RT-qPCR METHOD, PREMIX REAGENT FOR RT-qPCR, AND REAGENT SET FOR RT-qPCR

Abstract

Provided is an RT-qPCR method wherein steps (1), (2), and (3) are continuously performed in the following order in a RT-qPCR reaction liquid which contains an enzyme (A) having double strand specific deoxyribonuclease activity, an enzyme (B) having reverse transcription activity and DNA polymerase activity, and Mn2+, and in which the Mn2+ concentration is 3.3-4.6 mM. Step (1) is incubation that causes the enzyme (A) to function. Step (2) is a heat treatment that inactivates the enzyme (A) but does not inactivate the enzyme (B). Step (3) is reverse transcription, PCR, and real-time quantification. Also provided is a premix reagent for RT-qPCR comprising an enzyme (A), an enzyme (B), and Mn2+, wherein the Mn2+ concentration is not less than 3.3 mM. Also provided is a reagent set for RT-qPCR comprising an enzyme (A), an enzyme (B), and a manganese salt solution in which the Mn2+ concentration is not less than 3.3 mM.

Description

RT-qPCR method, RT-qPCR premix reagents, and RT-qPCR reagent sets

[0001] This disclosure relates to an RT-qPCR method, a premix reagent for RT-qPCR, and a reagent set for RT-qPCR.

[0002] Cell and gene therapies generally need to be administered to patients within 48 to 96 hours of manufacture. This means that cell and gene therapies have a shelf life of only 48 to 96 hours after manufacture. Microbiological testing by culture (for example, a 14-day culture test to determine the presence of bacteria and fungi) is not applicable to pharmaceuticals with such short shelf lives. There is a need for rapid microbiological testing that is applicable to pharmaceuticals with short shelf lives.

[0003] Nucleic acid amplification tests (NATs) are rapid tests that can detect nucleic acids quickly and are promising as rapid microbiological tests. Ribosomal RNA (rRNA) is a promising target for NAT microbiological testing. Because rRNA is the RNA (ribonucleic acid) that makes up ribosomes and exists in multiple copies within cells, it can be a highly sensitive target for NATs. In addition, rRNA sequences have been analyzed in a wide variety of organisms, and a comprehensive sequence database has been established. Therefore, rRNA sequences that are specific to and common to microorganisms are promising targets for NAT microbiological testing.

[0004] Rapid RNA-targeted NAT is achieved by RT-qPCR. RT-qPCR is prone to false positives if even a small amount of microorganisms or contaminating DNA (deoxyribonucleic acid) is present in the test tube. To suppress false positives, the number of times the test tube is opened and closed, and the number of pipetting operations, should be minimized. Conventionally, there is a technique called "one-step RT-PCR," in which reverse transcription and PCR are performed consecutively in a single tube. ("Two-step RT-PCR" involves adding the cDNA, the product of reverse transcription, to the PCR reaction mixture, or adding the PCR enzyme and materials to the reaction mixture after reverse transcription.)

[0005] Furthermore, as an advanced form of one-step RT-PCR, there is a technique that performs hydrolysis of contaminating DNA in the same tube prior to reverse transcription and PCR. For example, Patent Document 1 discloses a ribonucleic acid amplification method in which the enzymes and materials necessary for a series of reactions are mixed in a single tube, and the hydrolysis of contaminating DNA, reverse transcription, and PCR are performed in succession. (Note that the quantification in the ribonucleic acid amplification method described in Patent Document 1 is not real-time quantification, but quantification by electrophoresis of the PCR product. Therefore, although this ribonucleic acid amplification method is RT-PCR, it is not RT-qPCR.)

[0006] Japanese Patent Publication No. 2005-304396

[0007] To prevent contamination by microorganisms and contaminating DNA during RT-qPCR, it is desirable to perform the hydrolysis of contaminating DNA, reverse transcription, and PCR in a single tube. However, for the reasons (a) to (c) below, it is not easy to successfully perform RT-qPCR by mixing the enzymes and materials necessary for the series of reactions in a single tube.

[0008] (a) Deoxyribonucleases (DNases), which are responsible for the hydrolysis of contaminating DNA, can also hydrolyze primers and probes present in the test tube, depending on the type. For example, DNase I, used in the ribonucleic acid amplification method of Patent Document 1, is an endonuclease that hydrolyzes both single-stranded and double-stranded DNA, and thus degrades not only contaminating DNA but also primers and probes. (b) If DNase remains active in the test tube, the reverse transcriptase and PCR product DNA will be hydrolyzed. If the test tube is heat-treated to inactivate DNase before reverse transcription and PCR, there is a risk that the reverse transcriptase and / or DNA polymerase will also be inactivated. (c) The reaction rate of enzymes is affected by ionic strength, and since the ionic strength in the test tube cannot be controlled by adding chemicals afterwards, it is necessary to set the initial ionic strength in the test tube to a range in which the reaction can proceed for all types of enzymes.

[0009] This disclosure is made under the circumstances described above. The object of this disclosure is to provide an RT-qPCR method that performs hydrolysis of contaminating DNA, reverse transcription, and PCR in a single tube, and an RT-qPCR premix reagent and an RT-qPCR reagent set used in the RT-qPCR method.

[0010] The following embodiments are included as specific means for solving the aforementioned problem: <1> An enzyme (A) having double-strand specific deoxyribonuclease activity, an enzyme (B) having reverse transcription activity and DNA polymerase activity, and Mn ２＋ It contains Mn ２＋ An RT-qPCR method in which the following (1), (2), and (3) are performed sequentially in an RT-qPCR reaction solution with a concentration of 3.3 mM to 4.6 mM: (1) incubation to activate enzyme (A), (2) heat treatment to inactivate enzyme (A) but not enzyme (B), (3) reverse transcription, PCR and real-time quantification. <2> Enzyme (A), enzyme (B), and Mn ２＋ The RT-qPCR method according to <1>, comprising mixing a sample with a premix reagent for RT-qPCR containing the above. <3> The RT-qPCR method according to <1> or <2>, wherein the enzyme (B) is a DNA polymerase derived from Thermus thermophilus. <4> An enzyme (A) having double-strand specific deoxyribonuclease activity, an enzyme (B) having reverse transcription activity and DNA polymerase activity, and Mn ２＋ It contains Mn ２＋A premix reagent for RT-qPCR having a concentration of 3.3 mM or higher. <5> The premix reagent for RT-qPCR according to <4>, further comprising a fluorescent dye-labeled probe or a double-stranded DNA-binding fluorescent dye. <6> The premix reagent for RT-qPCR according to <4> or <5>, further comprising a first primer set for amplifying a conserved sequence in bacterial rRNA and a second primer set for amplifying a conserved sequence in fungal rRNA. <7> The premix reagent for RT-qPCR according to <6>, further comprising a first probe for detecting a conserved sequence in bacterial rRNA and a second probe for detecting a conserved sequence in fungal rRNA. <8> The premix reagent for RT-qPCR according to any one of <4> to <7>, wherein the enzyme (B) is a DNA polymerase derived from Thermus thermophilus. <9> An enzyme (A) having double-strand specific deoxyribonuclease activity, an enzyme (B) having reverse transcription activity and DNA polymerase activity, and Mn ２＋ A reagent set for RT-qPCR comprising a manganese salt solution with a concentration of 3.3 mM or higher. <10> The reagent set for RT-qPCR according to <9>, wherein the enzyme (B) is a DNA polymerase derived from Thermus thermophilus.

