CN116391039A

CN116391039A - Method for simultaneous detection of multiple target nucleic acids

Info

Publication number: CN116391039A
Application number: CN202180074034.3A
Authority: CN
Inventors: 道渕真史; 上仓佳子; 铃木广道
Original assignee: Toyobo Co Ltd
Current assignee: Toyobo Co Ltd
Priority date: 2020-10-30
Filing date: 2021-10-25
Publication date: 2023-07-04
Also published as: WO2022092012A1; JPWO2022092012A1

Abstract

The present invention addresses the problem of providing a method for simultaneously identifying a plurality of target nucleic acids, which is simple and inexpensive. The present invention provides a method for detecting a plurality of target nucleic acids by melting curve analysis in a single reaction solution, comprising a step of adding a first target probe for detecting a first target nucleic acid and a second target probe for detecting a second target nucleic acid to the reaction solution, wherein the first target probe and the second target probe satisfy T1 > T2 when the respective detection temperatures in the melting curve analysis are T1 and T2, and are labeled with labels capable of being detected at the same wavelength.

Description

Method for simultaneous detection of multiple target nucleic acids

Technical Field

The present invention relates to a method for simultaneously detecting a plurality of target nucleic acids contained in a sample by a simple method, and a reagent composition used in the method.

Background

Many of the clinical symptoms in various infections are very similar and difficult to identify. For example, the identification of chlamydia infection in sexually transmitted diseases is very similar to the symptoms of gonococcal infection and the like, and is becoming clinically important. Accordingly, methods for simultaneous identification using various detection methods typified by a PCR method have been developed.

In gene detection by the PCR method or the like, examples of the case where it is necessary to measure a plurality of target genes simultaneously include not only chlamydia and gonococcus which are sexually transmitted diseases as described above, but also influenza A and influenza B. In the examination of such infections with very similar clinical symptoms, it is required that the simultaneous identification can be performed by gene detection from the same subject.

As one of the reasons for this, since the symptoms of the above-described various infections are very similar, there is a possibility that a missed diagnosis of one infection may occur even when co-infection occurs. In this case, not only the treatment is delayed, but also there is a concern that the infection spreads to the surroundings. In this case, if pathogenic microorganisms whose symptoms are very similar can be identified at the same time, not only early treatment but also prevention of infection to the surroundings can be performed.

Heretofore, as a method for detecting a plurality of target nucleic acids simultaneously, the following methods have been widely conducted: a method of performing measurement using different reaction liquids (patent document 1); alternatively, a method of identifying by using labels having different wavelengths for the respective detection (patent document 2) and the like.

However, in the case of performing measurement using different reaction solutions as in patent document 1, there are problems such as a preparation time for preparing an analytical reagent, and a cost increase of the reagent.

In addition, in the case of measuring using labels having different wavelengths for the respective detection as in patent document 2, time and cost are required for selection of an analysis device having a plurality of fluorescence detection channels, setting of the wavelength to be detected, and the like.

Furthermore, the following methods are also known: by combining the annealing temperature condition setting and the temperature change profile of the probe, a single fluorescent label is used to detect a plurality of nucleic acids simultaneously in one reaction vessel (patent document 3). However, this method has the following problems: the base length of the probe needs to be changed greatly for each probe, and the design, condition setting, and the like of the probe become complicated, resulting in an increase in cost.

Prior art literature

Patent literature

Patent document 1: japanese patent laid-open No. 2002-136300

Patent document 2: japanese patent application laid-open No. 2004-203

Patent document 3: japanese patent laid-open No. 2017-521758

Disclosure of Invention

Problems to be solved by the invention

The present invention has been completed with the above-described conventional problems as a background. That is, the object is to develop a novel method capable of simultaneously recognizing a plurality of target nucleic acids in a simple manner at a lower cost.

Solution for solving the problem

The present inventors have conducted intensive studies and as a result, have unexpectedly found that: in melting curve analysis using probes, if the detection temperatures of the plurality of target probes are designed so as to deviate from each other to a certain extent, even if the targets are labeled with markers that can detect them at the same wavelength, 2 peaks represented by primary differential values of fluorescence/quenching change amounts of the respective target probes can be clearly observed, and a plurality of target nucleic acids can be identified by detection at the same wavelength. In order to design the detection temperatures of the target probes to deviate from each other to some extent, for example, a mismatched base may be introduced into any one of the target probes. The present invention has been completed based on this finding and further studies.

That is, the outline of the present invention is as follows.

[ item 1] A method for detecting a plurality of target nucleic acids by melting curve analysis in a single reaction solution, comprising a step of adding a first target probe for detecting a first target nucleic acid and a second target probe for detecting a second target nucleic acid to the reaction solution, wherein the first target probe and the second target probe satisfy T1 > T2 when the respective detection temperatures in the melting curve analysis are T1 and T2, and are labeled with labels capable of being detected at the same wavelength, respectively.

[ item 2] the method of item 1, further comprising a step of amplifying the first target nucleic acid and the second target nucleic acid by primers that are: a first target primer capable of amplifying 1 or more regions containing a first target nucleic acid sequence to which a first target probe can bind; and a second target primer capable of amplifying 1 or more regions containing a second target nucleic acid sequence to which the second target probe can bind.

[ item 3] the method according to item 1 or 2, wherein the label of the first target probe and the label of the second target probe are the same label.

The method according to any one of items 1 to 3, wherein the second target probe comprises mismatched bases.

The method according to any one of items 1 to 4, wherein the mismatched base is at least 1 selected from the group consisting of an adenine base, a cytosine base, a guanine base, a thymine base, and a universal base.

The method according to any one of items 1 to 5, wherein the universal base is at least 1 selected from the group consisting of hypoxanthine, 9- (beta-D-ribofuranose) purine (Nebularine), and 5-nitroindole.

The method according to any one of items 1 to 6, wherein the detection temperature T2 of the second target probe is adjusted to be lower than the detection temperature T1 of the first target probe in the melting curve analysis by making the first target probe contain no mismatched base and making the second target probe contain mismatched base.

The method according to any one of items 1 to 7, wherein in the melting curve analysis, a temperature difference between the detection temperature T1 of the first target probe and the detection temperature T2 of the second target probe is 5 to 30 ℃.

The method according to any one of items 1 to 8, wherein the difference between the length of the nucleobase of the first target probe and the length of the nucleobase of the second target probe is 8mer or less.

The method according to any one of items 1 to 10, wherein the label of the first target probe and the label of the second target probe are QProbe (registered trademark) or Eprobe (registered trademark).

