WO2022092012A1

WO2022092012A1 - Method for simultaneously detecting plurality of target nucleic acids

Info

Publication number: WO2022092012A1
Application number: PCT/JP2021/039267
Authority: WO
Inventors: 真史道渕; 佳子上倉; 広道鈴木
Original assignee: 東洋紡株式会社
Priority date: 2020-10-30
Filing date: 2021-10-25
Publication date: 2022-05-05
Also published as: JPWO2022092012A1; CN116391039A

Abstract

The present invention addresses the problem of providing a simple, low-cost method capable of simultaneously differentiating a plurality of target nucleic acids. The present invention provides a method for detecting a plurality of target nucleic acids by melting curve analysis in a one reaction solution, the method being characterized by including a process for adding, to the reaction solution, 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 satisfy T1>T2 when the detection temperatures in the melting curve analysis are T1 and T2, respectively, and each of the target probes is labeled with a label that can be detected at the same wavelength.

Description

Simultaneous detection method for multiple target nucleic acids

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

Many of the various infectious diseases have very similar clinical symptoms and are not easy to distinguish. For example, chlamydia infection and gonococcal infection, which are sexually transmitted diseases, have very similar symptoms, and their differentiation is clinically important. Therefore, a method for simultaneously differentiating by various test methods including the PCR method has been developed.

Examples of situations where it is necessary to measure multiple target genes at the same time in a genetic test such as the PCR method include not only chlamydia and gonorrhea, which are sexually transmitted diseases as described above, but also influenza A and B. Be done. In the tests for infectious diseases with very similar clinical symptoms, it is required to be able to distinguish them at the same time by genetic tests from the same sample.

One of the reasons is that the symptoms of multiple infectious diseases as described above are very similar, and therefore, when co-infection occurs, one of the infections may be overlooked. In this case, not only the treatment is delayed, but also there is a concern that the infection spreads to the surroundings. In such a case, if it becomes possible to simultaneously distinguish pathogenic microorganisms having similar symptoms, not only early treatment can be performed, but also infection to the surroundings can be prevented.

So far, as a method for simultaneously detecting a plurality of target nucleic acids, a method of performing measurements separately using different reaction solutions (Patent Document 1), or by using labeled substances having different wavelengths for each detection. A method for identification (Patent Document 2) and the like are widely used.

However, when the measurement is performed separately using different reaction solutions as in Patent Document 1, there is a problem that the time for preparing the preparation of the analytical reagent and the cost of the reagent are doubled.

Further, in the case of measuring using a labeled substance having a different wavelength for each detection as in Patent Document 2, it takes time to select an analyzer equipped with various types of fluorescence detection channels and set the wavelength to be detected. costly.

Further, there is also known a method of simultaneously detecting a plurality of nucleic acids using a single fluorescent label in one reaction vessel by combining the annealing temperature condition setting of the probe and the temperature change profile (Patent Document 3). However, this method has a problem that the design of the probe and the setting of conditions are complicated and costly because it is necessary to greatly change the base length of the probe for each probe.

Japanese Unexamined Patent Publication No. 2002-136300 Japanese Unexamined Patent Publication No. 2004-203 Japanese Unexamined Patent Publication No. 2017-521758

The present invention has been made against the background of the above-mentioned conventional problems. That is, the purpose is to develop a new method that can simultaneously identify a plurality of target nucleic acids at a simple and low cost.

As a result of diligent research, the present inventor surprisingly, if the detection temperatures of a plurality of target probes are designed to be separated from each other by a certain degree in the melting curve analysis using the probe, they are labeled with a labeled substance that can detect them at the same wavelength. Even in this case, it was found that two peaks indicated by the first-order differential value of the fluorescence / quenching change amount of each target probe were clearly observed, and that multiple target nucleic acids could be identified by detecting the same wavelength. In order to design the detection temperatures of the target probes to be separated from each other by a certain degree, it may be possible, for example, by introducing a mismatched base into one of a plurality of target probes. The present invention has been further studied and completed based on this finding.

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

[Item 1] A method for detecting a plurality of target nucleic acids in one reaction solution by melting curve analysis, a first target probe for detecting the first target nucleic acid and a second target for detecting the second target nucleic acid. Including the step of adding the probe to the reaction solution, 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. Moreover, the method is characterized in that each is labeled with a labeled substance that can be detected at the same wavelength.

[Item 2] One or more primary target primers capable of amplifying a region containing a sequence of the first target nucleic acid to which the first target probe can bind, and a second target to which the second target probe can bind. Item 2. The method according to Item 1, further comprising a step of amplifying the first target nucleic acid and the second target nucleic acid with one or more second target primers capable of amplifying the region containing the nucleic acid sequence.

[Item 3] The method according to Item 1 or 2, wherein the labeled substance of the first target probe and the labeled substance of the second target probe are the same labeled substance.

[Item 4] The method according to any one of Items 1 to 3, wherein the second target probe contains a mismatched base.

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

[Item 6] The method according to any one of Items 1 to 5, wherein the universal base is at least one selected from the group consisting of hypoxanthine, nebulaline, and 5-nitroindole.

[Item 7] The first target probe does not contain a mismatched base, and the second target probe contains a mismatched base, so that the detection temperature T1 of the first target probe is higher than that of the second target probe in the melting curve analysis. Item 6. The method according to any one of Items 1 to 6, wherein the detection temperature T2 is adjusted to be on the low temperature side.

[Item 8] Item 2. The item 1 to 7, wherein the 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 ° C. the method of.

[Item 9] 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 8 mer or less.

[Item 10] The method according to any one of Items 1 to 10, wherein the labeled substance of the first target probe and the labeled substance of the second target probe are Qprobe (registered trademark) or Eprobe (registered trademark).

[Item 11] The method according to any one of Items 1 to 10, wherein either the first target nucleic acid or the second target nucleic acid is the target nucleic acid in the Chlamydia endogenous plasmid and the other is the target nucleic acid in the gonococcus.

Item 12. The method according to any one of Items 1 to 10, wherein either the first target nucleic acid or the second target nucleic acid is the target nucleic acid in the nuc gene and the other is the target nucleic acid in the mecA gene.