[0011] This disclosure provides an RT-qPCR method in which the hydrolysis of contaminating DNA, reverse transcription, and PCR are performed sequentially in a single tube, as well as an RT-qPCR premix reagent and an RT-qPCR reagent set used in the RT-qPCR method.

[0012] Experiment 2: Amplification curve of RT-qPCR Experiment 3: Amplification curve of RT-qPCR Experiment 4: Amplification curve of RT-qPCR Experiment 4: Amplification curve of RT-qPCR Experiment 5: Amplification curve of RT-qPCR

[0013] The embodiments of this disclosure are described below. These descriptions and embodiments are illustrative and do not limit the scope of the embodiments.

[0014] In the present disclosure, the numerical range indicated by using "~" represents a range that includes the numerical values described before and after "~" as the minimum value and the maximum value, respectively. In the numerical ranges described step by step in the present disclosure, the upper limit value or the lower limit value described in one numerical range may be replaced with the upper limit value or the lower limit value of the numerical ranges described in other step-by-step descriptions. Also, in the numerical ranges described in the present disclosure, the upper limit value or the lower limit value of the numerical range may be replaced with the value shown in the examples.

[0015] In the present disclosure, "A and / or B" is synonymous with "at least one of A and B". That is, "A and / or B" means that it may be only A, only B, or a combination of A and B.

[0016] In the present disclosure, the term "step" includes not only an independent step but also cases where it cannot be clearly distinguished from other steps as long as the purpose of the step is achieved.

[0017] When referring to the amount of each component in a composition in the present disclosure, when there are multiple types of substances corresponding to each component in the composition, unless otherwise specified, it means the total amount of the multiple types of substances present in the composition.

[0018] Regarding substance concentration, "M" represents molar concentration, and 1M = 1 mol / L. Regarding substance concentration, unless otherwise specified, "%" is based on mass.

[0019] Write the full spelling of the abbreviations used in this disclosure. PCR: Polymerase Chain Reaction qPCR: quantitative Polymerase Chain Reaction RT-PCR: Reverse Transcription-Polymerase Chain Reaction RT-qPCR: Reverse Transcription-quantitative Polymerase Chain Reaction

[0020] qPCR is also called rtPCR (real-time Polymerase Chain Reaction). To avoid confusion between rt (real-time) and RT (Reverse Transcription), this disclosure will refer to it as qPCR, not rtPCR.

[0021] <Experiment> The RT-qPCR method described herein was created based on the results of experiments conducted by the inventors. The experiments conducted by the inventors and their results are described below.

[0022] [Experiment 1: Examination of buffers for RT-qPCR reaction solution] Based on the rRNA sequence database information of 22 bacterial species shown in Table 1, highly homologous conserved sequences were identified among the 22 bacterial species. A PCR primer set and probe were designed to amplify and detect these conserved sequences. Hereinafter referred to as the "bacterial primer / probe set". The conserved sequences were aligned, and a consensus sequence was identified.

[0023] Based on the rRNA sequence database information for three fungal species shown in Table 1, highly homologous conserved sequences were identified among the three fungal species. A PCR primer set and probe were designed to amplify and detect these conserved sequences. Hereinafter referred to as the "fungal primer / probe set."

[0024]

[0025] The buffer solutions shown in Table 2 were prepared, and the RT-qPCR reaction solutions shown in Table 3 were prepared using these buffer solutions.

[0026]

[0027]

[0028] The template is synthetic RNA containing the aforementioned consensus sequences of 22 types of bacteria. "Hot Start TTx DNA Polymerase" (Toyobo Co., Ltd.) is a DNA polymerase derived from Thermus thermophilus HB8 strain and is a DNA polymerase with reverse transcription activity. Hot Start TTx DNA Polymerase is a DNA polymerase used in so-called "hot start PCR" that has been inactivated by a neutralizing antibody.

[0029] RT-qPCR was performed using the RT-qPCR reaction mixture shown in Table 3, following the cycle described below. Of the cycles below, <1> is a heat treatment to inactivate the neutralizing antibody and restore the activity of Hot Start TTx DNA Polymerase, <2> is incubation for reverse transcription, <3> is a heat treatment to denature the cDNA into single strands, and <4>-<5> is a two-step PCR (denaturation / annealing and extension).

[0030] <1> 90°C / 30 seconds <2> 60°C / 10 minutes <3> 95°C / 1 minute <4> 95°C / 10 seconds <5> 60°C / 15 seconds...fluorescence detection Repeat <4>-<5> 45 times

[0031] Table 4 shows the Ct values ​​(Threshold Cycle) for RT-qPCR. RT-qPCR was performed after diluting the template to seven different concentrations. NTC (No Template Control) is a negative control without a template.

[0032]

[0033] The appropriate reaction efficiency for RT-qPCR is generally between 90% and 110%. Reaction solutions containing bicine, HEPPSO, or tricinine achieved an efficiency of 90% to 110%. Of these, reaction solutions containing bicine or tricinine showed sufficient sensitivity.

[0034] The results of Experiment 1 showed that a reaction solution using Hot Start TTx DNA Polymerase with a buffer of bicine or tricinine is preferable. Therefore, in subsequent experiments using Hot Start TTx DNA Polymerase, bicine or tricinine was used as the buffer for the RT-qPCR reaction solution.

[0035] [Experiment 2: Verification of the effectiveness of DNaseI] DNaseI is an endonuclease that hydrolyzes both single-stranded and double-stranded DNA. We verified whether RT-qPCR could be performed continuously in a single tube using DNaseI.

