. When the signal detected at the relatively middle detection temperature is not less than the MTT, the next step

^■■•i- ·, *

460 proceeds; while when the signal detected at the relatively middle detection i * temperature is less than the MTT, the step 470 proceeds. This step 450 corresponds to step 120 of identifying whether the first criterion defined by the HTT is, satisfied

{

according to the embodiment of the present invention 100. The MTT is a threshold for determining the significance of the signal detected at the relatively middle detection temperature, which corresponds to one example of the above-described HTT. Since the MTT is used in a same manner to the above-mentioned .HTT, a detailed description thereof will be referred to the description of _:the HTT. When the signal detected at the relatively middle detection temperature is not less than the MTT, it can be determined that the second target nucleic acid sequence is present in a sample; while when the signal detected at the relatively middle detection temperature has signal values less than the MTT, it can be determined that the second target nucleic acid sequence is absent in a sample.

When step 420 is not satisfied but step 450 is satisfied, it is performed to identify whether the End-RatiOu _M is not less than the

in order to determine whether the third target nucleic acid sequence is present in a sample 460. The End- represents a difference, specifically a ratio, between the signal detected at the relatively low detection temperature and the signal detected at the relatively middle temperature detection temperature, and corresponds to one example of the signal difference described above. Since the

is used for a similar purpose to the above-described signal difference, the description of the above-described signal difference is specifically described. Although the End-Ratio^ is described as an example of the signal difference in step 460 of this embodiment 400, it will be appreciated by those skilled in the art that various other signal differences may be used as well by reference to the embodiment of the . present invention 100. further, the RT_UM is a threshold for distinguishing a sample containing a third target nucleic acid sequence from a sample containing no third target nucleic acid sequence, which corresponds to one example of the aforementioned SDT_; Since the RTL/_M is used in a similar manner to the above-described SDT, a detailed description thereof will be referred to the description of the SDT. Although the RT M is. described as an example of the SDT in step 460 of this embodiment 400, it will be appreciated by those skilled in the art the various signal-difference thresholds may be used as well by reference to the embodiment of the present invention 100. Specifically, the SDT may be RTL/M_/ and the RTL _M is any value between the ratio of the signal at the relatively low detection temperature to the signal at the relatively middle detection temperature for the control sample containing only the second target nucleic acid sequence and the ratio of the signal at the relatively low detection temperature to the signal at the relatively middle detection temperature for the control sample containing the second target nucleic acid sequence and the third target nucleic acid sequence to the signal · at the relatively middle detection temperature. According to an embodiment of the present invention, the TL _M used in the step 432, the step 440 or the step 460 is a value determined by the signal values from the controls in consideration of the combination of the target nucleic acid sequences present in the sample. According to another embodiment of the present invention, some or all of the RT_UM used in the step 432, the step 440 or the step 460 is a same value. The selection of the value can be made in consideration of the error ratio of the result interpretation process and the values determined from the controls containing only a combinations of the target nucleic acid sequences present in the sample. The TY/M used in the step 460 may be the same as or different from the RT_UM used in the step 440 or 432.

When the End-RatiOL/M is not less than the RTL/_M, it can be determined that the third target nucleic acid sequence is present in a sample; while when the End-Ratio^ is less than the RTL/_M, it can be determined that the third target nucleic acid sequence is absent in a sample. When the step 420 is not satisfied, the step 450 is satisfied and the step 460 is satisfied, any signal indicative of the absence of a target nucleic acid sequence (or the signal detected at the relatively high detection temperature) is provided, without signal extraction, as the signal for the absence of the first target nuclejc acid sequence; the signal detected at the relatively middle detection temperature is provided, without signal extraction, as the signal for the second target nucleic acid sequence; and the signal for the third target nucleic add sequence is provided by signal extraction process of eliminating the second target nucleic acid sequence from the signal detected at the relatively low detection temperature 462 (F-type of

the relatively low detection temperature.

