US20180155764A1 - Kit for together detecting multiple target nucleic acids differing from each other and detection method using the same - Google Patents

Kit for together detecting multiple target nucleic acids differing from each other and detection method using the same Download PDF

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US20180155764A1
US20180155764A1 US15/576,923 US201615576923A US2018155764A1 US 20180155764 A1 US20180155764 A1 US 20180155764A1 US 201615576923 A US201615576923 A US 201615576923A US 2018155764 A1 US2018155764 A1 US 2018155764A1
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target nucleic
nucleic acid
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Takashi Nagano
Kensuke MIYAJIMA
Kenji Narahara
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Mizuho Medy Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2527/00—Reactions demanding special reaction conditions
    • C12Q2527/101—Temperature
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2527/00—Reactions demanding special reaction conditions
    • C12Q2527/107—Temperature of melting, i.e. Tm
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/107—Nucleic acid detection characterized by the use of physical, structural and functional properties fluorescence

Definitions

  • the present invention relates to a detection kit for improving the PCR method and further for measuring multiple target nucleic acids, and art related to the same.
  • the multiple target nucleic acids may be: a first pair of influenza A viruses and influenza B viruses; a second pair of influenza and RSV/human metapneumovirus; a third pair of Chlamydia trachomatis and Nisseria gonorrhoeae , which may cause sexually transmitted diseases; a fourth pair of Mycoplasma pneumoniae and resistance factor thereof; and so on.
  • the conditions of patients are similar to each other and also infection of the same expands simultaneously. So, it is conceivable to distinguish the multiple target nucleic acids from each other to make a diagnosis, thereby deciding on a course of treatment.
  • Considering the resistance factor is effective for selecting/judging medication against thereto, or the like.
  • a first process of performing independent measurement for each of the multiple target nucleic acids, respectively, and a second process of distinguishing the multiple target nucleic acids from each other by means of different pilot dyes or the like, respectively can be conceived.
  • the melting curve analysis method after having simultaneously processed amplification reaction.
  • temperature is gradually increased/decreased to make distinction and judgment with respect to the multiple target nucleic acids based on both of first temperature wherein a change rate of fluorescent signals shows a peak and second temperature wherein the target nucleic acid is melt (the melting curve analysis method).
  • the first process of performing the independent measurement requires long time and high costs.
  • the second process of distinguishing different labeling substances from each other costs too much because the second process needs not only preparing plural kinds of labeled reagents but also complicated wavelength-setting in an analyzer.
  • a case by means of the melting curve analysis method costs less than the above. However, since the temperature must be gradually changed, it is necessary to take about 5 to 10 minutes for changing the temperature in order to conduct precise analysis.
  • Reference 1 Japanese application Laid-open No. 2002-136300 discloses: preparing a plurality of reaction vessels containing different reaction solution from each other; and performing amplification and detection with the plurality of reaction vessels, respectively.
  • Reference 2 Japanese application Laid-open No. 2004-203 discloses simultaneously amplifying multiple genes by means of one reaction vessel containing one kind of reaction solution.
  • Distinction is carried out by using different pilot dyes for each of the multiple genes. So, a plurality of optical systems installed in an analyzer are needed as many as the used pilot dyes. In other words, the analyzer costs too much. This is a serious problem.
  • Reference 3 Japanese application Laid-open No. 2008-173127 discloses simultaneously amplifying multiple genes by means of one reaction vessel containing one kind of reaction solution.
  • Dissociation temperature of PCR products and labeled probes should be changed for every gene. After amplification, while temperature is gradually increased from a lower temperature side to a higher temperature side, dissociation curve analysis, which is a synonym of “melting curve analysis”, of monitoring fluorescence values is carried out. Distinction is carried out by monitoring existence or nonexistence of a peak depending on base sequence at the respective dissociation temperature.
  • Reference 4 Japanese unexamined patent application publication ⁇ Translation of PCT application> No. 2012-513215 discloses simultaneously amplifying multiple genes by means of one reaction vessel.
  • Dissociation temperature of PCR products and melting temperature of primers are changed for every gene. Distinction is carried out by monitoring fluorescence at the respective melting temperature.
  • one cycle includes: a denaturation step; and an annealing and elongation step.
  • the melting temperature of PCR products is changed for every gene. Accordingly, too many conditions should be taken into consideration, design of the profile is also difficult, and time for measurement must be too long.