[0036] The RT-qPCR reaction solutions shown in Table 5 were prepared. The templates were synthetic RNAs containing the aforementioned consensus sequences from 22 types of bacteria.

[0037]

[0038] RT-qPCR was performed using the RT-qPCR reaction mixture shown in Table 5, following the cycle below. Of the cycles below, <01> is the incubation to activate DNaseI. DNaseI is inactivated by the heat treatment in <1>.

[0039] <01> 37°C / 10 min <1> 90°C / 30 sec <2> 60°C / 10 min <3> 95°C / 1 min <4> 95°C / 10 sec <5> 60°C / 15 sec...Fluorescence detection Repeat <4>-<5> 45 times

[0040] The amplification curves for RT-qPCR are shown in Figure 1. In both the reaction mixture with bicine buffer and the reaction mixture with tricinine buffer, DNA amplification did not occur with DNaseI(+). It was presumed that DNaseI hydrolyzed the primers and probes in the reaction mixture.

[0041] The results of Experiment 2 showed that deoxyribonucleases lacking double-strand specificity hydrolyze primers and probes, making them unsuitable for continuous processing in a single tube.

[0042] [Experiment 3: Confirmation of HL-DSN activity] Experiment 2 showed that using DNase without double-strand specificity for the hydrolysis of contaminating DNA is inappropriate. Therefore, it was considered to use double-strand specific DNase for the hydrolysis of contaminating DNA. We investigated whether double-strand specific DNase functions in the RT-qPCR reaction mixture.

[0043] The RT-qPCR reaction solutions shown in Table 6 were prepared. HL-DSN (ArcticZymes) was used as the double-stranded DNA-specific DNase. HL-DSN is an endonuclease for double-stranded DNA that is inactivated by being placed at approximately 60°C for 5 minutes. The templates were circular double-stranded plasmid DNAs containing the aforementioned consensus sequences from 22 types of bacteria.

[0044]

[0045] RT-qPCR was performed using the RT-qPCR reaction mixture shown in Table 6, following the cycle described below. Of the cycles below, <01> is the incubation to activate HL-DSN, and <02> is the heat treatment to deactivate HL-DSN.

[0046] <01> 37°C / 10 min <02> 95°C / 5 min <1> 90°C / 30 sec <2> 60°C / 10 min <3> 95°C / 1 min <4> 95°C / 10 sec <5> 60°C / 15 sec...Fluorescence detection Repeat <4>-<5> 45 times

[0047] Figure 2 shows the amplification curves for RT-qPCR. In the reaction mixture with bicine as the buffer, the Ct value for HL-DSN(-) was 16.0 and the Ct value for HL-DSN(+) was 17.5, showing little difference. In the reaction mixture with tricinine as the buffer, the Ct value for HL-DSN(-) was 17.0 and the Ct value for HL-DSN(+) was 21.5, a difference of 4.5 points. It was presumed that the difference in Ct values ​​occurred because a portion of the circular double-stranded plasmid DNA used as the template was hydrolyzed by HL-DSN (an endonuclease for double-stranded DNA).

[0048] From the results of Experiment 3, it was confirmed that HL-DSN functions in the RT-qPCR reaction solution. Also, it was shown that a reaction solution with Tricine as the buffer is suitable for HL-DSN. Hereinafter, in experiments using HL-DSN, Tricine was used as the buffer of the buffer solution used for preparing the RT-qPCR reaction solution.

[0049] [Experiment 4: Screening of the Mn concentration at which HL-DSN and DNA polymerase derived from Thermus thermophilus function] HL-DSN has been reported to be activated by Mn. DNA polymerase derived from Thermus thermophilus exhibits reverse transcription activity in the presence of Mn. Therefore, the Mn concentration range at which these two enzymes function was examined. ２＋ HL-DSN has been reported to be activated by Mn. ２＋ DNA polymerase derived from Thermus thermophilus exhibits reverse transcription activity in the presence of Mn. ２＋ Therefore, the Mn concentration range at which these two enzymes function was examined. ２＋

[0050] The RT-qPCR reaction solutions shown in Table 7 were prepared. The template is circular double-stranded plasmid DNA or synthetic RNA having the consensus sequence of 22 types of bacteria. The final concentration of manganese(II) acetate (Mn(OAc)) in the reaction solution is either 2.5 mM, 3.3 mM, 3.6 mM, 3.9 mM, 4.3 mM, or 4.6 mM. ２

[0051]

[0052] Using the RT-qPCR reaction solutions shown in Table 7, RT-qPCR was performed with the following cycles. Among the following cycles, <01> is the incubation to function HL-DSN, and <02> is the heat treatment to inactivate HL-DSN.

[0053] <01> 37°C / 10 minutes <02> 95°C / 5 minutes <1> 90°C / 30 seconds <2> 60°C / 5 minutes <3> 95°C / 1 minute <4> 95°C / 10 seconds <5> 60°C / 15 seconds... Fluorescence detection <4> - <5> was repeated 45 times

[0054] The amplification curve of RT-qPCR when circular double-stranded plasmid DNA was used as the template is shown in Figure 3, and the Ct value (average value of 2 samples) is shown in Table 8.

[0055] ​​

[0056] Figure 4 shows the amplification curve of RT-qPCR using synthetic RNA as a template, and Table 9 shows the reaction efficiency and the correlation coefficient of the calibration curve. The template concentration was set to eight levels (1.0 × 10⁻⁶). ７ Copy, 1.0 x 10 ６ Copy, 1.0 x 10 ５ Copy, 1.0 x 10 ４ Copy, 1.0 x 10 ３ Copy, 1.0 x 10 ２ Copy, 1.0 x 10 １ Copy, 1.0 x 10 ０ The sample was diluted (in a copy) and RT-qPCR was performed.

[0057]

[0058] As shown in Table 8, Mn ２＋ At concentrations of 3.3 mM to 4.6 mM, the Ct value of HL-DSN(+) was more than 1 point greater than the Ct value of HL-DSN(-). ２＋ In the reaction solution at the specified concentration, it was hypothesized that the Ct value increased because a portion of the template circular double-stranded plasmid DNA was hydrolyzed by HL-DSN (an endonuclease for double-stranded DNA).

[0059] As shown in Table 9, Mn ２＋ For concentrations between 3.3 mM and 4.6 mM, the correlation coefficient of the calibration curve was 0.975 or higher. ２＋ In the case of reaction solutions of varying concentrations, quantitative analysis was shown to be relatively appropriate.