The method according to any one of items 1 to 10, wherein either one of the first target nucleic acid and the second target nucleic acid is a target nucleic acid in an endogenous plasmid of Chlamydia, and the other is a target nucleic acid in gonococcus.

The method according to any one of items 1 to 10, wherein either one of the first target nucleic acid and the second target nucleic acid is a target nucleic acid in the nuc gene and the other is a target nucleic acid in the mecA gene.

The method according to any one of items 1 to 10, wherein any one of the first target nucleic acid and the second target nucleic acid is a target nucleic acid in influenza A, and the other is a target nucleic acid in influenza B.

[ 14] A kit for detecting a plurality of target nucleic acids by melting curve analysis in one reaction solution, comprising at least a first target probe for detecting a first target nucleic acid and a second target probe for detecting a second target nucleic acid, wherein the first target probe and the second target probe are designed to satisfy T1 > T2 when the respective detection temperatures in the melting curve analysis are T1 and T2, and are labeled with labels capable of detecting at the same wavelength.

[ 15] the kit of item 14, further comprising the following primers: a first target primer capable of amplifying 1 or more regions containing a first target nucleic acid sequence to which a first target probe can bind; and a second target primer capable of amplifying 1 or more regions containing a second target nucleic acid sequence to which the second target probe can bind.

ADVANTAGEOUS EFFECTS OF INVENTION

According to the present invention, complicated probe designs, condition settings, etc., which have been conventionally required for identifying a plurality of target nucleic acids by gene detection and which require high costs and long time, are not required, and a plurality of target nucleic acids can be measured simultaneously with ease and at a lower cost.

Drawings

Fig. 1 is a graph showing the results of example 1. The results of comparison of the detection temperatures of mismatched probes and non-mismatched probes targeting gonococci are shown.

Fig. 2 is a graph showing the result of example 2. Shows the results of simultaneous detection of chlamydia endogenous plasmid and gonococcus as target nucleic acids.

Fig. 3 is a graph showing the result of example 3. Shows the results of simultaneous detection of mecA gene and nuc gene as target nucleic acids.

Fig. 4 is a graph showing the result of example 4. The results of simultaneous detection of influenza A and influenza B genes as target nucleic acids are shown.

Detailed Description

The following shows embodiments of the present invention and further details of the present invention, but the present invention is not limited to these.

[ method for simultaneously detecting multiple target nucleic acids ]

One embodiment of the present invention is a method for detecting a plurality of target nucleic acids by melting curve analysis in a single reaction solution, the method comprising at least a step of adding a first target probe for detecting a first target nucleic acid and a second target probe for detecting a second target nucleic acid to the reaction solution, wherein the first target probe and the second target probe satisfy T1 > T2 when the respective detection temperatures in the melting curve analysis are T1 and T2, and are labeled with labels capable of being detected at the same wavelength.

According to the method of the present invention, in simultaneous detection of a plurality of target nucleic acids, a plurality of reaction solutions, detection of a plurality of labeling wavelengths, complicated probe design, etc., which have been conventionally considered to be indispensable, are not necessary.

The aforementioned method of the present invention has the following features:

characterized in that, in a melting curve analysis using a probe, a plurality of target nucleic acids possibly contained in a sample can be detected simultaneously in one reaction solution, and a label for detecting a first target nucleic acid and a label for detecting a second target nucleic acid are labels detected by the same wavelength.

In this specification, a label that can be detected at the same wavelength may include: all labels that can be assayed with the same fluorescence detection channel in an assay device for melt curve analysis. For example, a label that can be detected at the same wavelength is referred to as a label when the difference in maximum fluorescence wavelengths from each other is within 40nm, preferably within 30nm, and more preferably within 25 nm. The difference in the maximum fluorescence wavelength of each label may be, for example, 20nm or less, 15nm or less, 10nm or less, or 5nm or less, and the maximum fluorescence wavelengths of labels detectable at the same wavelength may be substantially the same or have no difference. Specifically, for example, a router-Gene-Q (QIAGEN Co.) has 6 types of fluorescence detection channels, each having a different detection wavelength. In addition, each detection wavelength is capable of detecting a plurality of fluorescent pigments. Specifically, as described in table 1.

TABLE 1

For example, as a marker having the same wavelength when 610.+ -.5 nm is selected as the detection wavelength using a router-Gene-Q (router-Gene Q MDx 5plex HRM) or the like, there can be mentioned ROX ^TM CAL Fluor Red 610, cy (registered trademark) 3.5, texas Red, alexa Fluor 568, etc. In addition, FAM and SYBR Green I can be detected under the same fluorescent channel, for example. The maximum fluorescence wavelength of FAM is 520nm,SYBR Green I, that of ela Green is 525nm, and that of eva Green is 521nm, and these fluorescent dyes cannot be distinguished when fluorescence is detected. In this case, FAM, SYBR Green I, eva Green can be the same Detection is performed at wavelength. In the present invention, the group of labels that can be detected in each channel as shown in table 1 above may be used as labels that can be detected at the same wavelength, but the present invention is not limited to these, and any label having the same maximum fluorescence wavelength may be used, and a person skilled in the art can arbitrarily select a label that can be detected at the same wavelength depending on the detection wavelength selected. In a particularly preferred embodiment, the same label is used as label that can be detected at the same wavelength. By using the same label, the invention can be implemented at lower cost and effectively. By using a label capable of detecting at the same wavelength as the present invention for a plurality of target probes, a plurality of target nucleic acids can be detected in one reaction solution without using an analytical device having a plurality of fluorescence detection channels. In addition, since only 1 fluorescence detection channel is required for detecting a plurality of target probes in the present invention, there is an advantage in that, in the case of an analytical device having a plurality of fluorescence detection channels, other fluorescence detection channels can be used more effectively for detecting other target nucleic acids.

The aforementioned method of the invention furthermore has the following features:

in the melting curve analysis, when the detection temperature of the first target probe is T1 and the detection temperature of the second target probe is T2, the condition that T1 > T2 is satisfied.

That is, in the method of the present invention, in order to generate a certain difference in the detection temperature between the first target probe and the second target probe in the melting curve analysis, each probe is designed so that the detection temperature of the second target probe is on the low temperature side compared to the detection temperature of the first target probe.