  • an object of the present invention is to provide a kit for detecting multiple target nucleic acids capable of simultaneously amplifying and detecting multiple genes by means of one reaction vessel containing one kind of reaction solution and one label.
  • a first aspect of the present invention provides a kit for together detecting multiple target nucleic acids differing from each other, comprising: solution, the multiple target nucleic acids including a first target nucleic acid and a second target nucleic acid; defining: T 0 as denaturation temperature; T 1 as annealing temperature; T 2 as elongation temperature; and T 3 as second target detection temperature, T 0 through T 3 being set up such that a condition that T 0 is higher than T 2 ; T 2 is not lower than T 1 ; and T 1 is higher than T 3 is satisfied, the solution being capable of containing the first target nucleic acid and the second target nucleic acid therein, at the denaturation temperature T 0 , first double-stranded hydrogen bond of the first target nucleic acid being cut off to be dissociate into first two single strands, second double-stranded hydrogen bond of the second target nucleic acid being cut off to be dissociate into second two single strands, respectively, the solution further containing therein:
  • each of the first labeling substance and the second labeling substance is selected from a group consisting of: a QProbe (registered trademark) probe; an Eprobe (registered trademark) probe; and a TaqMan (registered trademark) probe.
  • the fluorescent signals may be shown by quenching light when the annealing occurs. Alternatively, the fluorescent signals may be shown by emitting light when the annealing occurs.
  • the first target nucleic acid is at least one of a Mycoplasma pneumoniae P gene and a Chlamydia trachomatis endogeneous plasmid gene
  • the second target nucleic acid is at least one of internal control composition and a Nisseria gonorrhoeae CMT gene, respectively.
  • the multiple target nucleic acids can be detected by means of one kind of mixed-solution and one kind of labeling substances.
  • Distinction of the multiple target nucleic acids can be carried out without melting curve analysis. Accordingly, measuring time can be remarkably shortened, thereby providing excellent practical performance.
  • the present inventors have developed a kit for detecting nucleic acids by means of one kind of fluorescent labels and one reaction vessel containing one kind of reaction solution. Condition setting with respect to annealing temperature of probes and temperature change profiles in the PCR method has been incorporated within the present kit.
  • the present kit uses four kinds of temperature including: denaturation temperature T 0 (95 Centigrade); annealing temperature T 1 (70 Centigrade); elongation temperature T 2 (72 Centigrade); and second target detection temperature T 3 (55 Centigrade).
  • the multiple target nucleic acids are a first target nucleic acid and a second target nucleic acid.
  • a third target nucleic acid or more can be added thereto by adding further setting such as third target detection temperature T 4 (T 3 >T 4 ), or the like.
  • a first target nucleic acid 10 and a second target nucleic acid 20 are targets to be detected.
  • the solution 2 does not contain the at least one of the first target nucleic acid 10 and the second target nucleic acid 20 . Therefore, fluorescent signals and amplification regarding the not contained target nucleic acid will not be carried out in the following explanation.
  • the temperature is decreased from the denaturation temperature T 0 to reach the annealing temperature T 1 , regarding the first target nucleic acid 10 , a first target's F-primer 13 specifically bonds with a complementary sequence of the first single strand 11 , and a first target's R-primer 14 specifically bonds with another complementary sequence of the second single strand 12 , respectively.
  • a second target's F-primer 23 specifically bonds with a complementary sequence of the first single strand 21
  • a second target's R-primer 24 specifically bonds with another complementary sequence of the second single strand 22 , respectively.
  • the first target's probe 15 labeled by means of the first labeling substance 16 specifically bonds with a specific part of the first single strand 11 derived from the first target nucleic acid 10 , thereby the first labeling substance 16 outputs first fluorescence signals.
  • the annealing temperature T 1 is higher than the second target detection temperature T 3 .
  • the second target's probe 25 labeled by means of the second labeling substance 26 does not bond with the first single strand 21 derived from the second target nucleic acid 20 , thereby the second labeling substance 26 outputs no fluorescence signal at this time.
  • the deoxyribonucleoside triphoshate 31 bonds therewith to elongate, respectively.
  • the deoxyribonucleoside triphoshate 31 bonds therewith to elongate, respectively.