[0060] From the results of Experiment 4, in order to express the hydrolytic activity of HL-DSN on double-stranded DNA and the reverse transcription activity and DNA polymerase activity of Hot Start TTx DNA Polymerase, and to perform appropriate quantification, the Mn of the reaction solution is necessary. ２＋ It was shown that a concentration of 3.3 mM to 4.6 mM is preferable, 3.6 mM to 4.6 mM is more preferable, and 3.9 mM to 4.3 mM is even more preferable.

[0061] [Experiment 5: Verification of False Positives] The same procedure as in Experiment 4 was followed, except that distilled water was used as the template, and HL-DSN(+) RT-qPCR was performed. The amplification curve of the RT-qPCR is shown in Figure 5, and the Ct values ​​are shown in Table 10. The RT-qPCR was performed for 45 cycles. "Not detected" in Table 10 refers to samples that did not reach the threshold by the 40th cycle. Samples that reached the threshold by the 40th cycle were judged to be false positives.

[0062]

[0063] In Experiment 4, it was confirmed that both types of enzymes functioned in Mn. ２＋ Concentrations of 3.3 mM to 4.6 mM result in 0 or 1 false positive sample, indicating a low risk of generating false positives. ２＋ It was shown to be a concentration.

[0064] From the results of experiments 1-5, it was found that the mixture contains double-strand specific DNase, a DNA polymerase with reverse transcription activity, and materials necessary for the RT-PCR reaction, and Mn ２＋ Using RT-PCR reaction solutions with concentrations of 3.3 mM to 4.6 mM, it was demonstrated that hydrolysis of double-stranded DNA (an impurity), reverse transcription, and PCR could be performed sequentially in a single tube, and that the composition of the reaction solution had a low risk of generating false positives.

[0065] Based on the experimental results described above, this disclosure provides a novel RT-qPCR method. The RT-qPCR method, RT-qPCR premix reagents, and RT-qPCR reagent set of this disclosure are described below.

[0066] <RT-qPCR method, RT-qPCR reaction solution> The RT-qPCR method disclosed herein comprises an enzyme (A) having double-strand specific deoxyribonuclease activity, an enzyme (B) having reverse transcription activity and DNA polymerase activity, and Mn ２＋ It contains Mn ２＋ In an RT-qPCR reaction solution with a concentration of 3.3 mM to 4.6 mM, steps (1), (2), and (3) below are carried out sequentially in this order.

[0067] Step (1): Incubation to activate enzyme (A). Step (2): Heat treatment to inactivate enzyme (A) but not enzyme (B). Step (3): Reverse transcription, PCR, and real-time quantification.

[0068] In step (1), enzyme (A) exhibits double-strand specific deoxyribonuclease activity, hydrolyzing double-stranded DNA (a substance that can be used as a template for PCR and therefore can cause false positives). Examples of double-stranded DNA include DNA contained in nucleic acid samples, DNA attached to test materials (e.g., tubes, pipette tips), DNA mixed in reagents, and DNA in the air inside the tubes. Next, in step (2), enzyme (A) is inactivated, so enzyme (A) does not hydrolyze the product in step (3). On the other hand, enzyme (B) is not inactivated in step (2). Then, in step (3), enzyme (B) functions, reverse transcription and replication of nucleic acids proceed, and the amplified product is quantified in real time.

[0069] The RT-qPCR reaction solution contains all the enzymes and materials necessary for RT-qPCR, and Mn ２＋ The concentration is 3.3 mM to 4.6 mM. Mn of the RT-qPCR reaction solution ２＋ A concentration of 3.3 mM to 4.6 mM is an appropriate ionic strength for both enzyme (A) and enzyme (B) to function.

[0070] Mn of RT-qPCR reaction solution ２＋ If the concentration is less than 3.3 mM, the double-strand specific deoxyribonuclease activity of enzyme (A) is not expressed or is low, and the contaminant double-stranded DNA is not sufficiently degraded. From the viewpoint of promoting the hydrolysis of the contaminant double-stranded DNA, the Mn of the RT-qPCR reaction solution ２＋ The concentration is 3.3 mM or higher, preferably 3.6 mM or higher, and more preferably 3.9 mM or higher. (Manic content of RT-qPCR reaction solution) ２＋ If the concentration exceeds 4.6 mM, the reverse transcription activity and DNA polymerase activity of enzyme (B) are not expressed or are at low activity, resulting in a small amount of RT-qPCR product and low sensitivity. From the perspective of increasing the sensitivity of RT-qPCR, the Mn of the RT-qPCR reaction solution ２＋The concentration is 4.6 mM or less, and preferably 4.3 mM or less.

[0071] In the RT-qPCR method of this disclosure, enzyme (A) functions in the reaction solution, followed by enzyme (B), making it possible to perform hydrolysis of contaminating DNA, reverse transcription, and PCR continuously in a single tube. According to the RT-qPCR method of this disclosure, there is no need to open and close test tubes or pipette operations during the hydrolysis of contaminating DNA, reverse transcription, and PCR, minimizing the opportunity for microorganisms and contaminating DNA to enter the test tube, and thus reducing the occurrence of false positives. Furthermore, according to the RT-qPCR method of this disclosure, the contaminating DNA that causes false positives is hydrolyzed before PCR, thus reducing the occurrence of false positives.

[0072] The components of the RT-qPCR reaction solution and the RT-qPCR procedure will be explained in detail below.

[0073] The RT-qPCR reaction solution contains enzyme (A), enzyme (B), and Mn ２＋ It contains and also contains all the materials necessary for the RT-qPCR reaction. These materials include nucleic acid samples, primer sets, fluorescent dye-labeled probes or double-stranded DNA-binding fluorescent dyes, and dNTPs.

[0074] [Enzyme (A)] Enzyme (A) is an enzyme that possesses double-chain specific deoxyribonuclease activity.

[0075] The enzyme (A) is preferably an enzyme that is irreversibly inactivated when left standing at 95°C for 5 minutes while contained in the RT-qPCR reaction solution. In this disclosure, this property is referred to as "non-heat-resistant," and an enzyme having this property is referred to as a "non-heat-resistant enzyme."

[0076] Enzyme (A) can be selected from any DNase. A suitable enzyme can be selected for enzyme (A) based on its double-strand specific deoxyribonuclease activity and heat resistance in the RT-qPCR reaction solution. Enzyme (A) may also be a known DNase modified to possess the above characteristics. An example of enzyme (A) is an enzyme modified to be heat-resistant from an enzyme exhibiting excellent double-strand specific deoxyribonuclease activity. Another example of enzyme (A) is an enzyme modified to be heat-resistant from a double-strand specific deoxyribonuclease of a marine organism (e.g., a crab).