When the step 420 is not satisfied, the step 450 is satisfied and the step 460 is not satisfied, any signal indicative of the absence of a target nucleic acid sequence (or the signal detected at the relatively high detection temperature) is provided, without signal extraction, as the signal for the absence of the first target nucleic acid sequence; the signal detected at the relatively middle detection temperature is be provided, without signal extraction, as the signal for the second target nucleic acid sequence; and any signal indicative of the absence of a target nucleic acid sequence may be provided, without signal extraction, as the signal for the absence of the third target nucleic acid sequence (B-type of sample) 464.

Meanwhile, when the step 420 is not satisfied and the step 450 is not satisfied, it is identified whether the signal detected at the relatively low detection temperature is not less than a low-temperature threshold (LTT) in order to determine whether the third target nucleic acid sequence is present in a sample 470. This step 470 corresponds to the step 120 of identifying whether the first criterion defined by the HTT is satisfied according to the embodiment of the present invention 100. The LTT is a threshold for determining the significance ofi the signal detected at the relatively low detection temperature. Since the LTT is used in a same manner as the above-mentioned LTT, a detailed description will be referred to the description of the LTT. When the signal detected at the relatively high detection temperature is pot less than the LTT, it can be determined that the third target nucleic acid sequence is present in a sample; while when the signal detected at the relatively high detection temperature has signal values less than the LTT, it can be determined that the third target nucleic acid sequence is absent in a sample.

When the step 420 is not satisfied, the step 450 is not satisfied and the step 470 is satisfied, any signal indicative of the absence of a target nucleic acid sequence (or the signal detected at the relatively high detection temperature) is provided, without signal extraction, as the signal for the absence of the first target nucleic acid sequence; any signal indicative of the absence of a target nucleic acid sequence (or the signal detected at the relatively middle detection temperature) is provided, without signal extraction, as the signal for the absence of the second target nucleic acid sequence; and the signal detected at the relatively low detection temperature is provided, without signal extraction, as the signal for the third target nucleic acid sequence (C-type of sample) 472.

When the step 420 is not satisfied, the step 450 is not satisfied and the step 470 is not satisfied, any signal indicative of the absence of a target nucleic acid sequence (or the signal detected at the relatively high detection temperature, the signal detected at the relatively high detection temperature, and the signal detected at the relatively low detection temperature) is provided, without signal extraction as the signal for the absence of the first target nucleic acid sequence, the signal for the absence of the second target nucleic acid sequence, or the signal for the absence of the third target nucleic acid sequence, respectively (H-type of sample) 474.

As described above, each signal for three or more target nucleic acid sequences can be accurately provided by applying the method of the present invention. The method of the present invention employs a signal extraction process only when particular criteria are satisfied, it can remarkably reduce the false positive results compared with the conventional method in which all samples are subjected to the signal extraction process.

The method described above is merely an example for providing a signal for at least one target nucleic acid sequence for a sample containing up to three target nucleic acid sequences. It should be understood by those skilled in the art that various modifications in sequence thereof are within, the scope of the present invention.

Meanwhile, practicing the method for providing a signal for at least one target nucleic acid sequence for a sample containing up to three target nucleic acid sequences may comprise practicing the method for providing a signal for at least one target nucleic acid sequenc for a sample containing up to two target nucleic acid sequences.

For example, where signals are detected at a first detection temperature, a second detection temperature and a third detection temperature (wherein the detection temperatures are high in the order of the first detection temperature, the second detection temperature, the third detection temperature) for a sample containing up to three target nucleic acid sequences, the process of processing the signals detected at the first detection temperature and the second detection temperature may be carried out by using a process of the signals detected at the first detection temperature and the second detection temperature.

Likewise, the process of processing the signals detected at a first detection temperature, a second detection temperature and a third detection temperature for a sample containing up to four target nucleic acid sequences may be carried out by using a process of processing the signals detected at the first detection temperature, the second detection temperature and the third detection temperature. The above method can be applied in a similar manner even when the number of the target nucleic acid sequences that may be present is increased. In the method of the present invention, the n signal generating means (n is an integer) used for detecting n target nucleic acid sequences (n is an integer) have the sequentially decreasing temperature ranges for providing signals, permitting n detection temperatures to be used.