The base sequence, base length, and the like constituting the first target probe and the second target probe are not particularly limited as long as they satisfy the aforementioned conditions. For example, by including a mismatched base in at least one of the first target probe and the second target probe, the detection temperature of the target probe including the mismatched base in the melting curve analysis can be adjusted to be lower than the detection temperature of the other target probe. Further, by any method such as providing a difference in the length of the base sequences of the first target probe and the second target probe, a difference in the detection temperature in the melting curve analysis of the first target probe and the second target probe can be provided. Preferably, from the standpoint of not requiring complicated probe design and reducing the possibility that the condition setting in nucleic acid amplification and melting curve analysis becomes complicated, it is preferable that at least one of the first target probe or the second target probe is designed to include mismatched bases.

In a specific embodiment, from the standpoint of providing a difference in detection temperature of 2 target probes by a simpler method, it is preferable that the second target probe contains mismatched bases, and from the standpoint of facilitating more reliable obtaining of the effect of the present invention, it is preferable that: the first target probe does not contain a mismatched base, and the second target probe does contain a mismatched base, so that the detection temperature T2 of the second target probe is adjusted to be lower than the detection temperature T1 of the first target probe in the melting curve analysis.

In the present specification, the term "mismatched base (or simply referred to as" mismatch ") refers to a base that is not complementary to a base sequence of a target nucleic acid (a base sequence of a nucleic acid that is either single-stranded after double-strand dissociation in the case where the target nucleic acid is double-stranded). For example, when a cytosine base is present in the base sequence of the target nucleic acid, the base at the position corresponding to the cytosine base in the target probe is a base other than guanine (for example, an adenine base, a cytosine base, a thymine base, or a universal base). The mismatched bases in the first target probe and/or the second target probe may be any of adenine bases, cytosine bases, guanine bases, thymine bases, universal bases (e.g., hypoxanthine, 9- (beta-D-ribofuranose) purine, 5-nitroindole, etc.), and those skilled in the art can appropriately select non-complementary bases to design the probe. For example, in the case of detecting a target nucleic acid that is liable to be mutated, a universal base can be selected at the position of the target probe corresponding to the base that is liable to be mutated.

When the first target probe and/or the second target probe contain mismatched bases, the position at which the mismatch is introduced is not particularly limited as long as the effect of the present invention is not impaired. From the viewpoint of facilitating more reliable detection of the target nucleic acid, it is preferable that the base not be the terminal base of each probe. For example, when the second target probe includes mismatched bases, the position of the mismatched base is preferably within 8mer from the center of the entire length of the base sequence constituting the second target probe, and more preferably within 5mer from the center of the entire length.

One of the features of the method of the present invention is that a difference in detection temperature between the first target probe and the second target probe in the melting curve analysis is generated to some extent. As described above, it has been verified that: if the detection temperatures of the first target probe and the second target probe do not differ from each other in the melting curve analysis, the detection peaks of the first target probe and the second target probe overlap with each other, and therefore it is not possible to identify whether either the first target nucleic acid or the second target nucleic acid is detected or both of them are detected. From the viewpoint of being able to recognize the first target nucleic acid and the second target nucleic acid with higher sensitivity, for example, the difference between the detection temperature T1 of the first target probe and the detection temperature T2 of the second target probe in the melting curve analysis is preferably 5 ℃ or higher, more preferably 7 ℃ or higher, and still more preferably 10 ℃ or higher. The upper limit of the temperature difference between T1 and T2 is not particularly limited, and is preferably 30℃or less, more preferably 25℃or less, from the viewpoint that the temperature range in the melting curve analysis can be controlled within a certain range and the time required for nucleic acid detection in the melting curve analysis can be further shortened.

The difference in base length between the first target probe and the second target probe used in the present invention is not particularly limited as long as the effect of the present invention is exhibited. In general, the base length of a probe affects the optimal annealing temperature for a target nucleic acid, and therefore, if the base lengths of a plurality of probes are extremely different, there is a large difference in the optimal annealing temperature for each probe. Therefore, when a plurality of probes having widely different base lengths are used in one reaction solution, a plurality of annealing temperatures need to be set, which complicates the reaction conditions and may increase the overall reaction time. Therefore, in the present invention using a plurality of target probes for detecting a plurality of target nucleic acids in one reaction solution, it is preferable that there is no extreme difference in base length between the first target probe and the second target probe so that it is not necessary to set a plurality of annealing temperatures for each probe. From such a viewpoint, the difference between the base length of the first target probe and the base length of the second target probe is preferably 8mer or less, more preferably 5mer or less, and still more preferably 3mer or less. The lower limit of the difference in base length between the first target probe and the second target probe may be 0mer or 1mer or 2mer or more, which is not different in base length.

[ method for detecting multiple target nucleic acids ]

One embodiment of the present invention is a method for detecting a plurality of target nucleic acids possibly contained in a sample, comprising at least the following steps (1) to (3):

(1) A step of preparing a reaction solution by mixing a sample that may contain a plurality of target nucleic acids (e.g., a sample that may contain a chlamydia endogenous plasmid and/or gonococcus, a sample that may contain influenza a and/or influenza b, etc.) with a reagent for nucleic acid amplification;

(2) A step of performing a nucleic acid amplification reaction using the reaction solution; and

(3) And (2) hybridizing the first target probe and the second target probe labeled with the label capable of detecting the same wavelength with the amplification product possibly obtained in the step (2), and measuring the fluorescence intensity of the reaction solution by melting curve analysis.

The present invention is characterized by comprising a step of adding the first target probe and the second target probe described above to the reaction solution. The timing of adding the first target probe and the second target probe may be at least the timing of adding the first target probe and the second target probe to the reaction solution in the step (3), and may be, for example, added to the reaction solution before the nucleic acid amplification reaction in the step (1) or (2) at the beginning, to the reaction solution during the nucleic acid amplification reaction in the step (2), or to the reaction solution in the step (3) after the completion of the nucleic acid amplification reaction in the step (2). The first target probe and the second target probe are not necessarily added simultaneously, but may be added separately to the reaction solution. From the viewpoint of enabling continuous amplification and detection reactions and enabling easier handling, it is preferable to add the first and second target probes to the reaction solution in the step (1). In the case of adding a nucleic acid probe before the nucleic acid amplification reaction, for example, it is preferable to ligate a fluorescent dye to the 3' -end thereof or ligate a phosphate group thereto.

In certain embodiments, it can also be mixed with the nucleic acid amplification product in a solvent. The solvent is not particularly limited, and examples thereof include buffers such as Tris-HCl; comprises KCl and MgCl ₂ 、MgSO ₄ Solvents such as glycerin; the PCR reaction solution and other solvents known in the art.