[0077] The enzyme (A) contained in the RT-qPCR reaction solution may be one type or two or more types.

[0078] The concentration of enzyme (A) in the RT-qPCR reaction solution should preferably be the recommended concentration for each type of enzyme. Examples of enzyme (A) concentrations include 0.025 U / μL to 0.175 U / μL.

[0079] [Enzyme (B)] Enzyme (B) is an enzyme that possesses both reverse transcription activity and DNA polymerase activity. Enzyme (B) has both reverse transcription activity and DNA polymerase activity in a single enzyme.

[0080] It is preferable that enzyme (B) is an enzyme that does not undergo irreversible inactivation even when left standing at 95°C for 5 minutes while contained in the RT-qPCR reaction solution. In this disclosure, this property is referred to as "heat resistance," and an enzyme having this property is referred to as a "heat-resistant enzyme."

[0081] Examples of enzyme (B) include DNA polymerase derived from the hyperthermophilic bacterium Thermus thermophilus. Examples of DNA polymerase derived from Thermus thermophilus include Thermus thermophilus DNA polymerase and DNA polymerase modified from said DNA polymerase.

[0082] An example of an embodiment of enzyme (B) is a hot-start PCR enzyme that has been inactivated by a neutralizing antibody.

[0083] The enzyme (B) contained in the RT-qPCR reaction solution may be one type or two or more types.

[0084] The concentration of enzyme (B) in the RT-qPCR reaction solution should preferably be the recommended concentration for each type of enzyme. Examples of enzyme (B) concentrations include 0.05 U / μL to 0.175 U / μL.

[0085] [Mn ２＋ ] Mn of RT-qPCR reaction solution ２＋ The concentration is 3.3 mM to 4.6 mM, preferably 3.6 mM to 4.6 mM, and more preferably 3.9 mM to 4.3 mM, from the viewpoint of ensuring that both enzyme (A) and enzyme (B) function in a balanced manner. (RT-qPCR reaction solution Mn) ２＋ Concentration refers to Mn ２＋ This refers to the final concentration, i.e., Mn at the start of process (1). ２＋ It means concentration.

[0086] Mn ２＋ This can be achieved, for example, by mixing manganese salts such as manganese(II) acetate, manganese(II) chloride, and manganese(II) sulfate, or aqueous solutions of these manganese salts, with enzymes or the like.

[0087] [Sample (Nucleic Acid Sample)] In this disclosure, "sample" means a sample prepared by subjecting an object to be tested to nucleic acid extraction. In this disclosure, such sample is also referred to as a "nucleic acid sample."

[0088] The origin of the nucleic acid sample is not limited. If the application of the RT-qPCR method of this disclosure is for microbiological and / or viral testing, the nucleic acid sample is derived from the subject of the microbiological and / or viral testing. Examples of such subjects include pharmaceuticals, quasi-drugs, cosmetics, active pharmaceutical ingredients, reagents, foods, food materials, health foods, supplements, animal feed, drinking water, tap water, medical gases, medical components, medical devices, sanitary materials, biological samples, blood, cultured cells, etc. Examples of pharmaceuticals include small molecule drugs, medium molecule drugs (e.g., peptide drugs, nucleic acid drugs), biopharmaceuticals (e.g., antibody drugs), cell therapies, gene therapies, etc.

[0089] The method for preparing nucleic acid samples is not limited, and any known nucleic acid extraction and purification methods are applicable. If the sample is a liquid or suspension, a portion of the liquid or suspension is taken and the nucleic acid is extracted. If the sample is a powder, for example, the powder is dissolved or suspended in sterile water and the nucleic acid is extracted. If the sample is a solid, for example, the solid surface is washed with sterile water and the nucleic acid is extracted from this washing solution. If the sample is a gas, for example, the gas is passed through a sterile filter, the filter is washed with sterile water, and the nucleic acid is extracted from this washing solution.

[0090] An example of a nucleic acid sample is nucleic acid extracted from a pharmaceutical product. The RT-qPCR method of this disclosure is suitable for performing microbiological and / or viral testing of pharmaceutical products with short shelf lives after manufacturing (e.g., cell preparations, gene preparations).

[0091] [Primer Set] A primer set is a set of forward primers and reverse primers for PCR. The reverse primer also serves as the reverse transcription primer.

[0092] Primer sets are sequence-specific and designed based on the base sequence of the nucleic acid to be quantified. Any known design technique can be applied to the primer design. Primer sets may be commercially available or newly designed and manufactured.

[0093] When the RT-qPCR method described herein is used for microbiological testing, the target for quantification is RNA present in bacterial and / or fungal cells. Examples of RNAs to be quantified in this case include mRNA (messenger RNA), rRNA (ribosomal RNA), and tRNA (transfer RNA). Sequence-specific primers can be designed based on the gene sequences of these RNAs.

[0094] When the RT-qPCR method described herein is used for viral testing, the target for quantification is genomic RNA present within the viral particle. Sequence-specific primers can be designed based on the genome sequence.

[0095] The RT-qPCR method of this disclosure also includes multiplex RT-qPCR. That is, it is possible to use two or more primer sets together in the RT-qPCR method of this disclosure. For example, a first primer set (forward primer and reverse primer) for amplifying a conserved sequence in bacterial rRNA and a second primer set (forward primer and reverse primer) for amplifying a conserved sequence in fungal rRNA can be used together.

[0096] [Fluorescently labeled probe or double-stranded DNA-binding fluorescent dye] The RT-qPCR method of this disclosure preferably performs real-time quantification by a fluorescence monitoring method. The fluorescence monitoring method may be a probe method using a fluorescently labeled probe or an intercalator method using a double-stranded DNA-binding fluorescent dye. Therefore, the RT-qPCR reaction solution preferably contains a fluorescently labeled probe or a double-stranded DNA-binding fluorescent dye.

[0097] Fluorescent dye-labeled probes are sequence-specific and designed based on the base sequence of the nucleic acid to be quantified. Any known design technique can be applied to the probe design. Fluorescent dye-labeled probes may be commercially available or newly designed and manufactured probes.