When signals are measured at n detection temperatures, the present invention includes sequentially selecting two detection temperatures among the detection temperatures and analyzing the signals detected at the two detection temperatures. Particularly, the present invention comprises extracting a signal for a specific target nucleic acid sequence' by using the signals obtained-' at the two detection temperatures and a reference value that can be selected in the step, only when the signal difference between the signals of the two selected detection temperatures satisfies a second criterion defined by a LTT.

A combination of target nucleic acid sequences to be considered in each step is logically derived in the course of sequentially selecting and analyzing two detection temperatures among the detection temperatures, and thereby, a SDT or a reference value to be applied at the corresponding step can be determined.

The method of the present invention provides a signal for a target nucleic acid sequence with a signal extraction process only when particular criteria are satisfied, and provides a signal for a target nucleic acid sequence without a signal extraction process when a particular criterion is not satisfied. The method of the present invention can minimize the signal extraction process, thereby significantly reducing false positive results which may occur due to undertaking a signal extraction process undesirable or unnecessary. Accordingly, the method of the present invention can provide a signal for a target nucleic acid sequence using two detection temperatures in a more accurate and efficient manner, from which the presence or absence of a target nucleic acid sequence can be determined. The present invention will now be described in further detail by examples. It

^■* · ^'. i

would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the presenUnvention as set forth in the appended claims is not limited to or by the examples.

EXAMPLES

Example 1: Preparation of templates and oligonucleotides

The PTOCE method (WO 2012/096523) was used as a real-time PCR approach for detecting signals in a real-time manner at different detection temperatures.

Taq DNA polymerase having a 5' nuclease activity was used for the extension of upstream primers and downstream primers, the cleavage of PTO (Probing and

· ^{■ ■■■ '} b

Tagging Oligonucleotide), and the extension of PTO fragment. Genomic DNA of Chlamydia trachomatis (CT) and genomic DNA of Neisseria gonorrhoeae (NG) were used as first and second target nucleic acid sequences, respectively.

The PTOs for detection of CT and NG by using the PTOCE method as signal- generating means comprise (i) a 5'-tagging portion comprising a nucleotide sequence non-complementary to the target nucleic acid sequence and (ii) a 3'-targeting portion comprising a hybridizing nucleotide sequence complementary to the target nucleic acid sequence. The CTOs (Capturing and Templating Oligonucleotides) used in the detection of CT and NG comprise, in a 3' to 5' direction, (i) a capturing portion comprising a nucleotide sequence complementary to the 5'-tagging portion or a part of the 5'-tagging portion of the PTOs and (ii) a templating portion comprising a nucleotide sequence non-complementary to the 5'-tagging portion and the 3'- targeting portion of the PTOs. The CTOs were labeled with a quencher molecule (BHQ-2) at their 5'-ends and a fluorescent reporter molecule (CAL Fluor Red 610) in their templating portions. The PTOs and CTOs are blocked with a carbon spacer at their 3'-ends to prohibit their extension.

Because the PTOCE real-time method uses a single type of fluorescence label (CAL Fluor Red 610), signals for the target nucleic acid sequences cannot be differentiated from each other in a single reaction vessel by a single detector.

The sequences of upstream primers, downstream primers, PTOs, and CTOs used in this Example are shown in Table 1.

TABLE 1

I : Deoxyinosine

PTO : Probing and Tagging Oligonucleotide

CTO : Capturing and Templating Oligonucleotide

BHQ : Quencher (Black Hole Quencher)

Underlined letter : 5'-tagging portion of PTO

Example 2: Provision of signals for two target nucleic acid sequences from signals detected at different temperatures

After detecting signals at different detection temperatures using a single detection channel in a single-reaction vessel, the signals were used to obtain signals for the target nucleic acid sequences in accordance _;with an embodiment of the present invention.

the signal for CT is detected.

Four types of samples were prepared and analyzed:

(a) a sample containing only CT (A-type);

(b) a sample containing only NG (B-type);

(c) a sample containing both CT and NG (C-type); and

(d) a sample containing no CT and NG (D-type; no target control).

<2-l> Preparation of templates and oligonucleotides

Genomic DNAs of CT and NG, and the primers (SEQ ID NOs: 1, 2, 5 and 6), PTOs (SEQ ID Nos: 3 and 7), and CTOs (SEQ ID NOs: 4 and 8) prepared in Example 1 were used.