[Item 13] 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 the target nucleic acid in influenza A and the other is the target nucleic acid in influenza B.

[Item 14] A nucleic acid detection kit used for detecting a plurality of target nucleic acids in one reaction solution by melting curve analysis, the first target probe for detecting the first target nucleic acid and the second target nucleic acid. 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. A kit characterized by being labeled with a labeled substance that can be detected at the same wavelength.

[Item 15] One or more primary target primers capable of amplifying a region containing a sequence of the first target nucleic acid to which the first target probe can bind, and a second target to which the second target probe can bind. Item 12. The kit of Item 14, further comprising one or more secondary targeting primers capable of amplifying the region containing the sequence of nucleic acid.

The present invention eliminates the need for designing and setting conditions for complicated probes that require high cost and long time, which were conventionally required when identifying a plurality of target nucleic acids by genetic testing, and is simpler and cheaper. It will be possible to measure multiple target nucleic acids at the same time.

It is a figure which shows the result of Example 1. FIG. The results of comparing the detection temperatures of those with and without mismatch in the probe, targeting gonococci, are shown. It is a figure which shows the result of Example 2. FIG. The results of simultaneous detection of Chlamydia endogenous plasmid and Neisseria gonorrhoeae as target nucleic acids are shown. It is a figure which shows the result of Example 3. FIG. The results of simultaneous detection of the mecA gene and the nuc gene as target nucleic acids are shown. It is a figure which shows the result of Example 4. FIG. The results of simultaneous detection of influenza A gene and influenza B gene as target nucleic acids are shown.

Hereinafter, the present invention will be described in more detail while showing embodiments of the present invention, but the present invention is not limited thereto.

[Simultaneous detection method for multiple target nucleic acids]

One of the embodiments of the present invention is a method of detecting a plurality of target nucleic acids in one reaction solution by melting curve analysis, a first target probe for detecting the first target nucleic acid and a second target nucleic acid. At least the step of adding the probe for the second target to detect is added to the reaction solution, where the probe for the first target and the probe for the second target are used when the detection temperatures in the melting curve analysis are T1 and T2, respectively. It is a method characterized in that T1> T2 is satisfied and each of them is labeled with a labeled substance that can be detected at the same wavelength.

According to the method of the present invention, it may be unnecessary to detect a plurality of reaction solutions, a plurality of labeled wavelengths, and design a complicated probe, which are conventionally considered necessary for simultaneous detection of a plurality of target nucleic acids.

The method of the present invention has the following features:

In melting curve analysis using a probe, multiple target nucleic acids that can be contained in a sample can be detected simultaneously in one reaction solution, and a label for detecting the first target nucleic acid and a label for detecting the second target nucleic acid can be detected. It is characterized in that the labeled substance is a labeled substance detected at the same wavelength.

As used herein, the labeled substance that can be detected at the same wavelength may include all labeled substances that can be measured by the same fluorescence detection channel in the measuring instrument used for melting curve analysis. For example, when the difference between the maximum fluorescence wavelengths is 40 nm or less, preferably 30 nm or less, and more preferably 25 nm or less, the labeled substance can be detected at the same wavelength. Further, the difference in the maximum fluorescence wavelength of each labeled substance may be, for example, within 20 nm, within 15 nm, within 10 nm or within 5 nm, and the labeled substances that can be detected at the same wavelength have substantially the maximum fluorescence wavelength of each other. They may be the same and have no difference. As a specific example, taking Rotor-Gene-Q (QIAGEN) as an example, Rotor-Gene-Q has six types of fluorescence detection channels, each of which has a different detection wavelength. Then, a plurality of fluorescent dyes can be detected for each detection wavelength. Specifically, it is as shown in Table 1.

For example, when Rotor-Gene-Q (Rotor Gene-Q MDx 5plex HRM) or the like is used and 610 ± 5 nm is selected as the detection wavelength, the labels having the same wavelength include ROX ^TM , CAL Fluor Red 610, and Cy (registered). Trademarks) 3.5, Texas Red, Alexa Fluor 568 and the like. Also, for example, FAM and SYBR Green I are detected on the same fluorescence channel. The maximum fluorescence wavelength of FAM is 520 nm, the maximum fluorescence wavelength of SYBR Green I is 521 nm, and the maximum fluorescence wavelength of Eva Green is 525 nm, but these fluorescent dyes are not distinguished when fluorescence is detected. In this case, FAM, SYBR Green I, and Eva Green can be detected at the same wavelength. In the present invention, a group of labeled substances that can be detected in each channel as shown in Table 1 above can be used as labeled substances that can be detected at the same wavelength, but the present invention is not limited to these, and an equivalent maximum fluorescence wavelength can be used. Any labeled substance having can be used, and those skilled in the art can arbitrarily select a labeled substance that can be detected at the same wavelength depending on the detection wavelength selected. In certain preferred embodiments, the same labeled material is used as the labeled material that can be detected at the same wavelength. By using the same labeled substance, it may be possible to carry out the present invention efficiently at a lower cost. By using a labeled substance that can be detected at the same wavelength as in the present invention for a plurality of target probes, a plurality of target nucleic acids can be detected with one reaction solution even if the analyzer is not equipped with many types of fluorescence detection channels. It can be possible. Further, since only one fluorescence detection channel is required to detect a plurality of target probes of the present invention, in the case of an analyzer equipped with many types of fluorescence detection channels, another fluorescence detection channel can be used as another target nucleic acid. There is a merit that it can be used more effectively for the detection of.

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

When the detection temperature of the first target probe in the melting curve analysis is T1 and the detection temperature of the second target probe is T2, the condition of T1> T2 is satisfied. That is, in the method of the present invention, in the melting curve analysis, the detection temperature of the first target probe and the second target probe is set to have a certain difference in the detection temperature, and the detection temperature of the second target probe is set to the second. (1) Design each probe so that it is on the lower temperature side than the detection temperature of the target probe.