[0098] Examples of fluorescent dye-labeled probes include hydrolysis probes that emit fluorescence when hydrolyzed by a DNA extension reaction initiated by a primer; probes that emit fluorescence when their hairpin structure opens upon binding to a complementary strand; and probe sets in which two types of probes emit fluorescence when they bind to a complementary strand.

[0099] When using two or more primer sets, use the respective probes for the amplification products of each primer set together. For example, use the first probe for detecting conserved sequences in bacterial rRNA together with the second probe for detecting conserved sequences in fungal rRNA.

[0100] An example of a double-stranded DNA-binding fluorescent dye is SYBR Green.

[0101] [dNTPs] dNTPs (deoxynucleoside triphosphates) are a mixture of dATP (deoxyadenosine triphosphate), dCTP (deoxycytidine triphosphate), dGTP (deoxyguanosine triphosphate), and dTTP (deoxythymidine triphosphate). Generally, dNTPs are an equimolar mixture of dATP, dCTP, dGTP, and dTTP.

[0102] The concentrations of nucleic acid sample, primer set, fluorescent dye-labeled probe, double-stranded DNA-binding fluorescent dye, and dNTPs in the RT-qPCR reaction solution can be the same as those in a typical RT-qPCR reaction solution.

[0103] [Other components] The solvent and dispersion medium of the RT-qPCR reaction solution is water. The RT-qPCR reaction solution may contain stabilizers for each component, pH adjusters, pH buffers, etc. The pH of the RT-qPCR reaction solution is preferably in the range of 7.4 to 9.1, and more preferably in the range of 8.0 to 8.2.

[0104] The type of pH buffer contained in the RT-qPCR reaction solution is not limited. Examples of pH buffers include Good's buffers. Preferred Good's buffers have an optimal pH range in the alkaline range, such as Tricinene, Bicinene, TAPS, TAPSO, EPPS, HEPPSO, and POPSO. pH adjustment is preferably performed with sodium hydroxide or potassium hydroxide.

[0105] [Step (1)] Step (1) is an incubation step to activate enzyme (A). Step (1) is achieved by maintaining the RT-qPCR reaction mixture at a temperature at which enzyme (A) functions for a certain period of time. For example, the RT-qPCR reaction mixture is left to stand at a temperature of 37°C for 10 to 30 minutes.

[0106] [Step (2)] Step (2) is a heat treatment step that inactivates enzyme (A) but does not inactivate enzyme (B). The temperature and time of the heat treatment in step (2) are set according to the heat resistance of enzyme (A) and enzyme (B). For example, the RT-qPCR reaction solution is left to stand at a temperature of 95°C for 5 minutes.

[0107] [Step (3)] Step (3) consists of reverse transcription, PCR, and real-time quantification.

[0108] RT-PCR is performed by a temperature cycle that activates enzyme (B). For example, if enzyme (B) is a hot-start PCR enzyme, the following steps are performed in order: heat treatment to inactivate the neutralizing antibody and restore enzyme activity, incubation for reverse transcription, heat treatment to denature the cDNA into a single strand, and the PCR cycle. The PCR may be a three-step PCR (denaturation / annealing / extension) or a two-step PCR (denaturation / annealing and extension). The appropriate step is performed for enzyme (B). If the heat treatment to inactivate the neutralizing antibody and restore enzyme activity is achieved in step (2), the heat treatment in step (3) may be omitted.

[0109] Real-time quantification is performed using a fluorescence monitoring method corresponding to the fluorescent dye-labeled probe or double-stranded DNA-binding fluorescent dye contained in the RT-qPCR reaction solution. PCR is repeated while monitoring the fluorescence intensity. When the fluorescence intensity reaches the threshold, it is determined to be positive. When the fluorescence intensity does not reach the threshold, it is determined to be negative. The PCR threshold and the number of cycles are predetermined by preliminary experiments.

[0110] <Premix Reagents for RT-qPCR> Premix reagents for RT-qPCR refer to reagents in which the components necessary for RT-qPCR, excluding the template sample, are pre-mixed. The RT-qPCR premix reagents disclosed herein include forms that contain all the components necessary for RT-qPCR, excluding the template sample, and forms that contain some of the components necessary for RT-qPCR.

[0111] The RT-qPCR premix reagent of this disclosure is a premix reagent for preparing an RT-qPCR reaction solution. The RT-qPCR premix reagent of this disclosure comprises at least an enzyme (A) having double-strand specific deoxyribonuclease activity, an enzyme (B) having reverse transcription activity and DNA polymerase activity, and Mn ２＋ It contains.

[0112] The RT-qPCR premix reagents disclosed herein contain at least enzyme (A), enzyme (B), and Mn ２＋ The present disclosure provides an RT-qPCR method comprising mixing a sample with an RT-qPCR premix reagent containing the above.

[0113] In other words, the above RT-qPCR method involves mixing the RT-qPCR premix reagent of this disclosure with the sample, and adding 3.3 mM to 4.6 mM Mn ２＋ This is an RT-qPCR method in which the following steps (1), (2), and (3) are performed sequentially in the presence of [a specific substance].

[0114] Step (1): Incubation to activate enzyme (A). Step (2): Heat treatment to inactivate enzyme (A) but not enzyme (B). Step (3): Reverse transcription, PCR, and real-time quantification.

[0115] Step (1), Step (2), Step (3), Enzyme (A), Enzyme (B), Mn ２＋ The significance and form of the sample are as previously described in the description of the RT-qPCR method in this disclosure. "3.3 mM to 4.6 mM Mn ２＋ "In the presence of" means the Mn of the RT-qPCR reaction solution. ２＋ This is equivalent to a concentration of 3.3 mM to 4.6 mM.

[0116] The RT-qPCR premix reagents disclosed herein may be reagents with a concentration of, for example, 1 to 10 times, 1.5 to 4 times, or 1.5 to 2 times the concentration of the RT-qPCR reaction solution. An example of an embodiment is a 1.5-times concentration RT-qPCR premix reagent.

[0117] The RT-qPCR premix reagent disclosed herein contains Mn of the RT-qPCR reaction solution. ２＋ To adjust the concentration to 3.3 mM to 4.6 mM, Mn ２＋ The concentration is 3.3 mM or higher. Mn of the RT-qPCR premix reagent disclosed herein. ２＋The concentration is preferably greater than 3.3 mM to 6.9 mM, more preferably between 4.9 mM and 6.9 mM, even more preferably between 5.4 mM and 6.9 mM, even more preferably between 5.8 mM and 6.9 mM, and even more preferably between 6.2 mM and 6.9 mM.