<2-2> Real-time PCR and signal detection at different temperatures The real-time PCR was conducted in the final volume of 20 μΙ containing a target nucleic acid (10 pg of NG genomic DNA, 10 pg of CT genomic DNA or a mixture of 10 pg of NG genomic DNA and 10 pg of CT genomic DNA), 5 pmole of upstream primer (SEQ ID NO: 1), 5 pmole of downstream primer (SEQ ID NO: 2), 3 pmole of PTO (SEQ ID NO: 3) and 1 pmole of CTO (SEQ ID NO: 4) for NG target amplification, 5 pmole of upstream primer (SEQ ID NO: 5) and 5 pmole of downstream primer (SEQ ID NO: 6), 3 pmole of PTO (SEQ ID NO: 7) and 1 pmole of CTO . (SEQ ID NO: 8) for CT target amplification, and 10 μΙ of 2X Master Mix [final, 200 uM dNTPs, 2 mM MgCI₂, 2 U of Tag DNA polymerase (Enzynomics, Korea)]. The tubes containing the reaction mixture were placed in the real-time thermocycler (CFX96, Bio-Rad) for 5 min at 50°C, denatured for 15 min at 95°C and subjected to 50 cycles^of 30 sec at 95°C, 60 sec at 60°C, 30 sec at 72°C. Detection of signals was performed at 60°C and 72°C at each cycle. The results are represented by Fig. 1.

As shown in Fig. 1, for the sample containing only CT (A-type) and the sample containing both CT and NG (C-type), significant signals were detected both at 60°C and at 72°C; for the sample containing only NG (B-type), a significant signal was detected at 60°C but not at 72°C; and for the sample containing no CT and NG (D- type), no significant signals were detected both at 60°C and at 72°C.

By analyzing the signals detected at different detection temperatures in Fig. 1, the signal information about each target nucleic acid sequence can be obtained, which can be used to determine the presence or absence of each target nucleic acid sequence. Usually, a signal for the first target nucleic acid sequence (CT) can be directly taken from the signal detected at the relatively high detection temperature, since the signal detected at the relatively high detection temperature consists of the signal for the first target nucleic acid sequence. In contrast, a signal for the second target nucleic acid sequence (NG) is not detected at the relatively high detection temperature but at the relatively low detection temperature together with a signal for the first target nucleic acid sequence (CT), and the signal for the second target nucleic acid sequence (NG) can be obtained by eliminating the signal for the first target nucleic acid sequence (CT) from the signal detected at the relatively low detection temperature. The process is called as a signalextraction process and can be performed by the mathematical equation I as follows:

< Equation I>

Signal for the second target nucleic acid sequence = [signal at the relatively low detection temperature in the step (a)] - [(signal at the relatively high detection temperature in the step (a)) x (the reference value for the first target nucleic acid sequence)]

The application of the signal extraction process to all samples can increase possibility of false positive results.

The present method provides a signal for each target nucleic acid sequence by applying the signal extractidn process only to a sample satisfying a particular criterion(s).

<2-3> Signal acquisition for target nucleic acid sequences according to an embodiment

In order to provide signals for two target nucleic acid sequences from signals detected at different temperatures, the signals detected were analyzed according to an embodiment as depicted in Fig. 2A.

Prior to this analysis, a threshold and a reference value were set as described below.

- High-temperature threshold (HTT) = RFU 500

- Signal-difference threshold (SDT) = Ratio 1.50

- Reference value for the first target nucleic acid sequence = 1.15

The HTT is a value for determining the significance of the signal detected at

72°C, which was set with reference to two signals detected at 72°C in the presence and absence of CT in Fig. 1.

The SDT is a value for distinguishing a sample containing only the first target nucleic acid sequence (A-type) from a sample containing both the first target nucleic acid sequence and the second target nucleic acid sequence, which was set as follows.

First, for each of the sample containing only CT and the sample containing both CT and NG, a ratio (End-Ratio) of RFUs at end cycles was obtained from the signals detected at 60°C and 72°C. The results are shown in Table 2.