The first target probe and the second target probe are not particularly limited as long as they satisfy the above conditions, such as the base sequence and the base length constituting them. For example, when at least one of the first target probe and the second target probe contains a mismatched base, the detection temperature in the melting curve analysis of the target probe containing the mismatched base is higher than the detection temperature of the other target probe. It can be adjusted to the low temperature side. In addition, detection in the melting curve analysis of the first target probe and the second target probe by any method such as making a difference in the lengths of the base sequences of the first target probe and the second target probe. It is possible to provide a difference in temperature. Preferably, at least one of the first target probe and the second target probe is preferable because it does not require complicated probe design and the condition setting in nucleic acid amplification and melting curve analysis is less likely to be complicated. It is preferably designed to contain mismatched bases.

In a specific embodiment, it is preferable that the second target probe contains a mismatched base from the viewpoint of providing a difference in the detection temperature between the two target probes by a simpler method, and the effect of the present invention can be obtained more reliably. From the viewpoint of easy susceptibility, the probe for the first target does not contain a mismatched base, and the probe for the second target contains a mismatched base, so that the probe for the second target is used for the second target rather than the detection temperature T1 of the probe for the first target in the melting curve analysis. It is preferable that the detection temperature T2 of the probe is adjusted to be on the low temperature side.

As used herein, the term "mismatched base" (or simply referred to as "mismatch") means that the base sequence of the target nucleic acid (if the target nucleic acid is double-stranded, the double-stranded dissociated one of the single strands). It means that it contains a base that is not complementary to the base sequence of nucleic acid). For example, when a cytosine base is present in the base sequence of a 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, and a universal base). It means that it is. The mismatched base in the first target probe and / or the second target probe is any of an adenine base, a cytosine base, a guanine base, a thymine base, and a universal base (for example, hypoxanthine, nebulaline, 5-nitroindole, etc.). However, those skilled in the art can design the probe by appropriately selecting a non-complementary base. For example, when detecting a easily mutated target nucleic acid, a universal base can be selected at the position of the target probe corresponding to the easily mutated base.

When the first target probe and / or the second target probe contains a mismatched base, the position where the mismatch is inserted is not particularly limited as long as the effect of the present invention is not impaired. From the viewpoint of making it easier to detect the target nucleic acid more reliably, it is preferable that it is not the terminal base of each probe. For example, when the probe for the second target contains a mismatched base, the position of the mismatched base is preferably within 8 mer from the center of the total length of the base sequence constituting the second target probe to the front and back, and within 5 mer from the center of the total length to the front and back. More preferred.

One of the features of the method of the present invention is to allow a certain degree of difference in the detection temperature in the melting curve analysis between the probe for the first target and the probe for the second target. If there is no difference in the detection temperature in the melting curve analysis between the first target probe and the second target probe in this way, both detection peaks overlap, so that the first target nucleic acid and the second target It has been verified that it is not possible to identify which of the nucleic acids was detected or both were detected. From the viewpoint of being able to discriminate between 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 can be determined. It is preferably 5 ° C. or higher, more preferably 7 ° C. or higher, and even more preferably 10 ° C. or higher. The upper limit of the temperature difference between T1 and T2 is not particularly limited, but the temperature range in the melting curve analysis can be suppressed to a certain range, and the time required for nucleic acid detection in the melting curve analysis can be shortened. From this point of view, it is preferably 30 ° C. or lower, and more preferably 25 ° C. or lower.

The difference in base length between the probe for the first target and the probe for the second target used in the present invention is not particularly limited as long as the effect of the present invention is obtained. In general, the base length of a probe affects the optimum annealing temperature for a target nucleic acid. Therefore, if the base lengths of a plurality of probes are extremely different, the optimum annealing temperature of each probe will be significantly different. Therefore, if multiple probes with significantly different base lengths are used in one reaction solution, it is necessary to set multiple annealing temperatures, which not only complicates the reaction conditions but also increases the overall reaction time. There's a problem. Therefore, in the present invention using a plurality of targeting probes that detect a plurality of target nucleic acids in one reaction solution, the first targeting probe and the second targeting probe do not need to set a plurality of annealing temperatures of each probe. It is preferable that there is no extreme difference in base length between the probe and the probe. From this point of view, the difference between the base length of the first target probe and the base length of the second target probe is preferably 8 mer or less, and the base length of the first target probe and the base of the second target probe. The difference in length is more preferably 5 mer or less, and further preferably 3 mer or less. The lower limit of the difference in base length between the first target probe and the second target probe may be 0 mer with no difference in base length, 1 mer or more, or 2 mer or more. May be good.

[Method for detecting multiple target nucleic acids]

One of the embodiments of the present invention is a method for detecting a plurality of target nucleic acids that can be contained in a sample, and is characterized by carrying out at least the following steps (1) to (3):

(1) A sample that can contain a plurality of target nucleic acids (for example, a sample that can contain a chlamydia endogenous plasmid and / or a gonococcus, a sample that can contain influenza A and / or influenza B, etc.) is mixed with a nucleic acid amplification reagent. Steps to prepare the reaction solution,

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

(3) The amplification product obtained in step (2) is hybridized with a probe for a first target and a probe for a second target labeled with a labeled substance that can be detected at the same wavelength, and the reaction solution is fluorescent by melting curve analysis. The process of measuring strength.

One of the features of the present invention is to include the steps of adding the first target probe and the second target probe to the reaction solution as described above. The timing of adding the first target probe and the second target probe may be at least added in the reaction solution of the step (3), for example, before the nucleic acid amplification reaction of the step (1) or (2). It may be added to the reaction solution of the above step (2) from the beginning, or it may be added to the reaction solution during the nucleic acid amplification reaction of the step (2), or the step after the nucleic acid amplification reaction of the step (2) is completed. It may be added to the reaction solution in (3). Further, it is not necessary to add the probe for the first target and the probe for the second target at the same time, and they may be added to the reaction solution separately. From the viewpoint that the amplification reaction and the detection reaction can be continuously performed and a simpler operation is possible, it is preferable to use the first target probe and the second target probe in the reaction solution in the step (1). It is preferable to add it. When the nucleic acid probe is added before the nucleic acid amplification reaction as described above, it is preferable to add a fluorescent dye or a phosphate group to the 3'end thereof, for example.