[0118] The RT-qPCR premix reagent disclosed herein contains Mn ２＋ This can be achieved, for example, by mixing manganese salts such as manganese(II) acetate, manganese(II) chloride, and manganese(II) sulfate, or aqueous solutions of these manganese salts, with enzymes or the like.

[0119] The solvent and dispersion medium of the RT-qPCR premix reagents disclosed herein are water.

[0120] The RT-qPCR premix reagents of this disclosure may further contain at least one selected from the group consisting of a primer set, a fluorescently labeled probe, a double-stranded DNA-binding fluorescent dye, and dNTPs. Typical examples of the RT-qPCR premix reagents of this disclosure include enzyme (A), enzyme (B), and Mn ２＋ In addition, it contains a primer set consisting of at least a forward primer and a reverse primer.

[0121] The forms of the primer set, fluorescently labeled probe, double-stranded DNA-binding fluorescent dye, and dNTPs applicable to the RT-qPCR premix reagent of this disclosure are as previously described in the description of the RT-qPCR method of this disclosure, and the preferred forms are also as previously described.

[0122] The RT-qPCR premix reagents disclosed herein may contain stabilizers, pH adjusters, pH buffers, etc. for each component. The pH of the RT-qPCR premix reagents disclosed herein is preferably in the range of 7.4 to 9.1, and more preferably in the range of 8.0 to 8.2.

[0123] The type of pH buffer contained in the RT-qPCR premix reagent of this disclosure is not limited. Examples of pH buffers include Good's buffers. Preferred Good's buffers have an optimal pH range in the alkaline range, and examples include Tricinene, Bicinene, TAPS, TAPSO, EPPS, HEPPSO, and POPSO. pH adjustment is preferably performed with sodium hydroxide or potassium hydroxide.

[0124] The RT-qPCR premix reagents of this disclosure are preferably distributed in a form that preserves enzyme activity, for example, through low-temperature storage.

[0125] <RT-qPCR Reagent Set> The RT-qPCR reagent set of this disclosure is a reagent set for preparing a reaction solution to be used in the RT-qPCR method of this disclosure.

[0126] The RT-qPCR reagent set of this disclosure comprises at least an enzyme (A) having double-strand specific deoxyribonuclease activity, an enzyme (B) having reverse transcription activity and DNA polymerase activity, and a manganese salt solution. Enzymes (A) and (B) may be contained in separate containers or may be mixed and contained in a single container. Enzymes (A) and (B) and the manganese salt solution are contained in separate containers. A set of reagents contained in each respective container constitutes a reagent set.

[0127] [Enzyme (A), Enzyme (B)] The forms of enzyme (A) and enzyme (B) are as previously described in the description of the RT-qPCR method of this disclosure, and the preferred forms are also as previously described.

[0128] Enzymes (A) and (B) are each contained in a container in a form dissolved or dispersed in, for example, 50% (v / v) glycerol / buffer solution.

[0129] [Manganese Salt Solution] Manganese salt solution is made from Mn ２＋ This reagent is for preparing RT-qPCR reaction solutions with concentrations of 3.3 mM to 4.6 mM. The amount of manganese salt solution used is adjusted to control the Mn concentration of the RT-qPCR reaction solution. ２＋ Adjust the concentration to 3.3 mM to 4.6 mM.

[0130] Manganese salt solution is Mn ２＋ The concentration is 3.3 mM or higher, preferably 3.3 mM to 50 mM. Mn of the manganese salt solution ２＋ From the viewpoint of minimizing the volume of the RT-qPCR reaction solution, the concentration is preferably greater than 3.3 mM to 50 mM, more preferably 4.9 mM to 50 mM, even more preferably 5.4 mM to 50 mM, even more preferably 5.8 mM to 50 mM, and even more preferably 6.2 mM to 50 mM.

[0131] A manganese salt solution contains at least one manganese salt in the solvent. Examples of manganese salts include manganese(II) acetate, manganese(II) chloride, and manganese(II) sulfate, with manganese(II) acetate being preferred. The solvent for the manganese salt solution is water.

[0132] The manganese salt solution may contain other components besides manganese salt. Examples of other components include pH adjusters and pH buffers.

[0133] [Other Reagents] The RT-qPCR reagent set of this disclosure may further include at least one reagent selected from the group consisting of a primer set, a fluorescent dye-labeled probe, a double-stranded DNA-binding fluorescent dye, and dNTPs. Each of these reagents is housed in a separate container from the enzyme and manganese salt solution.

[0134] The morphology of the primer set is as previously described in the description of the RT-qPCR method in this disclosure, and the preferred morphology is also as previously described.

[0135] The forward and reverse primers that make up a primer set may be housed in separate containers or mixed together in a single container. The primers may be housed in a container in a form dissolved or dispersed in a buffer solution, for example.

[0136] The form of the fluorescent dye-labeled probe is as previously described in the description of the RT-qPCR method of this disclosure, and the preferred form is also as previously described. The fluorescent dye-labeled probe is, for example, contained in a light-shielding container in a form dissolved or dispersed in a buffer.

[0137] The RT-qPCR reagent set disclosed herein may include all the reagents necessary for RT-qPCR. The RT-qPCR reagent set disclosed herein may also include the instruments used for RT-qPCR.

[0138] The RT-qPCR reagent set of this disclosure is preferably distributed in a form that preserves enzyme activity, for example, through low-temperature storage.

[0139] The RT-qPCR method of this disclosure will be explained below with reference to examples. The following examples illustrate the application of the RT-qPCR method of this disclosure to rapid microbiological testing of pharmaceuticals. The materials, amounts used, concentrations, processing procedures, etc., shown in the following examples can be modified as appropriate, as long as they do not deviate from the spirit of this disclosure. Therefore, the scope of the RT-qPCR method of this disclosure should not be interpreted as being limited by the specific examples shown below.

[0140] <Rapid Microbiological Testing of Cell Therapies> [Preparation of Primer Sets and Probes] Prepare a bacterial-specific primer set, a bacterial-specific fluorescent dye-labeled probe, a fungal-specific primer set, and a fungal-specific fluorescent dye-labeled probe for use in RT-qPCR. The above primer sets and probes may be commercially available products or newly designed and manufactured primer sets and probes. An example of a method for designing primer sets and probes is shown below.