  • the first target's probe 15 labeled by the first labeling substance 16 specifically bonds with a specific part of the first single strand 11 derived from the first target nucleic acid 10 , and the first fluorescent signals of the first labeling substance 16 change. This is the same as that of FIG. 37 ( b ) .
  • the temperature is the second target detection temperature T 3 . Accordingly, also the second target's probe 25 labeled by the second labeling substance 26 bonds with the first single strand 21 derived from the second target nucleic acid 20 , and the second fluorescent signals of the second labeling substance 26 also change.
  • first labeling substance 16 and the second labeling substance 26 are identical.
  • the multiple target nucleic acids can be respectively detected during a series of continuing steps by means of the reaction vessel containing one kind of reaction solution.
  • Embodiments of detecting multiple nucleic acids by means of three kinds of probes will now be explained more concretely.
  • the QProbe (registered trademark) method the Eprobe (registered trademark) method, and the TaqMan (registered trademark) method have been used.
  • Necessary information for operation including: primer sequences; base sequences of the respectivEprobes; and material of nucleic acid samples will be also shown.
  • a pair of primers used for the PCR method in Embodiment 1 are as shown in Table 1.
  • sequence No. 1 and sequence No. 2 (primer pair for P adhesin gene) sequences recited in the report (The Journal Of Infectious Diseases, 1996; 173; 1445-52) by leven et al. have been used.
  • Mycoplasma pneumnoniae is a pathogenic organism of Mycoplasma pneumonia.
  • P protein is membrane protein derived from Mycoplasma pneumoniae .
  • Gene fragments (sequences amplified by a pair of primers of SEQ ID NO: 1 and SEQ ID NO: 2) encode the P1 protein.
  • pMYC is a plasmid DNA produced by artificial synthesizing the gene fragments to be incorporated into a pMD20T vector. Production of the plasmid DNA has been performed by requesting custom synthesis to the Takara Bio Inc.
  • pICM5 is a plasmid DNA produced by artificial synthesizing a sequence including a complementary sequence to the pair of the primers to be incorporated into a pMD20T vector. Production of the plasmid DNA has been performed by requesting custom synthesis to the Takara Bio Inc.
  • First length of the pMYC plasmid is 2942 [bp]
  • second length of the pICM5 plasmid is 2886 [bp].
  • the number of copies per 1 [ ⁇ l] has been calculated. After that, by means of TE buffer solution (10 [mM] Tris-HCl, 1.0 [mM] EDTA pH: 8.0), the pMYC plasmid has been diluted to be 1 ⁇ 10 [copies/ ⁇ l], and the pICM5 plasmid has been diluted to be 1 ⁇ 10 [copies/ ⁇ l], respectively.
  • MYC QP is one QProbe specifically annealing the pMYC plasmid
  • IC QP is another QProbe specifically annealing the pICM5 plasmid.
  • fluorescent dye for both of “MYC QP” and “IC QP” “BODIPY FL” (registered trademark) has been used to label “C” at 3′ ends, respectively.
  • Production of the QProbes has been performed by requesting custom synthesis to the NIPPON STEEL & SUMIKIN Eco-Tech Corporation.
  • a first Tm value for “MYC QP” that is the QProbe specifically annealing the pMYC plasmid of the first target is set up to be higher than another Tm value for the primers to perform first detection during amplification reaction.
  • a second Tm value for “IC QP” that is the QProbe specifically annealing the pICM5 plasmid of the second target is set up to be lower than changes (70 Centigrade to 95 Centigrade) of temperature during the amplification reaction to perform second detection after the amplification reaction.
  • Table 3 and Table 4 show composition of the PCR reaction solution and the reaction conditions respectively, and FIG. 1 shows a graph of the changes of temperature of the mixed solution.
  • Light Cycler nano system (registered trademark of the Roche Diagnostics K.K.) has been used.
  • the combination of 510 to 528 [nm] has been selected for excitation wavelength and fluorescent wavelength upon fluorescence measurement. Fluorescence values have been measured at the denaturation step (95 Centigrade) and the elongation step (72 Centigrade) for every cycle. Furthermore, after the amplification reaction has been completed, at a step for detecting the second target (95 Centigrade and 55 Centigrade), fluorescence measurement has been performed.
  • FIG. 2 shows fluorescent measurement conditions at the final cycle of the amplification reaction and at the second target detection step.