In certain embodiments, it may be mixed with a nucleic acid amplification product in a solvent. Examples of such a solvent are not particularly limited, and examples thereof include conventionally known solvents such as a buffer solution such as Tris-HCl, a solvent containing KCl, MgCl ₂ , ו ₄ , glycerol and the like, and a PCR reaction solution.

In certain preferred embodiments, the method of the invention for detecting multiple target nucleic acids is one or more primary targets capable of amplifying a region containing a sequence of primary target nucleic acids to which the primary target probe can bind. Primer (or primer set) and one or more secondary target primers (or primer set) capable of amplifying the region containing the sequence of the second target nucleic acid to which the second target probe can bind. It may further include the step of amplifying one target nucleic acid and a second target nucleic acid. Here, the first target primer and the second target primer are two single strands in which the double strands are dissociated when both the first target nucleic acid and the second target nucleic acid are double strands. It specifically binds to either and amplifies 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. Preferably, a set of two or more first target primers that 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 and 2 Use a set of one or more second target primers.

As a specific embodiment of the method for detecting a plurality of nucleic acids according to 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) A step of preparing one or more primary target primers that specifically bind to any of the two single strands in which the first target nucleic acid is dissociated.

(Ii) A step of preparing one or more secondary target primers that specifically bind to any of the two single strands in which the second target nucleic acid is dissociated.

(Iii) A step of preparing a probe for a first target and a probe for a second target capable of emitting light at the same wavelength as a label for detection in melting curve analysis.

(Iv) A step of preparing a reaction solution containing a sample containing a plurality of target nucleic acids, the first target primer, the second target primer, the first target probe, and the second target probe.

(V) A step of amplifying the first target nucleic acid and the second target nucleic acid in the sample using the reaction solution.

(Vi) The nucleic acid amplification product obtained by step (v) is hybridized 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. The process of soaking and forming a complex, as well as

(Vii) A step of detecting the complex obtained in the step (vi).

In the above method, the order of the steps (i) to (iii) does not matter.

In one embodiment, the method for detecting a plurality of target nucleic acids of the present invention is derived from each of the plurality of infectious microorganisms in a test for a plurality of infectious microorganisms having very similar symptoms or conditions and not easy to distinguish. It may be useful for detecting the first target nucleic acid and the second target nucleic acid to be obtained. Examples of multiple target nucleic acids that can be derived from such multiple pathogenic microorganisms include, for example, the target nucleic acid in the Chlamydia endogenous plasmid and the target nucleic acid in gonococcus; the target nucleic acid in influenza A and the target nucleic acid in influenza B; Target nucleic acid in corona virus and target nucleic acid in influenza virus; target nucleic acid in tuberculosis bacterium, target nucleic acid in non-tuberculous acid bacillus, and the like, but are not limited thereto. The present invention makes it possible to discriminate infectious diseases having very similar symptoms or conditions as described above by a simple method, so that the risk of overlooking one of the infections in cases where co-infection occurs is reduced, and the risk of overlooking one of the infections is reduced. It can also lead to the treatment of the disease and the prevention of the spread of infection to the surrounding area.

In another embodiment, the method for detecting a plurality of target nucleic acids of the present invention is also useful in detecting a plurality of target nucleic acids that may be derived from one microorganism or the like. Examples of such a plurality of target nucleic acids include, for example, a target nucleic acid in the nuc gene (nuclease gene) derived from staphylococcus aureus and a target nucleic acid in the mecA gene (methicillin resistance gene); tcdA derived from Clostridioides dificil. Target nucleic acid in the gene and target nucleic acid in the tcdB gene; target nucleic acid in the vanA gene derived from vancomycin-resistant bacillus, target nucleic acid in the vanB gene, and the like, but are not limited thereto.

[sample]

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

Examples of biological samples include, but are not limited to, animal and plant tissues, body fluids, excrement, cells, bacteria, viruses and the like. Further, blood, blood culture medium, urine, pus, spinal fluid, pleural fluid, pharyngeal swab, nasal swab, sputum, tissue section, skin, vomitus, feces, isolated culture colony, catheter lavage fluid, cervical scraping , Urethral scrapes, male urethral scrapes, urine and the like.

Examples of foods include water, alcoholic beverages, soft drinks, processed foods, vegetables, livestock products, marine products, eggs, dairy products, raw meat, raw fish, prepared foods and the like. When a food is used as a measurement sample, not only a part or all of the food can be used, but also a food whose surface has been wiped off can be used. Further, a material for wiping off a cooking utensil or a doorknob or a cleaning liquid for cleaning them can also be used as a sample.

Examples of environmental samples include water, ice, soil, air and aerosols. Examples of water here include tap water, seawater, and water collected from rivers, waterfalls, lakes, ponds, and the like. Further, a cleaning solution obtained by wiping the wall surface, floor surface, equipment / equipment, toilet bowl, etc. of the facility or cleaning them can also be used as a sample.

Any sample as described above can be used in the present invention, but the present invention is particularly directed to a sample collected from a subject or subject suspected of being infected or co-infected with any of a plurality of infectious microorganisms. Is valid. As the type of sample to be collected, it is preferable to select an appropriate sample each time based on the symptoms of the infectious disease of the subject, the condition of the subject, and the like. For example, when detecting Chlamydia endogenous plasmids and / or gonococci as target nucleic acids, biological samples (eg, animal and plant tissues, body fluids, excreta, pharyngeal swabs, tissue sections, cervical scrapes, urethral scrapes, male urethral scrapes). (Urine, vomitus, urine, isolated cultured colony) is preferred, excrement, urine, cervical scraped material, urethral scraped material, male urethral scraped urine, fractionated cultured colony is more preferable, and urine. , Cervical scrapes, urethral scrapes, male urethral scrapes urine, pharyngeal swabs are more preferred. Further, for example, when detecting influenza A and / or influenza B as a target nucleic acid, it is preferable to use biological samples such as nasopharyngeal swab, nasal swab, sputum, and saliva. Further, for example, when the nuc gene and / or the mecA gene is detected as a target nucleic acid, it is preferable to use a biological sample such as a blood culture medium. According to the present invention, a plurality of target nucleic acids can be simultaneously detected even when such a sample is used, and a plurality of target nucleic acids can be simultaneously detected at low cost with a single reaction vessel with simple condition setting. It is possible.