In a particularly preferred embodiment, the method of the present invention for detecting a plurality of target nucleic acids may further comprise a step of amplifying the first target nucleic acid and the second target nucleic acid by primers of: a first target primer (or primer set) capable of amplifying 1 or more regions containing a first target nucleic acid sequence to which the first target probe can bind, and a second target primer (or primer set) capable of amplifying 1 or more regions containing a second target nucleic acid sequence to which the second target probe can bind. Wherein, when the first target primer and the second target primer are double-stranded, the first target nucleic acid or the second target nucleic acid specifically binds to any of the 2 single strands after the double strands are dissociated, and a region of the first target nucleic acid or the second target nucleic acid to which the first target probe or the second target probe can bind is amplified. Preferably, the following are used: the set of 2 or more first target primers and the set of 2 or more second target primers can be amplified so as to sandwich the region of the first target nucleic acid or the second target nucleic acid to which the first target probe or the second target probe can bind.

As a specific embodiment of the method for detecting a plurality of nucleic acids of the present invention, a method including at least the following steps (i) to (vii) can be exemplified, but the method is not limited thereto.

(i) Preparing 1 or more first target primers that specifically bind to any 1 strand out of 2 strands after dissociation of the first target nucleic acid;

(ii) Preparing 1 or more second target primers that specifically bind to any 1 strand out of 2 strands after dissociation of the second target nucleic acid;

(iii) A step of preparing a first target probe and a second target probe for detection in a melting curve analysis, each of which has a label capable of emitting light at the same wavelength;

(iv) A step of preparing a reaction solution containing a sample that may contain a plurality of target nucleic acids, the first target primer, the second target primer, the first target probe, and the second target probe;

(v) Amplifying the first target nucleic acid and the second target nucleic acid in the sample using the reaction solution;

(vi) A step of hybridizing the nucleic acid amplification product obtained in the step (v) with a first target probe and/or a second target probe designed to form a complex with a part of the nucleic acid amplification product, thereby forming a complex; and

(vii) And (3) detecting the complex obtained in step (vi).

The order of the steps (i) to (iii) in the above-described method is not limited.

In one embodiment, the method of detecting a plurality of target nucleic acids of the present invention may be used to: in the examination of a plurality of infectious microorganisms whose symptoms or conditions are very similar and which cannot be easily identified, a first target nucleic acid and a second target nucleic acid, which may be derived from the plurality of infectious microorganisms, respectively, are detected. Examples of the plurality of target nucleic acids which may be derived from such a plurality of pathogenic microorganisms include, for example, a target nucleic acid in an endogenous plasmid of chlamydia and a target nucleic acid in gonococcus; a target nucleic acid in influenza a and a target nucleic acid in influenza b; target nucleic acid in coronavirus and target nucleic acid in influenza virus; target nucleic acid in tubercle bacillus and target nucleic acid in nontuberculous acid-fast bacteria, etc., but are not limited thereto. The present invention can discriminate the infection with very similar symptoms or states in a simple way, so that the risk of missing one infection in the case of co-infection is reduced, and the early treatment and prevention of the spread of infection to the surrounding can be realized.

In another embodiment, the method for detecting a plurality of target nucleic acids of the present invention can also be used for detecting a plurality of target nucleic acids possibly derived from one microorganism or the like. Examples of such a plurality of target nucleic acids include a target nucleic acid in nuc gene (nuclease gene) derived from staphylococcus aureus and a target nucleic acid in mecA gene (methicillin-resistant gene); target nucleic acid in tcdA gene and target nucleic acid in tcdB gene derived from Clostridium difficile; target nucleic acids in vanA gene and vanB gene derived from vancomycin-resistant cocci, etc., but are not limited thereto.

[ sample ]

The sample usable in the present invention is not particularly limited as long as it is possible to contain a plurality of target nucleic acids. For example, not only biological samples, foods, environmental samples, but also purified nucleic acids and the like can be mentioned. Alternatively, the sample may be subjected to nucleic acid extraction or some pretreatment. Nucleic acid extraction and pretreatment of samples are commonly performed in the art. Examples of the pretreatment include filtration, centrifugation, dilution, heat treatment, acid treatment, alkali treatment, organic solvent treatment, suspension treatment, crushing treatment, and grinding treatment, but the present invention is not limited thereto.

Examples of the biological sample include, but are not particularly limited to, animal and plant tissues, body fluids, excretions, cells, bacteria, viruses, and the like. Further examples include blood, blood culture fluid, urine, pus, spinal fluid, chest fluid, throat swab fluid, nasal swab fluid, sputum, tissue sections, skin, vomit, feces, isolated culture colonies, catheter washings, cervical canal wipes, urethral wipes, male urethra wipes, and urine.

Examples of the food include water, alcoholic beverages, soft drink water, processed foods, vegetables, livestock products, seafood, eggs, dairy products, raw meats, raw fish, side dishes, and the like. In the case of using a food as a measurement sample, not only a part or all of the food but also a product obtained by wiping the surface of the food may be used. Further, a cleaning liquid obtained by wiping the conditioning tool, the material after the door handle, or cleaning the same may be used as the sample.

Examples of the environmental sample include water, ice, soil, air, and aerosol. The water used herein includes, for example, water collected from tap water, sea water, river water, waterfall, lake, pool, or the like. In addition, a material after wiping a wall surface, floor surface, equipment, spare parts, toilet stool, or the like or a cleaning liquid obtained by cleaning them may be used as a sample.

The present invention can be applied to any sample as described above, but is particularly effective to a subject suspected of being infected or co-infected with any of a plurality of infectious microorganisms or a sample collected from a subject. The type of sample to be collected is preferably selected to be suitable for each sample, depending on the symptoms of the infection of the subject, the state of the subject, and the like. For example, when detecting a chlamydia endogenous plasmid and/or gonococcus as a target nucleic acid, a biological sample (for example, animal or plant tissue, body fluid, excrement, throat swab liquid, tissue slice, cervical canal wiper, urethra wiper urine, vomit, urine, isolated culture colony) is preferably used, excrement, urine, cervical canal wiper, urethra wiper, male urethra wiper urine, isolated culture colony is more preferably used, and urine, cervical canal wiper, urethra wiper, male urethra wiper urine, throat swab liquid is more preferably used. In addition, for example, when influenza a and/or influenza b is detected as a target nucleic acid, a biological sample such as nasopharyngeal swab liquid, nasal swab liquid, sputum, saliva or the like is preferably used. In addition, for example, when detecting nuc gene and/or mecA gene as a target nucleic acid, a biological sample such as a blood culture solution is preferably used. According to the present invention, even when such a sample is used, a plurality of target nucleic acids can be detected simultaneously, and a plurality of target nucleic acids can be detected simultaneously at low cost by a simple condition setting in one reaction vessel.