  • fn fluorescence intensity value in n cycle calculated according to Formula 1
  • fhyb.n fluorescence intensity value at the elongation step in n-th cycle
  • fden.n fluorescence intensity value at the denaturation step in n-th cycle
  • fe fluorescence intensity value at the second target detection step calculated according to Formula 1′
  • fhyb.e fluorescence intensity value (55 Centigrade) at the second target detection step
  • fden.e fluorescence intensity value (95 Centigrade) at the second target detection step.
  • Fn relative value in n-th cycle assuming that the fluorescence intensity value in the tenth cycle obtained according to Formula 1 is equal to a value of “1”
  • Fe relative value at the second target detection step assuming that the fluorescence intensity value in the tenth cycle obtained according to Formula 1′ is equal to a value of “1”.
  • F44 which is a value of Fn at the final cycle of the amplification reaction has been used for judgment of the existence or nonexistence of the first target nucleic acid.
  • Fs measured value regarding the second target.
  • Fs has been used for determination of the existence or nonexistence of the second target nucleic acid.
  • the F44 value of the measurement sample has been compared with Threshold 1. And, when the F44 value of the measurement sample is lower than Threshold 1, it has been determined that the first target nucleic acid is positive (existence), otherwise negative (nonexistence).
  • Threshold 1 an average of F44 values of the negative reference (reagent TE buffer added thereto instead of DNA) minus three times the standard deviation (hereinafter, called as “mean ⁇ 3SD”) has been used.
  • the Fs value has been compared with Threshold 2, and when the Fs value is lower than Threshold 2 it has been determined that the second target nucleic acid is positive (existence), otherwise negative (nonexistence).
  • FIG. 3 shows a flow chart of the determination method related thereto.
  • the pMYC plasmid has been used for Mycoplasma pneumoniae P1 genes
  • the pICM5 plasmid has been used for the internal control composition
  • the PCR has been performed thereon.
  • a first Tm value of 72.5 Centigrade has been set for a sequence of MYC QP, and a second Tm value of 57.5 Centigrade has been set for a sequence of IC QP. For this reason, it has been estimated that probes anneal only MYC QP during the temperature range (from 70 Centigrade to 95 Centigrade) in the PCR.
  • Table 5 shows the value of the Fs.
  • the amplification curve is shown in FIG. 6 .
  • the value ⁇ 0.116 of Fs is lower than Threshold 2, and it has been revealed that the 1.5 objective region of the pICM5 plasmid has been amplified.
  • Table 5 shows the value of the Fs.
  • the amplification curve related thereto is shown in FIG. 7 .
  • Mycoplasma pneumoniae P1 genes and internal control composition can be detected by means of one reaction vessel containing one kind of reaction solution and one kind of fluorescent labels when the method of designing probes and temperature profiles are incorporated with the QProbe method.
  • the primers, probes, reaction solution composition, and nucleic acids have been used for the PCR method as the same as Embodiment 1.
  • Table 6 shows reaction conditions in Embodiment 2.
  • FIG. 8 shows fluorescent measurement conditions at the final cycle of the amplification reaction and at the second target detection step.
  • Embodiment 2 fe has been calculated as follows. Calculation other than fe has been carried out as the same as Embodiment 1.
  • fe fluorescence intensity value at the second target detection step
  • fhyb.e fluorescence intensity value (55 Centigrade) at the second target detection step
  • fden. 44 fluorescence intensity value (95 Centigrade) at the final cycle of amplification reaction.
  • the F44 value of the measurement sample has been compared with Threshold 3. And, when the F44 value of the measurement sample is lower than Threshold 3, it has been determined that the first target nucleic acid is positive (existence), otherwise negative (nonexistence).
  • the Fs value has been compared with Threshold 4, and when the Fs value is lower than Threshold 4 it has been determined that the second target nucleic acid is positive (existence), otherwise negative (nonexistence).
  • Mycoplasma pneumoniae P1 genes and internal control composition have been detected based on the Eprobe method.
  • Embodiment 3 The same primers and nucleic acid samples for PCR as Embodiment 1 have been used.
  • Table 8 shows probe information used for the PCR in Embodiment 3.
  • a specific region for Eprobe has been selected referring to Tm values calculated by means of “Edesign” software produced by the K. K. Dnaform.