The method for collecting a sample, the method for preparing a sample, and the like are not particularly limited, and a known method can 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 nucleic acid amplification. Examples of such nucleic acid amplification methods include PCR method, LAMP method, LCR method, TMA method, SDA method, RT-PCR method, RT-LAMP method, NASBA method, TRC method, TMA method and the like. These techniques have already been established in the relevant technical field, and the method can be selected according to the purpose. The nucleic acid amplification method performed in the present invention is preferably, but is not limited to, the PCR method (including the RT-PCR method) from the viewpoint that a higher effect can be more reliably obtained.

[PCR method]

The PCR reaction is a reaction mainly catalyzed by DNA polymerase, [1] DNA denaturation by heat treatment (dissociation from double-stranded DNA to single-stranded DNA), and [2] primer to template single-stranded DNA. The three steps of annealing and [3] extension of the primer using DNA polymerase are set as one cycle, and the target nucleic acid is amplified by repeating this cycle. Examples of the DNA polymerase include Taq, Tth, Bst, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, DEEPVENT and their variants. From the viewpoint of enabling simpler and more specific nucleic acid amplification, it is preferable to use a DNA polymerase belonging to Family B in the present invention.

In the present specification, the variant of DNA polymerase is, for example, 85% or more, preferably 90% or more, more preferably 95% or more, still more preferably, with respect to the amino acid sequence of the wild-type DNA polymerase from which it is derived. Refers to those having 98% or more, particularly preferably 99% or more of sequence identity, and having an activity of amplifying DNA in the same manner as wild-type DNA polymerase. Here, as a method for calculating the identity of the amino acid sequence, any means known in the art can be used. For example, it can be calculated using analysis tools available on the market or through telecommunications lines (Internet), for example, the National Center for Biotechnology Information (NCBI) homology algorithm BLAST (Basic local alignment search tool) http. : // www. ncbi. nlm. nih. By using the default (initial setting) parameters in gov / BLAST /, it is possible to calculate the identity of the amino acid sequence. In addition, the mutant that can be used in the present invention has one or several amino acids substituted, deleted, inserted and / or added in the amino acid sequence of the wild-type DNA polymerase from which it is derived. It may be a polypeptide consisting of an amino acid sequence (also referred to as “mutation”) and may have an activity of amplifying DNA in the same manner as wild-type DNA polymerase. Here, 1 or several may be, for example, 1 to 80, preferably 1 to 40, more preferably 1 to 10, and even more preferably 1 to 5, but the number is not particularly limited.

[DNA polymerase belonging to Family B]

The DNA polymerase used in the present invention is preferably, but is not limited to, a DNA polymerase belonging to Family B. The DNA polymerase belonging to the family B is not particularly limited, but is preferably a DNA polymerase derived from archaea.

[DNA polymerase derived from archaea]

Examples of archaeal-derived DNA polymerases belonging to Family B include DNA polymerases isolated from bacteria of the genus Pyrococcus and Thermococcus. The present invention also includes variants thereof that have not lost their DNA polymerase activity from archaea belonging to Family B. Variants of DNA polymerase include, but are not limited to, mutants for the purpose of enhancing polymerase activity, deficiency of exonuclease activity, adjusting substrate specificity, and the like.

Examples of the DNA polymerase derived from the genus Pyrococcus include Pyrococcus furiosus and Pyrococcus sp. DNA polymerases isolated from GB-D, Pyrococcus womeni, Pyrococcus abyssi, Pyrococcus horikoshii, and variants thereof that have not lost their DNA polymerase activity, but are not limited thereto.

Examples of the DNA polymerase derived from the genus Thermococcus include Thermococcus kodakaraensis, Thermococcus gogonarius, Thermococcus litoralis, and Thermococcus sp. JDF-3, Thermococcus sp. 9 degrees North-7 (Thermococcus sp. 9 ° N-7), DNA polymerase isolated from Thermococcus siculi, and variants thereof that have not lost their DNA polymerase activity, but are not limited thereto.

PCR enzymes using these DNA polymerases are commercially available, Pfu (Staragene), KOD (Toyobo), Pfx (Life Technologies), Vent (New England Biolabs), DeepBent (New) , Tgo (Roche), Pwo (Roche) and the like, all of which can be used in the present invention.

Among them, KOD DNA polymerase having excellent extensibility and thermal stability and a variant thereof (for example, KOD DNA polymerase lacking 3'→ 5'exonuclease activity) are preferable.

KOD DNA polymerase is superior in accuracy, amplification efficiency, extensibility, and crude sample resistance to Taq DNA polymerase, which is a DNA polymerase belonging to Family A. In the present invention, by using such a KOD DNA polymerase, as shown in Examples described later, detection using a mismatched detection probe is also possible, and simultaneous detection of a plurality of target nucleic acids is possible. Become.

The conditions of the nucleic acid amplification step (for example, temperature, pH, cation concentration, presence of organic solvent in the solution, etc.) may be optimized in combination with the nucleic acid primer or the hybridization condition of the nucleic acid probe. If there is, it can be set as appropriate.