TABLE 2

As shown in Table 2, the End-ratio was 1.15 for the sample containing only

^' ■ .._. ... .... .^{■ : '}¾ 1 _ . .·¾: :

CT (A-type) and 2.09 for the sample containing both CT and N0 (C-type). Thus, a SDT was set to 1.50, a value between 1.15 and 2.09, for distinguishing the two samples.

Meanwhile, the reference value is the value used to extract a signal for NG by eliminating a signal for CT from the signal detected at 60°C, which was set to 1.15 corresponding to the End-Ratio calculated from the sample containing only CT (A- type).

< Identification step 1> Identifying whether the signal detected at the relatively high detection temperature satisfies the first criterion defined by the high- temperature threshold (HIT)

In this step, it was identified whether the signal detected at the relatively high detection temperature obtained from the unknown sample satisfies the first criterion defined by the HTT. The first criterion was as follows:

(i) Satisfied: the signal detected at 72°C > HTT (=RFU 500)

(ii) Unsatisfied: the signal detected at 72°C < HTT (=RFU 500)

If the first criterion was unsatisfied, the signal detected at 72°C was provided without signal extraction as the signal for CT and the signal detected at 60°C was provided without signal extraction as the signal for NG. On the other hand, if the '^:'^ν - " . ■ " ■!*

first criterion was satisfied, the process proceeded to„the_:subsequent step.

< Identification step 2> Identifying whether a signal difference between the signal detected at the relatively low detection temperature and the signal detected at the relatively high detection temperature satisfies the second criterion defined by the signal-difference threshold (SDT)

In this step, it was identified whether a signal difference between the signal detected at the relatively low detection temperature and the signal detected at the relatively high detection temperature satisfies the second^criterion defined by the SDT. The second criterion was as follows:

(i) Satisfied: End-Ratio > SDT (= Ratio 1.50)

(ii) Unsatisfied: End-Ratio < SDT (= Ratio 1.50)

If the second criterion was unsatisfied, the signal detected at 72°C was provided without signal extraction as the signal for CT and any signal indicative of the absence of a target nucleic acid sequence, e.g., a signal of approximately RFU 0, was provided without signal extraction as the signal for the absence of NG. In this case, the signal detected at both 60°C and 72°C cannot be adopted as the signal for NG, because these signals are both significant. Instead, any other signal indicative of the absence of a target nucleic acid sequence was provided as the signal for the absence of NG.

On the other hand, if the second criterion was satisfied, the process proceeded to the subsequent step.

The signal for NG was extracted eliminating the signal for CT from the signal detected at 60°C as follows:

The signal for NG = signal detected at 60°C - (signal detected at 72°C x the reference value for CT (RV 1.15))

The above process was also referred to as the signal extraction process. The results are shown in Figure 2B.

As shown in Fig. 2B, for the sample containing only NG (B-type) and the sample containing no CT and NG (D-type), the signal for CT and the signal for NG can be provided without signal extraction process from <Identification step 1>. And, for the sample containing only CT (A-type), the signal for CT and the signal for NG can be provided without signal extraction process from < Identification .step 2>. Further, for sample containing both the CT and NG (C-type), the signal for CT and the signal for NG can be provided from <Signal extraction step>.

As described above, it was confirmed that the signal for each target nucleic ϊ ... ^' Ί ^{■ ' Γ ■}

acid sequence can be accurately provided by the method of the present invention.

The signals for CT and NG provided can be used to determine the presence or absence of CT and NG, respectively.

As such, the method as described herein can (i) provide a signal for each target nucleic acid sequence from the signals detected at different temperatures, (ii) minimize the signal extraction process for detecting the presence of the target nucleic acid sequence having the relatively low detection temperature by sequential steps, and (iii) therefore reduce errors that may occur during signal extraction. Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.