The method of collecting the sample, the method of preparing the sample, and the like are not particularly limited, and a known method may be used depending on the type and purpose of the sample.

[ nucleic acid amplification reaction ]

In the step (2), any nucleic acid amplification method known in the art can be used for amplifying nucleic acids. Examples of such nucleic acid amplification methods include PCR, LAMP, LCR, TMA, SDA, RT-PCR, RT-LAMP, NASBA, TRC, TMA, and the like. These techniques have been established in the art and methods may be selected according to purposes. The nucleic acid amplification method to be carried out in the present invention is preferably a PCR method (including RT-PCR method) from the viewpoint of more reliably and easily obtaining a high effect, but is not limited thereto.

[ PCR method ]

The PCR reaction is mainly catalyzed by DNA polymerase, and takes the following three steps as one cycle: [1] denaturation of DNA based on heat treatment (dissociation of double-stranded DNA into single-stranded DNA); [2] annealing the template single-stranded DNA by the primer; [3] the cycle is repeated by extension of the primer using a DNA polymerase to amplify the target nucleic acid. Examples of the DNA polymerase include Taq, tth, bst, KOD, pfu, pwo, tbr, tfi, tfl, tma, tne, vent, DEEPVENT and mutants thereof. In the present invention, it is preferable to use a DNA polymerase belonging to family B, from the viewpoint that highly specific nucleic acid amplification can be achieved more easily.

In the present specification, the mutant of the DNA polymerase means: the DNA-amplifying activity is similar to that of the wild-type DNA polymerase, and has a sequence identity of, for example, 85% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, and particularly preferably 99% or more with the amino acid sequence of the wild-type DNA polymerase from which the DNA-amplifying activity is derived. The method for calculating the identity of amino acid sequences may be performed by any means known in the art. For example, it can be calculated using analysis tools that are commercially available or can be utilized via telecommunication lines (internet), as an example, by using the homology algorithm BLAST (local sequence alignment search basic tool) of the National Center for Biotechnology Information (NCBI) http: the default (initial setting) parameters of// www.ncbi.nlm.nih.gov/BLAST/can be used to calculate the identity of amino acid sequences. In addition, mutants that can be used in the present invention may be: an enzyme which comprises an amino acid sequence having 1 or more amino acids substituted, deleted, inserted and/or added (hereinafter, these will be collectively referred to as "mutation") in the amino acid sequence of a wild-type DNA polymerase from which it is derived, and which has DNA amplification activity similar to that of the wild-type DNA polymerase. The number of 1 or more may be, for example, 1 to 80, preferably 1 to 40, more preferably 1 to 10, still more preferably 1 to 5, and is not particularly limited.

[ DNA polymerase belonging to family B ]

The DNA polymerase used in the present invention is preferably a DNA polymerase belonging to family B, but is not limited thereto. The aforementioned DNA polymerase belonging to family B is not particularly limited, and a DNA polymerase derived from archaebacteria (Archia) is preferable.

[ archaebacteria-derived DNA polymerase ]

Examples of the archaebacteria-derived DNA polymerase belonging to family B include DNA polymerases isolated from bacteria belonging to the genus Pyrococcus (Pyrococcus) and the genus Thermococcus (Thermococcus). In addition, mutants thereof belonging to family B, in which the activity of the archaebacteria-derived DNA polymerase is not lost, are also included in the present invention. Examples of mutants of DNA polymerase include, but are not limited to, mutants aimed at enhancement of polymerase activity, deletion of exonuclease activity, and adjustment of substrate specificity.

Examples of the DNA polymerase derived from Pyrococcus include, but are not limited to, DNA polymerases isolated from Pyrococcus furiosus (Pyrococcus furiosus), pyrococcus GB-D, pyrococcus vortioides (Pyrococcus woesei), pyrococcus furiosus (Pyrococcus abyssi) and Pyrococcus hyperthermophiles (Pyrococcus horikoshii), and mutants thereof derived from these.

Examples of the DNA polymerase derived from Pyrococcus include, but are not limited to, DNA polymerases isolated from Pyrococcus hyperthermophiles (Thermococcus kodakaraensis), pyrococcus erythropolis (Thermococcus gorgonarius), pyrococcus maritimus (Thermococcus litoralis), pyrococcus hyperthermophiles JDF-3, pyrococcus hyperthermophiles 9degrees North-7 (Pyrococcus 9DEG N-7), pyrococcus hyperthermophiles (Thermococcus siculi), and mutants thereof derived from these.

Examples of PCR enzymes using these DNA polymerases are commercially available, and Pfu (Stargene Co.), KOD (Toyobo Co.), pfx (Life Technologies Co.), vent (New England Biolabs Co.), deep Vent (New England Biolabs Co.), tgo (Roche Co.), pwo (Roche Co.), etc., may be used in the present invention.

Among them, KOD DNA polymerase and its mutants (e.g., KOD DNA polymerase lacking 3 '. Fwdarw.5' exonuclease activity, etc.) excellent in extensibility and thermostability are preferable.

KOD DNA polymerase is excellent in accuracy, amplification efficiency, extensibility, and crude sample resistance as compared with Taq DNA polymerase, which is a DNA polymerase belonging to family A. In the present invention, by using such KOD DNA polymerase, detection can be performed using a detection probe having a mismatch introduced, and simultaneous detection of a plurality of target nucleic acids can be achieved as shown in examples described later.

The conditions (e.g., temperature, pH, cation concentration, presence of an organic solvent in a solution, etc.) of the nucleic acid amplification step may be optimized according to hybridization conditions of the nucleic acid primer or the nucleic acid probe, etc., and may be appropriately set by those skilled in the art.

[ reagent for nucleic acid amplification ]

In the present invention, in performing the nucleic acid amplification reaction, the nucleic acid amplification reagent used in the nucleic acid amplification reaction may be appropriately selected according to the nucleic acid amplification reaction to be performed. For example, the nucleic acid amplification reagent includes components necessary for a nucleic acid amplification reaction in addition to the first target primer and the second target primer, and the first target probe and the second target probe, which are characteristic described above. The components required for the nucleic acid amplification reaction vary depending on the nucleic acid amplification reaction to be carried out, and known components can be used. For example, when a plurality of target nucleic acids contained in a sample are detected by a PCR method, it is preferable that the sample contains at least inorganic salts such as DNA polymerase, deoxyribonucleoside triphosphates (dNTPs), and magnesium salts. The concentration of each component can be appropriately adjusted, and for example, the oligonucleotide probe (first target probe or second target probe) is preferably 0.1 to 1. Mu.M, more preferably 0.2 to 0.5. Mu.M. The DNA polymerase is preferably 0.01 to 1U/uL, more preferably 0.1 to 0.5U/uL. The oligonucleotide primer (first target primer or second target primer) is preferably 0.1 to 10. Mu.M. Deoxyribonucleoside triphosphates (dNTPs) are preferably 0.02 to 1mM, more preferably 0.1 to 0.5mM. Inorganic salts such as magnesium salts are preferably 0.1 to 6mM, more preferably 1 to 5mM.