  • MYC EP is one Eprobe specifically annealing the pMYC plasmid
  • IC EP is another Eprobe specifically annealing the pICM5 plasmid.
  • fluorescent dye for both of “MYC EP” and “IC EP” “D514” has been used.
  • the nineteenth “T” from 5′ end of “MYC EP” and the ninth “T” from 5′ end of “IC EP” have been labeled, respectively.
  • Embodiment 3 has been calculated with the following Formula. The other calculations have been carried out as the same as Embodiment 1.
  • Embodiment 3 Determination in Embodiment 3 has been performed using the method shown below.
  • the F40 value of the measurement sample has been compared with Threshold 5. And, when the F40 value of the measurement sample is higher than Threshold 5, it has been determined that the first target nucleic acid is positive (existence), otherwise negative (nonexistence).
  • the Fs value has been compared with Threshold 6, and when the Fs value is higher than Threshold 6, it has been determined that the second target nucleic acid is positive (existence), otherwise negative (nonexistence).
  • FIG. 13 shows a flow chart of the determination method related thereto.
  • a first Tm value of 76.4 Centigrade has been set for a sequence of MYC EP, and a second Tm value of 59.8 Centigrade has been set for a sequence of IC EP. For this reason, it has been estimated that probes anneal only MYC EP during the temperature range (from 70 Centigrade to 95 Centigrade) in the PCR.
  • a value 4.254 of Fs is higher than Threshold 6, and it has been revealed that the objective region of the pICM5 plasmid has been amplified.
  • Table 11 shows the value of the Fs.
  • the amplification curve related thereto is shown in FIG. 17 .
  • Mycoplasma pneumoniae P1 genes and internal control composition can be detected by means of one reaction vessel containing one kind of reaction solution and one kind of fluorescent labels when the method of designing probes and temperature profiles are combined even with the Eprobe method.
  • Table 12 shows information of the probes used for the PCR method in Embodiment 4.
  • Probes used for PCR in this Embodiment Probe Length name Sequence (5′ ⁇ 3′) (bp) MYC Taq CCCTCGACCAAGCCAACCTCCAGCTC 26 (SEQ ID No. 3) IC Taq AGTGGGACTCACCAACC 17 (SEQ ID No. 4)
  • a specific region for the TaqMan probe has been selected referring to Tm values calculated according to the nearest neighbor method.
  • MYC Taq is one TaqMan probe specifically annealing the pMYC plasmid
  • IC Taq is another TaqMan probe specifically annealing the pICM5 plasmid.
  • fluorescent dye for both of “MYC Taq” and “IC Taq” “FAM” (registered trademark) has been used to be labeled at 5′ end and “TAMRA” (registered trademark) has been used to be labeled at 3′ end, respectively.
  • Embodiment 4 thyb.e has been defined as a fluorescence intensity value (50 Centigrade), and the same calculation except this point as Embodiment 1 has been conducted.
  • the F44 value of the measurement sample has been compared with Threshold 7. And, when the F44 value of the measurement sample is higher than Threshold 7, it has been determined that the first target nucleic acid is positive (existence), otherwise negative (nonexistence).
  • the Fs value has been compared with Threshold 8, and when the Fs value is higher than Threshold 8, it has been determined that the second target nucleic acid is positive (existence), otherwise negative (nonexistence).
  • a first Tm value of 75.8 Centigrade has been set for a sequence of MYC Taq, and a second Tm value of 53.7 Centigrade has been set for a sequence of IC Taq. For this reason, it has been estimated that probes anneal only MYC Taq during the temperature range (from 70 Centigrade to 95 Centigrade) in the PCR.
  • Table 15 shows the value of the Fs.
  • the amplification curve is shown in FIG. 20 .
  • a value 0.177 of Fs is higher than Threshold 8, and it has been revealed that the objective region of the pICM5 plasmid has been amplified.
  • Table 15 shows the value of the Fs.
  • the amplification curve related thereto is shown in FIG. 21 .
  • Mycoplasma pneumoniae P1 genes and internal control composition can be detected by means of one reaction vessel containing one kind of reaction solution and one kind of fluorescent labels when the method of designing probes and temperature profiles are incorporated with even the TaqMan probe method.
  • Mycoplasma pneumoniae P1 genes and internal control composition have been detected by means of actual specimens.