Claims

What is claimed is:

1. A method for providing a signal for at least one target nucleic acid sequence in a sample from signals detected at different temperatures, which comprises the steps of:

(a) incubating the sample with a first signal-generating means capable of generating a signal for a first target nucleic acid sequence and a second signal- generating means capable of generating a signal for a second target nucleic acid sequence in a single reaction vessel, and detecting signals at a relatively high detection temperature and a relatively low detection temperature by a single type of detector; wherein the first signal-generating means generates signals at both the relatively high detection temperature and the relatively low detection temperature in the presence of the first target nucleic acid sequence in the sample and the second signal-generating means generates a signal at the relatively low detection temperature in the presence of the second target nucleic acid sequence in the sample; wherein the two signals to be generated by the two signal-generating means are not differentiated by the single type of detector;

(d) extracting the signal for the second target nucleic acid sequence by using the signal detected at the relatively high detection temperature in the step (a), the signal detected at the relatively low detection temperature in the step (a) and a reference value for the first target nucleic acid sequence, thereby providing the signal for the second target nucleic acid sequence; wherein the reference value for the first target nucleic acid sequence is a value representing a relationship of change in signals provided by the first Signal-generating means at the relatively high detection temperature and the relatively low detection temperature; wherein the reference value for the first target nucleic acid sequence is determined from a control reaction using the first target nucleic acid sequence and the first signal-generating means; wherein the steps (b) and (c), may be performed in reverse order.

2. The method of claim 1, wherein when the signal detected at the relatively high detection temperature does not satisfy the first criterion in the step (b), the signal detected at the relatively low detection temperature in the step (a] is provided as the signal for the second target nucleic acid sequence.

3. The method of claim 1, wherein when the signal detected at the relatively high detection temperature does not satisfy the first criterion in the step (b), the method further comprises a step of identifying whether the signal detected at the relatively low detection temperature satisfies a third criterion defined by a low-temperature threshold (LTT).

4. The method of claim 3, wherein when the signal detected at the relatively low detection temperature does not satisfy the third criterion, any signal indicative of the absence of a target nucleic acid sequence is provided as the signal for the absence of the second target nucleic acid sequence.

5. The method of claim 3, wherein when the signal detected at the relatively low detection temperature satisfies the third criterion, the signal detected at the relatively low detection temperature in the step (a) is provided as the signal for the second target nucleic acid sequence.

6. The method of claim 1, which further comprises identifying whether the signal detected at the relatively low detection temperature satisfies a third criterion defined by a low-temperature threshold (LTT) prior to the step (b).

7. The method of claim 6, wherein when the signal detected at the relatively low detection temperature does not satisfy the third criterion, any signal indicative of the

. 4.

absence of a target nucleic acid sequence is provided as the signal for the absence of the second target nucleic acid sequence.

8. The method of claim 6, wherein when the signal detected at the relatively low detection temperature satisfies the third criterion, the next step (b) proceeds.

9. The method of claim 1, wherein the signal difference in the step (c) is calculated by mathematically processing the signals detected at the relatively low detection temperature and the relatively high detection temperature.

10. The method of claim 9, wherein the mathematical processing is a subtraction or ratio between the signals detected at the relatively low detection temperature and the relatively high detection temperature.

11. The method of claim 1, wherein when the signal difference in step (c) does not satisfy the second criterion, any signal indicative of the absence of a target nucleic acid sequence is provided as the signal for the absence of the second target nucleic acid sequence.

12. The method of claim 1, wherein the signal for the second target nucleic acid sequence in the step (d) is provided by eliminating a signal generated by the first signal-generating means from the signal detected at the relatively low detection temperature or the relatively high detection temperature in the step (a), by using the reference value for the first target nucleic acid sequence.

13. The method of claim 1, wherein the signal for the second target nucleic acid sequence in the step (d) is provided by the following mathematical equation I:

< Equation I>

Signal for the second target nucleic acid sequence - [signal detected at the relatively low detection temperature in the step (a)] - [(signal detected at the relatively high detection temperature in the step (a)) x (the reference value for the first target nucleic acid sequence)]

14. The method of claim 1, wherein the signal for the second target nucleic acid sequence in the step (d) is provided by the following mathematical equation II:

< Equation II >

Signal for the second target nucleic acid sequence = [signal detected at the relatively high detection temperature in the step (a)] - [(signal detected at the relatively low detection temperature in the step (a)) ÷ (the reference value for the first target nucleic acid sequence)]

wherein the reference value for the first target nucleic acid sequence is a ratio of a signal provided by the first signal-generating means at the relatively low detection temperature to a signal provided by the first signal -generating means at the relatively high detection temperature.