[Nucleic acid amplification reagent]

When the nucleic acid amplification reaction is carried out in the present invention, the nucleic acid amplification reagent used for the nucleic acid amplification reaction can be appropriately selected according to the nucleic acid amplification reaction to be carried out. For example, the nucleic acid amplification reagent contains the above-mentioned characteristics in addition to the first target primer and the second target primer, the first target probe and the second target probe, and the components necessary for the nucleic acid amplification reaction. The components required for the nucleic acid amplification reaction differ depending on the nucleic acid amplification reaction to be carried out, and known components can be used for each. For example, when detecting a plurality of target nucleic acids contained in a sample by using the PCR method, it is preferable to contain at least an inorganic salt such as DNA polymerase, deoxyribonucleoside triphosphates (dNTPs), and magnesium salt. The concentration of each component can be adjusted as appropriate, but for example, the oligonucleotide probe (first target probe or second target probe) is preferably 0.1 to 1 μM, more preferably 0.2 to 0.5 μM. The DNA polymerase is preferably 0.01 to 1 U / uL, more preferably 0.1 to 0.5 U / uL. The oligonucleotide primer (primer for first target or primer for second target) is preferably 0.1 to 10 μM. Deoxyribonucleoside triphosphates (dNTPs) are preferably 0.02 to 1 mM, more preferably 0.1 to 0.5 mM. The inorganic salt such as magnesium salt is preferably 0.1 to 6 mM, more preferably 1 to 5 mM.

The nucleic acid amplification reagent may further contain additives and the like known in the art for the purpose of suppressing non-specific amplification and promoting the reaction. Examples of additives for the purpose of suppressing non-specific amplification include anti-DNA polymerase antibodies and phosphoric acid. Additives aimed at promoting the reaction include bovine serum albumin (BSA), protease inhibitors, single strand binding protein (SSB), T4 gene 32 protein, tRNA, sulfur or acetic acid-containing compounds, dimethylsulfoxide (DMSO), glycerol, Ethylene glycol, propylene glycol, trimethylene glycol, formamide, acetamide, betaine, ectin, trehalose, dextran, polyvinylpyrrolidone (PVP), gelatin, tetramethylammonium chloride (TMC), tetramethylammonium hydroxide (TMAH), tetramethylacetate Examples thereof include ammonium (TMA), polyethylene glycol, Triton, Tween 20 and Nonidet P40. In the present invention, one or more of these additives may be used in combination, but the present invention is not limited thereto.

Further, when the nucleic acid amplification step is carried out by the PCR method, the thermal cycle conditions are not particularly limited and may be appropriately set by those skilled in the art. As an example, in the nucleic acid amplification reaction, the first thermal deformation step is 80 to 100 ° C. for 10 seconds to 15 minutes, the repeated thermal deformation step is 80 to 100 ° C. for 0.5 to 300 seconds, and Annie Link is 40 to 80 ° C. The extension reaction step is preferably carried out at 60 to 85 ° C. for about 1 to 300 seconds, and this repetition is preferably repeated 30 to 70 times.

[Melting curve analysis]

Melting curve analysis is widely used in the analysis of target nucleic acids. The temperature at which half of the double-stranded nucleic acid becomes single-stranded is called the melting temperature. Since the melting temperature basically depends on the content of the base contained in the nucleic acid, the predetermined base sequence has a unique melting temperature depending on the content of the base contained therein. Melting curve analysis is an analysis method that utilizes the property that nucleic acids dissociate from double strands to single strands in response to temperature changes.

[Detected temperature in melting curve analysis]

In melting curve analysis, the labeled probe monitors the intensity of the fluorescence or quenching signal associated with dissociation from the target sequence in real time with respect to temperature changes. Then, when the first derivative value obtained by differentiating the change in the emission or extinguishing signal intensity with the rise in temperature by the value of the temperature change is plotted, the melting temperature is displayed as a peak in the obtained melting curve (temperature-first derivative curve). You will be able to do it. In the present invention, the melting temperature displayed as a peak when the melting curve analysis is performed using a predetermined target probe is referred to as the detection temperature of the target probe.

Conventionally, as a specific application method utilizing the characteristics of melting curve analysis, detection of mutation in a target nucleic acid (for example, SNP analysis) has been performed. When a mutation occurs in the target nucleic acid, PCR is performed to form a double-stranded gene with a mismatched portion. As described above, the double-stranded gene having a mismatched portion easily undergoes denaturation (dissociation) of the double-stranded structure, and the detection temperature is lower than that of the wild-type gene in the melting curve analysis. Therefore, when a mutation occurs in the target nucleic acid, a change in the detection temperature can be detected.

The use of conventional melting curve analysis as described above has been useful for detecting the presence of mismatched bases on the target nucleic acid side. The present invention is different from the conventional one in that the detection temperature of the melting curve analysis is adjusted by adding the mismatched base to the target probe side, not due to the presence of the mismatched base in the target nucleic acid.

In the present invention, the nucleic acid amplification product can be detected by any analysis method as long as it is a melting curve analysis using a probe, for example, Qprobe (registered trademark) (also referred to as Q-probe), Eprobe (registered trademark). (Also referred to as an E probe), a TaqMan probe, a molecular beacon probe, a FRET hybridization probe, a scorpion probe, or a method in which these are arbitrarily combined can be used for detection. From the viewpoint of enabling simultaneous detection of multiple target nucleic acids with higher sensitivity, it is preferable to detect by melting curve analysis using Q probe or E probe, and in particular, detection by melting curve analysis using Q probe. Is more preferable.

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

For example, the fluorescent extinguishing dye used in the Q probe is not particularly limited, but is fluorescein or a derivative thereof (for example, fluorescein isothiocyanate), rhodamine or a derivative thereof (for example, tetramethylrhodamine, tetramethylrhodamine isothiocyanate, carboxylodamine, x). -Rhodamine, Rhodamine 101 Acid Chloride), BODIPY or derivatives thereof (eg, BODIPY-FL, 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. Details of the fluorescent quenching dye are described in Japanese Patent No. 581263 and the like, and the present invention can also refer to the technique.

When a Q probe is used as a labeled substance in the present invention, a probe having a base sequence in which at least one terminal base is cytosine and cytosine at the terminal base is labeled with the fluorescent quenching dye is preferable. When such a probe hybridizes to an amplification product, it can be quenched by forming a base pair with the guanine base in the amplification product and interacting with it, so it is very easy to measure changes in the fluorescence intensity of the reaction solution. can do.

When the probe hybridizes, even if the cytosine base of the probe and the guanine base in the amplification product do not form a base pair, the fluorescence can be quenched if the distance between the bases is short. For example, details are described in Japanese Patent No. 5354216, and the present invention can also refer to the technique. That is, when the probe hybridizes, it can be extinguished if the guanine base in the amplification product is present in the range of, for example, 1 to 3 bases with respect to the cytosine base of the probe (forms a base pair with the cytosine base). Base is 1).