Furthermore, for the purpose of suppressing nonspecific amplification and promoting reaction, a nucleic acid amplification reagent may be added with additives known in the art. Examples of the additives for the purpose of suppressing non-specific amplification include an anti-DNA polymerase antibody and phosphoric acid. Examples of the additives for the purpose of promoting the reaction include Bovine Serum Albumin (BSA), protease inhibitors, single-chain binding proteins (SSB), T4 gene 32 proteins, tRNA, sulfur-or acetic acid-containing compounds, dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, trimethylene glycol, formamide, acetamide, betaine, tetrahydropyrimidine, trehalose, dextran, polyvinylpyrrolidone (PVP), gelatin, tetramethylammonium chloride (TMAC), tetramethylammonium hydroxide (TMAH), tetramethylammonium acetate (TMAA), polyethylene glycol, triton, tween20, and Nori detergent P40. In the present invention, 1 or more kinds of these additives may be used in combination, but the present invention is not limited to these.

In addition, in the nucleic acid amplification step by the PCR method, the thermal cycle conditions are not particularly limited, and can be appropriately set by those skilled in the art. As an example, the nucleic acid amplification reaction is preferably: the initial thermal denaturation step is performed at 80 to 100℃for 10 seconds to 15 minutes, the repeated thermal denaturation step is performed at 80 to 100℃for 0.5 to 300 seconds, the annealing step is performed at 40 to 80℃for 1 to 300 seconds, the elongation reaction step is performed at 60 to 85℃for about 1 to 300 seconds, and the cycle is repeated 30 to 70 times.

[ melting Curve analysis ]

Melting curve analysis can be widely used for analysis of target nucleic acids. The temperature at which half of the double-stranded nucleic acid becomes single-stranded is referred to as the melting temperature. The melting temperature is basically dependent on the content of bases contained in the nucleic acid thereof, and thus the prescribed base sequence has an inherent melting temperature corresponding to the content of bases contained therein. Melting curve analysis is an analysis method that uses the property of a nucleic acid that dissociates from a double strand into a single strand according to a temperature change.

[ detection temperature in melting Curve analysis ]

In melting curve analysis, the intensity of fluorescence or quenching signal generated with dissociation of the labeled probe from the target sequence is monitored in real time for temperature changes. When a primary differential value obtained by differentiating a value of a change in luminescence or quenching signal intensity with a temperature rise by a temperature change is plotted, a melting temperature can be expressed as a peak in a melting curve (temperature-primary differential value curve) obtained. In the present invention, when a melting curve analysis is performed using a predetermined target probe, the melting temperature indicated as a peak is referred to as the detection temperature of the target probe.

Currently, as a specific application method utilizing the characteristics of melting curve analysis, detection of mutation in a target nucleic acid (for example, SNP analysis) and the like have been performed. If the target nucleic acid is mutated, a double-stranded gene having a mismatched portion is formed by performing PCR. Thus, the double-stranded gene having the mismatched portion is liable to cause denaturation (dissociation) of the double-stranded structure, and the detection temperature becomes lower than that of the wild-type gene at the time of melting curve analysis. Thus, if the target nucleic acid is mutated, a change in the detection temperature can be detected.

The use of the conventional melting curve analysis as described above is useful for detecting the presence of mismatched bases on the target nucleic acid side. The present invention is different from the conventional method in that the detection temperature of the melting curve analysis is adjusted by introducing a mismatched base into a target probe, not based on the presence of a mismatched base in a target nucleic acid.

In the present invention, the nucleic acid amplification product can be detected by any analysis method using melting curve analysis of a probe, and for example, detection can be performed by a method using Qprobe (registered trademark) (also referred to as Q probe), epobe (registered trademark) (also referred to as E probe), taqMan probe, molecular beacon probe, FRET hybridization probe, scope probe, or any combination thereof. From the viewpoint of being able to detect a plurality of target nucleic acids simultaneously with higher sensitivity, detection is preferably performed by melting curve analysis using a Q probe or an E probe, and among these, detection is more preferably performed by melting curve analysis using a Q probe.

The Q probe (also referred to as "guanine quenching probe") is a fluorescent probe (fluorescence quenching probe) developed by KURATA et al (Japanese patent No. 5354216). The probe is a hybridization probe labeled with a fluorescence quenching dye quenched by the interaction of at least one terminal base with guanine.

For example, the fluorescence quenching dye used in the Q probe is not particularly limited, and fluorescein or its derivative (e.g., fluorescein isothiocyanate), rhodamine or its derivative (e.g., tetramethylrhodamine isothiocyanate, carboxyrhodamine, x-rhodamine, sulforhodamine 101 acid chloride), BODIPY or its derivative (e.g., BODIPY-FL/C3, BODIPY-FL/C6, BODIPY-5-FAM, BODIPY-TMR, BODIPY-TR, BODIPY-R6G, BODIPY-564, BODIPY-581, BODIPY-591, BODIPY-630, BODIPY-650, BODIPY-665) and the like can be exemplified. Details of the fluorescent quenching dye are described in Japanese patent No. 5813263, etc., and the present invention is also applicable to this technique.

In the case of using a Q probe as a label in the present invention, a probe composed of a base sequence in which at least one terminal base is cytosine and in which the cytosine of the terminal base is labeled with the above-mentioned fluorescence quenching dye is preferable. When such a probe hybridizes to an amplification product, it can be quenched by forming a base pair with a guanine base in the amplification product, and therefore, the change in fluorescence intensity of the reaction solution can be measured very easily.

In the hybridization of the probe, even if the cytosine base of the probe and the guanine base in the amplified product do not form a base pair, fluorescence can be quenched if the bases are closely spaced. For example, details are described in japanese patent No. 5354216, and the present invention can refer to this technology. That is, when hybridization occurs with the probe, the guanine base in the amplified product can be quenched (the base forming a base pair with the cytosine base is 1) if the guanine base is present in the range of 1 to 3 bases, for example.