  • first total DNA has been used, the first total DNA having been extracted from first pharynx wiping liquid of which Mycoplasma pneumoniae has been determined to be positive (tested by the BML, Inc.) according to the LAMP method (Japanese registered patent No. 3313358) by means of a “QIAamp” DNA mini Kit (registered trademark of the QIAGEN).
  • the F44 value of the measurement sample has been compared with Threshold 9. And, when the F44 value of the measurement sample is lower than Threshold 9, it has been determined that the first target nucleic acid is positive (existence), otherwise negative (nonexistence).
  • the Fs value has been compared with Threshold 10, and when the Fs value is lower than Threshold 10, it has been determined that the second target nucleic acid is positive (existence), otherwise negative (nonexistence).
  • Threshold 10 the value of “mean ⁇ 3SD” with respect to Fs of the positive sample has been used.
  • a value ⁇ 0.235 of Fs is lower than Threshold 10, and it has been revealed that the objective region of the pICM5 plasmid has been amplified.
  • Table 16 shows the value of the Fs.
  • the amplification curve related thereto is shown in FIG. 25 .
  • Mycoplasma pneumoniae P1 genes and internal control composition can be detected by means of one reaction vessel containing one kind of reaction solution and one kind of fluorescent labels when the method of designing probes and temperature profiles are incorporated with the actual clinical specimens.
  • the present invention is also applicable for identifying subtypes and/or single nucleotide polymorphism of pathogenic organisms causing infectious diseases.
  • pCT plasmids Production of pCT plasmids has been performed by requesting custom synthesis to the Hokkaido System Science Co., Ltd.
  • Gene fragments of common endogenous plasmids (pLGV440) of Chlamydia trachomatis which are pathogenic organisms causing Chlamydia infection have been artificially synthesized into plasmid DNA to be incorporated into a pUC57 vector, thereby having prepared the pCT plasmids.
  • Production of pNG plasmids has also been performed by requesting custom synthesis to the Hokkaido System Science Co., Ltd.
  • Gene fragments of cytosine DNA methyl transferase (CMT) of Neisseria gonorrhoeae which is a pathogenic organism causing gonorrhea have been artificially synthesized into plasmid DNA to be incorporated into a pUCS7 vector, thereby having prepared the pNG plasmids.
  • CMT cytosine DNA methyl transferase
  • First length of the pCT plasmid is 3064 [bp]
  • second length of the pNG plasmid is 3059 [bp].
  • Primer pairs used in the PCR method in Embodiment 6 are as shown in Table 17.
  • CT QP is one QProbe specifically annealing the pCT plasmid
  • NG QP is another QProbe specifically annealing the pNG plasmid.
  • BODIPY FL registered trademark
  • the combination of 510 to 528 [nm] has been selected for excitation wavelength and fluorescent wavelength upon fluorescence measurement.
  • Fluorescence values have been measured at the denaturation step (95 Centigrade) and the elongation step (68 Centigrade) for every cycle. Furthermore, after the amplification reaction has been completed, at a step for detecting the second target (95 Centigrade and 55 Centigrade), fluorescence measurement has been performed.
  • the F44 value of the measurement sample has been compared with Threshold 1. And, when the F44 value of the measurement sample is lower than Threshold 11, it has been determined that the first target nucleic acid is positive (existence), otherwise negative (nonexistence).
  • the Fs value has been compared with Threshold 12, and when the Fs value is lower than Threshold 12, it has been determined that the second target nucleic acid is positive (existence), otherwise negative (nonexistence).
  • a first Tm value of 73.4 Centigrade has been set for a sequence of CT QP, and a second Tm value of 62.5 Centigrade has been set for a sequence of NG QP. For this reason, it has been estimated that probes anneal only CT QP during the temperature range (from 68 Centigrade to 95 Centigrade) in the PCR.
  • a value ⁇ 0.111 of Fs is lower than Threshold 12, and it has been revealed that the objective region of the pNG plasmid has been amplified.
  • Table 21 shows the value of the Fs.
  • the amplification curve related thereto is shown in FIG. 29 .
  • Mycoplasma pneumoniae P1 genes and internal control composition can be detected by means of one reaction vessel containing one kind of reaction solution and one kind of fluorescent labels when the method of designing probes and temperature profiles are incorporated with two items of genes.