[0141] Based on the rRNA sequence database information for 21 bacterial species shown in Table 11, highly homologous conserved sequences are identified among the 21 bacterial species. A PCR primer set and probes are designed to amplify and detect these conserved sequences. The reverse primers also function as reverse transcription primers. Based on the rRNA sequence database information for 3 fungal species shown in Table 11, highly homologous conserved sequences are identified among the 3 fungal species. A PCR primer set and probes are designed to amplify and detect these conserved sequences. The reverse primers also function as reverse transcription primers.

[0142] The bacteria and fungi listed in Table 11 are microorganisms listed in ISO 24190:2023 "Biotechnology - Analytical methods - Risk-based approach for method selection and validation for rapid microbial detection in bioprocesses".

[0143]

[0144] Bacteria-specific probes and fungal-specific probes are labeled with fluorescent dyes for fluorescence monitoring. Preferably, the fluorescent dyes of the bacteria-specific probe and the fungal-specific probe have the same principle of controlling fluorescence emission. The fluorescent dyes of the bacteria-specific probe and the fungal-specific probe may be the same or different. If the fluorescent dyes are different, bacteria and fungi can be detected separately.

[0145] [Preparation of nucleic acid samples] Cell preparations are autologous cell preparations for cancer treatment, manufactured, for example, by introducing specific genes into T cells collected from the blood of cancer patients.

[0146] A portion of the cell preparation is collected, and the cells are separated by centrifugation, or by filtration using a filter that allows microorganisms to pass through. After separating the cells, ultracentrifugation may be performed to concentrate the microorganisms. The recovered microorganisms are suspended in a buffer solution to obtain a suspension.

[0147] Cell wall-degrading enzymes are added to the suspension to degrade the microbial cell walls. Next, proteolytic enzymes are added to the suspension to degrade the proteins contained in the suspension. Then, nucleic acid extraction is performed using the Boom method. The extracted nucleic acids are washed and concentrated to obtain a nucleic acid sample.

[0148] [Preparation of RT-qPCR premix reagents] Enzyme (A), Enzyme (B), Mn ２＋A premix reagent for RT-qPCR is prepared containing a bacterial-specific primer set, a bacterial-specific fluorescent dye-labeled probe, a fungal-specific primer set, a fungal-specific fluorescent dye-labeled probe, and dNTPs. The Mn of the RT-qPCR premix reagent is also prepared. ２＋ The concentration is, for example, 6.9 mM.

[0149] [RT-qPCR (Multiplex RT-qPCR)] Mix the RT-qPCR premix reagent with the nucleic acid sample, for example, Mn ２＋ Prepare an RT-qPCR reaction solution with a concentration of 4.0 mM. Use this RT-qPCR reaction solution to perform RT-qPCR, for example, in the following cycle.

[0150] <01> 37°C / 10 min <02> 95°C / 5 min <1> 90°C / 30 sec <2> 60°C / 5 min <3> 95°C / 1 min <4> 95°C / 10 sec <5> 60°C / 15 sec...Fluorescence detection

[0151] <01> is step (1), <02> is step (2), and <1> to <5> are steps (3). <1> is a heat treatment performed when enzyme (B) is a hot-start PCR enzyme to inactivate the neutralizing antibody and restore enzyme activity. However, since the neutralizing antibody is inactivated in <02>, <1> can be omitted. <2> is incubation for reverse transcription, <3> is a heat treatment to denature the cDNA into a single strand, and <4>-<5> are two-step PCR.

[0152] Real-time quantification is performed using a fluorescence monitoring method. Steps <4> and <5> are repeated a predetermined number of times while monitoring the fluorescence intensity. When the fluorescence intensity reaches the threshold, it is determined to be positive (i.e., the cell preparation is contaminated with bacteria and / or fungi). When the fluorescence intensity does not reach the threshold, it is determined to be negative (i.e., the cell preparation is not contaminated with bacteria and / or fungi). The PCR threshold and the number of cycles are predetermined by preliminary experiments.

[0153] By automating the preparation of nucleic acid samples and RT-qPCR, it is possible to perform rapid microbial testing of cell preparations in a sterile room without human intervention.

[0154] All documents, patent applications, and technical standards described herein are incorporated by reference to the same extent as if each individual document, patent application, and technical standard were specifically and individually noted to be incorporated by reference.

[0155] The disclosure of Japanese application number 2024-171188, filed on 30 September 2024, is incorporated herein by reference in its entirety.

Claims

1. An enzyme (A) having double-strand specific deoxyribonuclease activity, an enzyme (B) having reverse transcription activity and DNA polymerase activity, and Mn ２＋ It contains Mn ２＋ An RT-qPCR method in which the following (1), (2), and (3) are performed sequentially in an RT-qPCR reaction solution with a concentration of 3.3 mM to 4.6 mM: (1) incubation to activate enzyme (A), (2) heat treatment to inactivate enzyme (A) but not enzyme (B), and (3) reverse transcription, PCR, and real-time quantification.

2. The enzyme (A), the enzyme (B), and Mn ２＋ The RT-qPCR method according to claim 1, comprising mixing a premix reagent for RT-qPCR containing a sample with a premix reagent containing a sample.

3. The RT-qPCR method according to claim 1 or claim 2, wherein the enzyme (B) is a DNA polymerase derived from Thermus thermophilus.

4. An enzyme (A) having double-strand specific deoxyribonuclease activity, an enzyme (B) having reverse transcription activity and DNA polymerase activity, and Mn ２＋ It contains Mn ２＋ A premix reagent for RT-qPCR with a concentration of 3.3 mM or higher.

5. The RT-qPCR premix reagent according to claim 4, further comprising a fluorescent dye-labeled probe or a double-stranded DNA-binding fluorescent dye.

6. The RT-qPCR premix reagent according to claim 4, further comprising: a first primer set for amplifying a conserved sequence in bacterial rRNA; and a second primer set for amplifying a conserved sequence in fungal rRNA.

7. The RT-qPCR premix reagent according to claim 6, further comprising a first probe for detecting a conserved sequence in bacterial rRNA and a second probe for detecting a conserved sequence in fungal rRNA.

8. The RT-qPCR premix reagent according to any one of claims 4 to 7, wherein the enzyme (B) is a DNA polymerase derived from Thermus thermophilus.

9. An enzyme (A) having double-strand specific deoxyribonuclease activity, an enzyme (B) having reverse transcription activity and DNA polymerase activity, and Mn ２＋ A reagent set for RT-qPCR, containing a manganese salt solution with a concentration of 3.3 mM or higher.

10. The RT-qPCR reagent set according to claim 9, wherein the enzyme (B) is a DNA polymerase derived from Thermus thermophilus.

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