In the method for simultaneously detecting a plurality of target nucleic acids of the present invention, the base sequence of the first target probe and/or the second target probe is not particularly limited as long as it can form a complex with a part of the amplification product of the first target nucleic acid and/or the second target nucleic acid. Preferably, the probe containing a mismatch in the base sequence of the second target probe is used, whereby the detection temperature of the second target nucleic acid is adjusted to a low temperature side, and as a result, a plurality of target nucleic acids can be detected simultaneously.

[ kit for simultaneously detecting multiple target nucleic acids ]

Further, as another embodiment of the present invention, a nucleic acid detection kit for detecting a plurality of target nucleic acids by melting curve analysis in one reaction solution is provided. The kit of the present invention is characterized by comprising at least a first target probe for detecting a first target nucleic acid and a second target probe for detecting a second target nucleic acid, wherein the first target probe and the second target probe are designed so that T1 > T2 is satisfied when the detection temperatures in the melting curve analysis are T1 and T2, and are labeled with labels that can be detected at the same wavelength. The kit of the present invention preferably further comprises: the primer set for a first target (or primer set) is capable of amplifying 1 or more regions containing a first target nucleic acid sequence to which a first target probe can bind, and the primer set for a second target (or primer set) is capable of amplifying 1 or more regions containing a second target nucleic acid sequence to which a second target probe can bind. The kit of the present invention is not particularly limited as long as it can detect a plurality of target nucleic acids simultaneously by detecting the temperature in the melting curve analysis. In order to efficiently amplify a highly specific nucleic acid, the kit preferably further comprises at least 1 component suitable for a nucleic acid amplification reaction, such as a DNA polymerase selected from the group consisting of Taq, tth, bst, KOD, pfu, pwo, tbr, tfi, tfl, tma, tne, vent, DEEPVENT and mutants thereof.

Examples

The present invention will be specifically described with reference to examples, but the present invention is not limited to the examples.

Example 1: variations in detected temperature between when a mismatch is introduced and when no mismatch is introduced in the probe

(1-1) method

The melting curve analysis was performed using the following primers and probes for targets (probe with no mismatch introduced and probe with mismatch added) as targets. In the following table, the shaded bases are mismatched bases.

TABLE 2

* The shaded bases are mismatched bases.

(1-2) reaction solution

Using GENECUBE (registered trademark) Test basic (manufactured by Toyobo Co., ltd.) and the primers and probes shown in Table 2, a reaction solution containing the following components was prepared.

0.3μM SNG F

2.0μM SNG R

0.3. Mu.M SNG QP (non-mismatched probe or mismatched probe)

(1-3) reaction

Using GENECUBE (registered trademark), the reaction mixture was subjected to a temperature cycle reaction as described below to amplify the nucleic acid. After the nucleic acid amplification reaction, a melting curve analysis was performed based on the following conditions.

94 ℃ for 30 seconds,

97 ℃ 1 second-58 ℃ 5 second-63 ℃ 2 seconds (cycle number 60 times)

94℃30 seconds 39℃30 seconds 40-75℃0.09 ℃/second

(1-4) results

Fig. 1 shows the results of melting curve analysis. The base lengths of the probe having no mismatch and the probe having mismatch used in this test example are the same. In general, in the case of probes having substantially the same base length, the detection temperature tends to be easily close in the melting curve analysis, but it is known that even if only 1 mismatched base is added, the detection temperature can be greatly adjusted to the low temperature side.

Example 2: simultaneous detection of Chlamydia endogenous plasmid and gonococcus

(2-1) method

In this example, a gene in an endogenous plasmid of Chlamydia was used as the first target nucleic acid, and a gene in gonococcus was used as the second target nucleic acid, and simultaneous detection in melting curve analysis at the same wavelength was attempted. The first target primer and the second target primer, the first target probe and the second target probe used in the PCR method in this example are as follows. Wherein the first target probe is labeled with BODIPY-FL at the 3' end and the second target probe is labeled with BODIPY-FL at the 5' end to phosphorylate the 3' end. In the case of this example, the SNG QP contains a mismatch. The shaded bases are mismatched bases.

TABLE 3

In the case of this example, the SNC QP contains a mismatch. The shaded bases are mismatched bases.

(2-2) reaction solution

Using GENECUBE (registered trademark) Test bases (manufactured by Toyobo Co., ltd.), the primers and probes shown in Table 3 were added at the concentrations shown below to prepare a reaction solution.

0.5μM SCT F

2.5μM SCT R

0.3μM SCT QP

0.3μM SNG F

2.0μM SNG R

0.3μM SNG QP

(2-3) reaction

94 ℃ for 30 seconds,

97 ℃ 1 second-58 ℃ 5 second-63 ℃ 2 seconds (cycle number 60 times)

94℃30 seconds 39℃30 seconds 40-75℃0.09 ℃/second

(2-4) results

Fig. 2 shows the results of the melting curve analysis. As shown in FIG. 2, the detection peak was confirmed at about 62℃for the first target probe (SCT QP, chlamydia detection probe) containing no mismatched base, and at about 46℃for the second target probe (SNG QP, gonococcus detection probe) containing mismatched base. The difference in base length between the first target probe and the second target probe is about 2mer, but the difference in detection temperature in the melting curve analysis is not only increased to about 16 ℃. From this result, it can be seen that: by introducing a mismatch into the probe for gonococcus target and adjusting the detection temperature to a low temperature side, the target nucleic acid in the endogenous plasmid of chlamydia and the target nucleic acid in gonococcus can be detected simultaneously in the melting curve analysis based on the detection temperature, and the target nucleic acid can be recognized by using a marker having the same wavelength.

Example 3: simultaneous detection of mecA Gene and nuc Gene

(3-1) method

In this example, the first target nucleic acid was designated as nuc gene and the second target nucleic acid was designated as mecA gene. The first target primer and the second target primer, the first target probe and the second target probe used in the PCR method in this example are as follows. Wherein the first target probe and the second target probe are labeled with BODIPY-FL at the 3' -end. In the case of this example, the mecA QP contains mismatches. The shaded bases are mismatched bases.

TABLE 4

In the case of this example, the mecA QP contains a mismatch. The shaded bases are mismatched bases.

(3-2) reaction solution

Using GENECUBE (registered trademark) Test bases (manufactured by Toyobo Co., ltd.), the primers and probes shown in Table 4 were added at the concentrations shown below to prepare a reaction solution.