  • the second target detection step has been carried out in an inserted manner, and detection of Mycoplasma pneumoniae P1 genes and internal control composition has been performed based on the QProbe method.
  • the primers, probes, reaction solution composition, and nucleic acids have been used for the PCR method as the same as Embodiment 1.
  • Table 22 shows reaction conditions thereof.
  • FIG. 30 is a graph of changes of temperature in the amplification reaction.
  • Embodiment 7 In analysis of Embodiment 7, the same steps as Embodiment 1 except the following calculation have been carried out.
  • fen fluorescence intensity value in n times second target detection step calculated according to Formula 4; fhyb.en: fluorescence intensity value (55 Centigrade) in n times second target detection step; and fden.en: fluorescence intensity value (95 Centigrade) in n times second target detection step.
  • Fen relative value in n times second target detection step assuming that the fluorescence intensity value in the tenth cycle obtained according to Formula 4 is equal to a value of “1.”
  • Fs1 measurement value in first time second target detection step
  • Fs2 measurement value in second time second target detection step
  • Fs3 measurement value in third time second target detection step
  • Fs4 measurement value in fourth time second target detection step
  • the F41 value of the measurement sample has been compared with Threshold 13. And, when the F41 value of the measurement sample is lower than Threshold 13, it has been determined that the first target nucleic acid is positive (existence), otherwise negative (nonexistence).
  • the Fs1 value has been compared with Threshold 14, and when the Fs1 value is lower than Threshold 14, it has been determined that the second target nucleic acid is positive (existence), otherwise negative (nonexistence).
  • the Fs2 value has been compared with Threshold 15, and when the Fs2 value is lower than Threshold 15 it has been determined that the second target nucleic acid is positive (existence), otherwise negative (nonexistence).
  • the Fs3 value has been compared with Threshold 16, and when the Fs3 value is lower than Threshold 16 it has been determined that the second target nucleic acid is positive (existence), otherwise negative (nonexistence).
  • the Fs4 value has been compared with Threshold 17, and when the Fs4 value is lower than Threshold 17 it has been determined that the second target nucleic acid is positive (existence), otherwise negative (nonexistence).
  • a value ⁇ 0.057 of Fs2 has been higher than Threshold 15, and it has been revealed that the objective region of the pICM5 plasmid has not been amplified up to the identification limit until the second time second target detection step.
  • a value ⁇ 0.084 of Fs3 has been lower than Threshold 16, and it has been revealed that the objective region of the pICM5 plasmid has been amplified beyond the identification limit until the third time second target detection step.
  • a value ⁇ 0.192 of Fs4 has also been lower than Threshold 17.
  • Table 23 shows the values of the Fs1, Fs2, Fs3 and Fs4. The amplification curves are shown in FIG. 33 .
  • a value ⁇ 0.058 of Fs1 has been higher than Threshold 14, and it has been revealed that the objective region of the pICM5 plasmid has not been amplified beyond the identification limit until the first time second target detection step.
  • a value ⁇ 0.068 of Fs2 has been also higher than Threshold 15, and it has been revealed that the objective region of the pICM5 plasmid has not been amplified beyond the identification limit until the second time second target detection step.
  • a value ⁇ 0.109 of Fs3 has been lower than Threshold 16, and it has been revealed that the objective region of the pICM5 plasmid has been amplified beyond the identification limit until the third time second target detection step.
  • a value ⁇ 0.154 of Fs4 has also been lower than Threshold 17.
  • Table 23 shows the values of the Fs1, Fs2, Fs3 and Fs4. The amplification curves are shown in FIG. 34 .
  • FIG. 1 is a graph showing changes of temperature within mixed-solution in Embodiment 1 according to the present invention.
  • FIG. 3 is a flow chart showing a determination method in Embodiment 1 according to the present invention.
  • FIG. 4 is a graph showing changes of fluorescence intensity regarding a negative reference in Embodiment 1 according to the present invention.
  • FIG. 5 is a graph showing changes of fluorescence intensity regarding a first target nucleic acid in Embodiment 1 according to the present invention.
  • FIG. 6 is a graph showing changes of fluorescence intensity regarding a second target nucleic acid in Embodiment 1 according to the present invention.
  • FIG. 7 is a graph showing changes of fluorescence intensity regarding the first target nucleic acid and the second target nucleic acid in Embodiment 1 according to the present invention.