In the method of 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 a part of the amplification product of the first target nucleic acid and / or the second target nucleic acid. The complex is not particularly limited as long as it can be formed. Preferably, by using a probe containing a mismatch in the base sequence of the second target nucleic acid, the detection temperature of the second target nucleic acid is adjusted to the low temperature side, and as a result, simultaneous detection of a plurality of target nucleic acids becomes possible.

[Kit for detecting multiple target nucleic acids at the same time]

Furthermore, as another embodiment of the present invention, there is provided a nucleic acid detection kit used for detecting a plurality of target nucleic acids by melting curve analysis in one reaction solution. The kit of the present invention includes 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, and the first target probe and the second target probe are included. It is designed to satisfy T1> T2 when the respective detection temperatures in the melting curve analysis are T1 and T2, and is characterized by being labeled with a labeled substance that can be detected at the same wavelength. The kit of the present invention further comprises one or more primary target primers (or primer sets) capable of amplifying the region containing the sequence of the primary target nucleic acid to which the primary target probe can bind, as described above. It is preferred to include one or more second target primers (or primer sets) capable of amplifying the region containing the sequence of the second target nucleic acid to which the second target probe can bind. Such a kit of the present invention is not particularly limited as long as it is configured so that a plurality of target nucleic acids can be simultaneously detected by the detection temperature in the melting curve analysis. The kit further comprises Taq, Tth, Bst, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, DEEPVENT and variants thereof for efficient and highly specific nucleic acid amplification. It is preferable to further contain a component suitable for a nucleic acid amplification reaction such as at least one DNA polymerase selected from the group.

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

Example 1: Change in detected temperature when mismatch is added to the probe and when it is not added

(1-1) Method

A melting curve analysis was performed using the following targeting primers and probes (probe without mismatch and probe with mismatch) targeting gonococci. In the table below, the shaded bases are mismatched bases.

(1-2) Reaction solution

A reaction solution containing the components shown below was prepared using the primers and probes shown in Table 2 using GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.).

0.3μM SNG F

2.0 μM SNG R

0.3μM SNG QP (probe without mismatch or probe with mismatch)

(1-3) Reaction

Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycle to perform nucleic acid amplification. After the nucleic acid amplification reaction, melting curve analysis was performed under the following conditions.

94 ° C for 30 seconds,

97 ° C 1 second-58 ° C 5 seconds -63 ° C 2 seconds (60 cycles)

94 ° C 30 seconds 39 ° C 30 seconds 40-75 ° C 0.09 ° C / sec

(1-4) Result

FIG. 1 shows the results of melting curve analysis. The base lengths of the probe without mismatch and the probe with mismatch used in this test example are the same. Normally, in the case of probes with almost the same base length, the detection temperatures in the melting curve analysis tend to be close to each other, but even if only one mismatched base is added, the detection temperature can be adjusted to the low temperature side. I understand.

Example 2: Simultaneous detection of Chlamydia endogenous plasmid and Neisseria gonorrhoeae

(2-1) Method

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

(2-2) Reaction solution

A reaction solution was prepared by adding the primers and probes shown in Table 3 at the concentrations shown below using GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.).

0.5 μM SCT F

2.5 μM SCT R

0.3 μM SCT QP

0.3μM SNG F

2.0 μM SNG R

0.3μM SNG QP

(2-3) Reaction

Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycle to perform nucleic acid amplification. After the nucleic acid amplification reaction, melting curve analysis was performed under the following conditions.

94 ° C for 30 seconds,

97 ° C 1 second-58 ° C 5 seconds -63 ° C 2 seconds (60 cycles)

94 ° C 30 seconds 39 ° C 30 seconds 40-75 ° C 0.09 ° C / sec

(2-4) Result

FIG. 2 shows the results of melting curve analysis. As shown in FIG. 2, the detection peak was observed at around 62 ° C in the first target probe (SCT QP, chlamydia detection probe) containing no mismatched base, whereas the detection peak was observed at around 62 ° C., whereas the second target containing a mismatched base was used. With the probe (SNG QP, gonococcal detection probe), a detection peak was observed at around 46 ° C. The difference in base length between the probe for the first target and the probe for the second target is about 2 mer, but not only the difference in the detection temperature in the melting curve analysis is widened to about 16 ° C, but also it can be detected at the same wavelength. Even in the labeled product, both genes could be distinguished as clear peaks in the melting curve analysis. From this result, the detection temperature was adjusted to the low temperature side by adding a mismatch to the probe for gonococcal target, and the target nucleic acid in Chlamydia endogenous plasmid and the target nucleic acid in gonococcus were simultaneously detected by melting curve analysis according to the detection temperature, and the same wavelength. It turned out that it is possible to identify by the label.

Example 3: Simultaneous detection of mecA gene and nuc gene

(3-1) Method

In this example, the first target nucleic acid was the nuc gene and the second target nucleic acid was the mecA gene. The primers for the first target and the primer for the second target, the probe for the first target and the probe for the second target used in the PCR method in this example are as follows. Here, both the first target probe and the second target probe were labeled with BODIPY-FL at the 3'end. In the case of this embodiment, the mecA QP contains a mismatch. Shaded bases are mismatched bases.

(3-2) Reaction solution

A reaction solution was prepared by adding the primers and probes shown in Table 4 at the concentrations shown below using GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.).

4.0 μM nuc F

0.9 μM nuc R

1.2 μM nuc QP

2.0 μM mecA F

0.4 μM mecA R

0.2 μM mecA QP

(3-3) Reaction

Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycle to perform nucleic acid amplification. After the nucleic acid amplification reaction, melting curve analysis was performed under the following conditions.