15. The method of claim 1, which further comprises: (e) identifying whether the signal for the second target nucleic acid sequence provided in the step (d) satisfies a fourth criterion defined by a verification threshold; when the signal for the second target nucleic acid sequence does not satisfy the fourth criterion, any signal indicative of the absence of a target nucleic acid sequence is provided as the signal for the absence of the second target nucleic acid sequence.

16. The method of any one of claims 1 to 15, wherein the signal detected at the relatively high detection temperature in the step (a) is provided as the signal for the first target nucleic acid sequence.

17. The method of any one of claims 2, 4, 5 and 7, wherein any signal indicative of the absence of a target nucleic acid sequence is provided as the signal for the absence of the first target nucleic acid sequence.

18. The method of claim 11 or 15, wherein the signal detected at the relatively high detection temperature in the step (a) or the signal detected at the relatively low detection temperature in the step (a) is provided as the signal for the first target nucleic acid sequence.

19. The method of claim 1, wherein the incubation in the step (a) is performed by using the first signal-generating means, the second signal-generating means and at least one additional signal-generating means capable of generating a signal for at least one additional target nucleic acid sequence; and wherein the detection by the single type of detector is performed for detecting signals at the relatively high detection temperature, the relatively low detection temperature and at least one additional detection temperature lower than the relatively high detection temperature and the relatively low detection temperature.

20. A computer readable storage medium containing instructions to configure a processor to perform a method for providing a signal for at least one target nucleic acid sequence in a sample from signals detected at different temperatures, which comprises the steps of: (a) incubating the sample with a first signal-generating means capable of generating a signal for a first target nucleic acid sequence and a second signal- generating means capable of generating a signal for a second target nucleic acid sequence in a single reaction vessel, and receiving signals detected at a relatively high detection temperature and a relatively low detection temperature by a single type of detector; wherein the first signal-generating means generates signals at both the relatively high detection temperature and the relatively low detection temperature in the presence of the first target nucleic acid sequence in the sample and the second signal-generating means generates a signal at the relatively low detection temperature in the presence of the second target nucleic acid sequence in the sample; wherein the two signals to be generated by the two signal-generating means are not differentiated by the single type of detector;

(b) identifying whether the signal detected at~the relatively high detection temperature satisfies a first criterion defined by a high-temperature threshold (HTT); when the signal detected at the relatively high detection temperature satisfies the first criterion, the next step (c) proceeds;

21. A device for providing a signal for at least one target nucleic acid sequence in a sample from signals detected at different temperatures; comprising: (a) a computer processor and (b) the computer readable storage medium of claim 20 coupled to the computer processor.

22. A computer program to be stored on a computer readable storage medium to configure a processor to perform a method for providing a signal for at least one target nucleic acid sequence in a sample from signals detected at different temperatures, which comprises the steps of:

(b) identifying whether the signal detected at the relatively high detection temperature satisfies a first criterion defined by a high-temperature threshold (HTT); when the signal detected at the relatively high detection temperature satisfies the first criterion, the next step (c) proceeds; (c) identifying whether a signal difference between the signal detected at the relatively low detection temperature and the signal detected at the relatively high detection temperature satisfies a second criterion defined by a signal-difference threshold (SDT); when the signal difference satisfies the second criterion, the next step (d) proceeds; and

(d) extracting the signal for the second target nucleic acid sequence by using the signal detected at the relatively high detection temperature in the step (a), the signal detected at the relatively low detection temperature in the step (a) and a reference value for the first target nucleic acid sequence, thereby providing the signal for the second target nucleic acid sequence; wherem th_¾e reference value for the first target nucleic acid sequence is a value representing a relationship of change in signals provided by the first signal-generating means at the relatively high detection temperature and the relatively low detection temperature; wherein the reference value for the first target nucleic acid sequence is determined from a control reaction using the first target nucleic acid sequence and the first signal-generating means; wherein the steps (b) and (c) may be performed in reverse order.