  • FIG. 8 is a timing explanatory diagram of fluorometry in Embodiment 2 according to the present invention.
  • FIG. 9 is a graph showing changes of fluorescence intensity regarding a negative reference in Embodiment 2 according to the present invention.
  • FIG. 10 is a graph showing changes of fluorescence intensity regarding a first target nucleic acid in Embodiment 2 according to the present invention.
  • FIG. 11 is a graph showing changes fluorescence intensity regarding a second target nucleic acid in Embodiment 2 according to the present invention.
  • FIG. 12 is a graph showing changes of fluorescence intensity regarding the first target nucleic acid and the second target nucleic acid in Embodiment 2 according to the present invention.
  • FIG. 13 is a flow chart showing a determination method in Embodiment 3 according to the present invention.
  • FIG. 14 is a graph showing changes of fluorescence intensity regarding a negative reference in Embodiment 3 according to the present invention.
  • FIG. 15 is a graph showing changes of fluorescence intensity regarding a first target nucleic acid in Embodiment 3 according to the present invention.
  • FIG. 16 is a graph showing changes of fluorescence intensity regarding a second target nucleic acid in Embodiment 3 according to the present invention.
  • FIG. 17 is a graph showing changes of fluorescence intensity regarding the first target nucleic acid and the second target nucleic acid in Embodiment 3 according to the present invention.
  • FIG. 18 is a graph showing changes of fluorescence intensity regarding a negative reference in Embodiment 4 according to the present invention.
  • FIG. 19 is a graph showing changes of fluorescence intensity regarding a first target nucleic acid in Embodiment 4 according to the present invention.
  • FIG. 20 is a graph showing changes of fluorescence intensity regarding a second target nucleic acid in Embodiment 4 according to the present invention.
  • FIG. 22 is a graph showing changes of fluorescence intensity regarding a negative reference in Embodiment 5 according to the present invention.
  • FIG. 23 is a graph showing changes of fluorescence intensity regarding a first target nucleic acid in Embodiment 5 according to the present invention.
  • FIG. 25 is a graph showing changes of fluorescence intensity regarding the first target nucleic acid and the second target nucleic acid in Embodiment 5 according to the present invention.
  • FIG. 26 is a graph showing changes of fluorescence intensity regarding a negative reference in Embodiment 6 according to the present invention.
  • FIG. 27 is a graph showing changes of fluorescence intensity regarding a first target nucleic acid in Embodiment 6 according to the present invention.
  • FIG. 28 is a graph showing changes of fluorescence intensity regarding a second target nucleic acid in Embodiment 6 according to the present invention.
  • FIG. 29 is a graph showing changes of fluorescence intensity regarding the first target nucleic acid and the second target nucleic acid in Embodiment 6 according to the present invention.
  • FIG. 30 is a graph showing changes of temperature within mixed-solution in Embodiment 7 according to the present invention.
  • FIG. 31 is a graph showing changes of fluorescence intensity regarding a negative reference in Embodiment 7 according to the present invention.
  • FIG. 32 is a graph showing changes of fluorescence intensity regarding a first target nucleic acid in Embodiment 7 according to the present invention.
  • FIG. 33 is a graph showing changes of fluorescence intensity regarding a second target nucleic acid in Embodiment 7 according to the present invention.
  • FIG. 34 is a graph showing changes of fluorescence intensity regarding the first target nucleic acid and the second target nucleic acid in Embodiment 7 according to the present invention.
  • FIG. 35 is an enlarged view of changes of temperature in Embodiment 1 according to the present invention.
  • FIG. 36 is an explanatory diagram of mixed-solution composition in Embodiment 1 according to the present invention.
  • FIG. 37( a ) is an explanatory diagram of a denaturation step in Embodiment 1 according to the present invention.
  • FIG. 37( b ) is an explanatory diagram of an annealing step in Embodiment 1 according to the present invention.
  • FIG. 37( c ) is an explanatory diagram of an elongation step in Embodiment 1 according to the present invention.
  • FIG. 37( d ) is an explanatory diagram of an elongation-completed step in Embodiment 1 according to the present invention.
  • FIG. 38( a ) is an explanatory diagram of the denaturation step in Embodiment 1 according to the present invention.
  • FIG. 38( b ) is an explanatory diagram of the annealing step in Embodiment 1 according to the present invention.

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