4.0μM nuc F

0.9μM nuc R

1.2μM nuc QP

2.0μM mecA F

0.4μM mecA R

0.2μM mecA QP

(3-3) reaction

94 ℃ for 30 seconds,

97 ℃ 1 second-58 ℃ 3 second-63 ℃ 5 seconds (cycle number 60 times)

94℃30 seconds 39℃30 seconds 40-75℃0.09 ℃/second

(3-4) results

Fig. 3 shows the results of the melting curve analysis. As shown in FIG. 3, the first target probe (nuc QP, nuc gene detection probe) containing no mismatched base detected a detection peak at about 58℃and the second target probe (mecAQP, mecA gene detection probe) containing mismatched base detected a detection peak at about 45 ℃. The base lengths of the first target probe and the second target probe are the same, but the difference in detection temperature in the melting curve analysis is increased to about 13 ℃. From this result, it can be seen that: by introducing a mismatch into the mecA gene target probe and adjusting the detection temperature to a low temperature side, nuc gene and mecA gene can be detected simultaneously in a melting curve analysis based on the detection temperature, and can be recognized by a marker having the same wavelength.

Example 4: simultaneous detection of influenza A and B genes

(4-1) method

In this example, the first target nucleic acid was designated as the influenza A gene and the second target nucleic acid was designated as the influenza B gene. The first target primer and the second target primer, the first target probe and the second target probe used in the PCR method in this example are as follows. Wherein the first target probe is labeled with BODIPY-FL at the 3' end and the second target probe is labeled with BODIPY-FL at the 5' end to phosphorylate the 3' end. In the case of this example, the FluB QP contains mismatches. The shaded bases are mismatched bases.

TABLE 5

In the case of this example, the FluB QP contained mismatches. The shaded bases are mismatched bases.

(4-2) reaction solution

Using GENECUBE (registered trademark) Test basic (manufactured by Toyobo Co., ltd.) and ReverTra Ace (registered trademark), the primers and probes shown in Table 5 were added at the concentrations shown below to prepare a reaction solution.

0.5μM FluA F

3.0μM FluA R

0.3μM FluA QP

0.5μM FluB F

3.0μM FluB R

0.3μM FluB QP

(4-3) reaction

Using GENECUBE (registered trademark), the reaction mixture was subjected to reverse transcription and nucleic acid amplification by circulating the reaction mixture at the following temperature. After the nucleic acid amplification reaction, a melting curve analysis was performed based on the following conditions.

120 seconds at 42℃,

97 ℃ for 15 seconds,

97 ℃ 1 second-58 ℃ 3 second-63 ℃ 5 seconds (cycle number 50 times)

94℃30 seconds 39℃30 seconds 40-75℃0.09 ℃/second

(4-4) results

Fig. 4 shows the results of the melting curve analysis. As shown in FIG. 4, the first target probe (FluAQP, influenza A gene detection probe) containing no mismatched base detected a detection peak at around 60℃and the second target probe (FluB QP, influenza B gene detection probe) containing mismatched base detected a detection peak at around 46 ℃. The difference in base length between the first target probe and the second target probe was about 2mer, but the difference in detection temperature in the melting curve analysis was increased to about 14 ℃. From this result, it can be seen that: by introducing a mismatch into the probe for influenza b gene target and adjusting the detection temperature to the low temperature side, the influenza a gene and the influenza b gene can be detected and identified simultaneously based on the detection temperature.

Sequence listing

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Claims

1. A method for detecting a plurality of target nucleic acids by melting curve analysis in a single reaction solution, characterized in that the method comprises a step of adding a first target probe for detecting a first target nucleic acid and a second target probe for detecting a second target nucleic acid to the reaction solution, wherein the first target probe and the second target probe satisfy T1 > T2 when the respective detection temperatures in the melting curve analysis are T1 and T2, and are labeled with labels capable of being detected at the same wavelength, respectively.

2. The method of claim 1, further comprising the step of amplifying the first target nucleic acid and the second target nucleic acid by primers that are: a first target primer capable of amplifying 1 or more regions containing a first target nucleic acid sequence to which a first target probe can bind; and a second target primer capable of amplifying 1 or more regions containing a second target nucleic acid sequence to which the second target probe can bind.

3. The method according to claim 1 or 2, wherein the label of the first target probe and the label of the second target probe are the same label.

4. The method according to any one of claims 1 to 3, wherein the second target probe comprises mismatched bases.

5. The method according to any one of claims 1 to 4, wherein the mismatched base is at least 1 selected from the group consisting of an adenine base, a cytosine base, a guanine base, a thymine base, and a universal base.

6. The method according to any one of claims 1 to 5, wherein the universal base is at least 1 selected from the group consisting of hypoxanthine, 9- (beta-D-ribofuranose) purine, and 5-nitroindole.

7. The method according to any one of claims 1 to 6, wherein the detection temperature T2 of the second target probe is adjusted to be lower than the detection temperature T1 of the first target probe in the melting curve analysis by making the first target probe contain no mismatched base and making the second target probe contain mismatched base.

8. The method according to any one of claims 1 to 7, wherein a temperature difference between the detection temperature T1 of the first target probe and the detection temperature T2 of the second target probe in the melting curve analysis is 5 to 30 ℃.

9. The method according to any one of claims 1 to 8, wherein the difference between the length of the nucleobase of the first target probe and the length of the nucleobase of the second target probe is 8mer or less.

10. The method according to any one of claims 1 to 10, wherein the label of the first target probe and the label of the second target probe are Qprobe (registered trademark) or epoprobe (registered trademark).

11. The method of any one of claims 1-10, wherein either one of the first target nucleic acid and the second target nucleic acid is a target nucleic acid in a chlamydia endogenous plasmid, and the other is a target nucleic acid in gonococcus.

12. The method of any one of claims 1-10, wherein either one of the first target nucleic acid and the second target nucleic acid is a target nucleic acid in a nuc gene and the other is a target nucleic acid in a mecA gene.

13. The method of any one of claims 1-10, wherein either one of the first and second target nucleic acids is a target nucleic acid in influenza a and the other is a target nucleic acid in influenza b.

14. A kit for detecting a plurality of target nucleic acids by melting curve analysis in a single reaction solution, comprising at least a first target probe for detecting a first target nucleic acid and a second target probe for detecting a second target nucleic acid, wherein the first target probe and the second target probe are designed to satisfy T1 > T2 when the respective detection temperatures in the melting curve analysis are T1 and T2, and are labeled with labels capable of being detected at the same wavelength.

15. The kit of claim 14, further comprising primers that are: a first target primer capable of amplifying 1 or more regions containing a first target nucleic acid sequence to which a first target probe can bind; and a second target primer capable of amplifying 1 or more regions containing a second target nucleic acid sequence to which the second target probe can bind.