94 ° C for 30 seconds,

97 ° C 1 second-58 ° C 3 seconds -63 ° C 5 seconds (60 cycles)

94 ° C 30 seconds 39 ° C 30 seconds 40-75 ° C 0.09 ° C / sec

(3-4) Result

FIG. 3 shows the results of melting curve analysis. As shown in FIG. 3, the detection peak was observed at around 58 ° C. in the first target probe (nuc QP, nuc gene detection probe) containing no mismatched base, whereas the second target containing the mismatched base was observed. A detection peak was observed at around 45 ° C. for the probe (mecA QP, mecA gene detection probe). Although the base lengths of the first target probe and the second target probe are the same, the difference in the detection temperature in the melting curve analysis is widened to about 13 ° C, and even labeled substances that can be detected at the same wavelength can be detected in the melting curve analysis. Both target genes could be clearly distinguished. From this result, the detection temperature is adjusted to the low temperature side by adding a mismatch to the probe for targeting the mecA gene, and the nuc gene and the mecA gene are simultaneously detected by melting curve analysis according to the detection temperature and identified by the labeled substance having the same wavelength. Turned out to be possible.

Example 4: Simultaneous detection of influenza A gene and influenza B gene

(4-1) Method

In this example, the first target nucleic acid was an influenza A gene and the second target nucleic acid was an influenza B gene. The primers for the first target and the primer for the second target, the probe for the first target and the probe for the second target used in the PCR method in this example are as follows. Here, the probe for the first target was labeled with BODIPY-FL at the 3'end, and the probe for the second target was labeled with BODIPY-FL at the end of 5'and phosphorylated at the end of 3'. In the case of this embodiment, the FluB QP contains a mismatch. Shaded bases are mismatched bases.

(4-2) Reaction solution

A reaction solution was prepared by adding the primers and probes shown in Table 5 at the concentrations shown below using GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) and RiverTra Ace (registered trademark).

0.5 μM FluA F

3.0 μM FluA R

0.3μM FluA QP

0.5 μM FluB F

3.0 μM FluB R

0.3μM FluB QP

(4-3) Reaction

Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycle to perform reverse transcription and nucleic acid amplification. After the nucleic acid amplification reaction, melting curve analysis was performed under the following conditions.

42 ° C for 120 seconds,

97 ° C for 15 seconds,

97 ° C 1 second-58 ° C 3 seconds -63 ° C 5 seconds (50 cycles)

94 ° C 30 seconds 39 ° C 30 seconds 40-75 ° C 0.09 ° C / sec

(4-4) Result

FIG. 4 shows the results of melting curve analysis. As shown in FIG. 4, the detection peak was observed at around 60 ° C. in the first target probe (FluA QP, influenza A gene detection probe) containing no mismatched base, whereas the number containing the mismatched base was observed. In the two target probes (FluB QP, influenza B gene detection probe), a detection peak was observed at around 46 ° C. The difference in base length between the probe for the first target and the probe for the second target is about 2 mer, but the difference in the detection temperature in the melting curve analysis is widened to about 14 ° C, and even labeled substances that can be detected at the same wavelength are melted. Both target genes could be clearly distinguished in the curve analysis. From this result, the detection temperature is adjusted to the low temperature side by adding a mismatch to the probe for influenza B gene target, and it is possible to simultaneously detect and distinguish the influenza A gene and the influenza B gene according to the detection temperature. I understand.

Claims

A method of detecting a plurality of target nucleic acids in one reaction solution by melting curve analysis, in which a first target probe for detecting the first target nucleic acid and a second target probe for detecting the second target nucleic acid are reacted. Including the step of adding to the liquid, 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 have the same wavelength. A method, characterized in that each is labeled with a label that can be detected in.
One or more primary target primers capable of amplifying the region containing the sequence of the first target nucleic acid to which the first target probe can bind, and the sequence of the second target nucleic acid to which the second target probe can bind. The method of claim 1, further comprising the step of amplifying the first target nucleic acid and the second target nucleic acid with one or more second target primers capable of amplifying the containing region.
The method according to claim 1 or 2, wherein the labeled substance of the first target probe and the labeled substance of the second target probe are the same labeled substance.
The method according to any one of claims 1 to 3, wherein the probe for the second target contains a mismatched base.
The method according to any one of claims 1 to 4, wherein the mismatched base is at least one 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 claims 1 to 5, wherein the universal base is at least one selected from the group consisting of hypoxanthine, nebulaline, and 5-nitroindole.
Since the first target probe does not contain a mismatched base and the second target probe contains a mismatched base, the detection temperature T2 of the second target probe is higher than the detection temperature T1 of the first target probe in the melting curve analysis. The method according to any one of claims 1 to 6, which is adjusted to be on the low temperature side.
The method according to any one of claims 1 to 7, wherein in the melting curve analysis, the 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 ° C.
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 8 mer or less.
The method according to any one of claims 1 to 10, wherein the labeled substance of the first target probe and the labeled substance of the second target probe are Qprobe (registered trademark) or Eprobe (registered trademark).
The method according to any one of claims 1 to 10, wherein either the first target nucleic acid or the second target nucleic acid is the target nucleic acid in the Chlamydia endogenous plasmid and the other is the target nucleic acid in Neisseria gonorrhoeae.
The method according to any one of claims 1 to 10, wherein either the first target nucleic acid or the second target nucleic acid is the target nucleic acid in the nuc gene and the other is the target nucleic acid in the mecA gene.
The method according to any one of claims 1 to 10, wherein either the first target nucleic acid or the second target nucleic acid is the target nucleic acid in influenza A and the other is the target nucleic acid in influenza B.
A nucleic acid detection kit used for detecting a plurality of target nucleic acids in one reaction solution by melting curve analysis, the first target probe for detecting the first target nucleic acid and the second target nucleic acid for detecting the second target nucleic acid. (2) The target probe includes at least the target probe, and the first target probe and the second target probe are designed to satisfy T1> T2 when the detection temperatures in the melting curve analysis are T1 and T2, respectively. A kit characterized by being labeled with a labeled substance that can be detected at the same wavelength.
One or more primary target primers capable of amplifying the region containing the sequence of the first target nucleic acid to which the first target probe can bind, and the sequence of the second target nucleic acid to which the second target probe can bind. 15. The kit of claim 14, further comprising one or more second target primers capable of amplifying the containing region.