CN106048014B - High-specificity probe for detecting NASBA product in real time - Google Patents

High-specificity probe for detecting NASBA product in real time Download PDF

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CN106048014B
CN106048014B CN201610397821.2A CN201610397821A CN106048014B CN 106048014 B CN106048014 B CN 106048014B CN 201610397821 A CN201610397821 A CN 201610397821A CN 106048014 B CN106048014 B CN 106048014B
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CN106048014A (en
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张岩
盖伟
邢婉丽
单万水
刘厚明
宋翠丹
程京
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CapitalBio Corp
Third Peoples Hospital of Shenzhen
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    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6865—Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]

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Abstract

The invention belongs to the technical field of nucleic acid amplification, and relates to a high-specificity probe for detecting NASBA products in real time and a method for detecting the NASBA products. The high-specificity probe consists of two adjacent nucleotide sequences, wherein the 1 st to 4 th bases at the 3' end of the two adjacent nucleotide sequences are ribonucleotide bases, and the rest bases are deoxyribonucleotide bases. The high-specificity probe is applied to the detection of the NASBA amplification product, has high sensitivity and strong specificity, shortens the detection time, and ensures that the NASBA detection technology can be better applied to the nucleic acid detection of pathogenic microorganisms.

Description

High-specificity probe for detecting NASBA product in real time
Technical Field
The invention belongs to the technical field of nucleic acid amplification, and relates to a high-specificity probe for detecting NASBA products in real time.
Background
In recent years, with the rapid development of molecular biology techniques, diagnostic methods based on nucleic acid detection have been largely established and widely applied to molecular diagnostic reagents. The isothermal amplification technology is a novel in vitro nucleic acid amplification technology developed after the PCR technology. The current constant temperature amplification technology mainly comprises the following steps: rolling circle nucleic acid amplification, loop-mediated isothermal amplification, strand displacement amplification, nucleic acid sequence dependent amplification, and melting enzyme amplification, among others. The isothermal amplification has the advantages of rapidness, high efficiency, good specificity and the like, and does not need special equipment.
The NASBA is a process of amplifying single-stranded RNA in an isothermal manner, which is specific in vitro and continuous and uniform, mediated by a pair of primers with a T7 promoter Sequence, under the isothermal condition of 41 ℃, the NASBA can amplify the template RNA by about 10 9 to 10 12 times in 1h under the reaction speed, and the common PCR reaction needs 2 to 3h, under the detection sensitivity, the NASBA can detect trace targets lower than 10gene copies/mu L in a solution, the detection limit of the PCR is about 100gene copies/mu L, therefore, the NASBA technology has higher Amplification efficiency and detection sensitivity than the RT-PCR technology, and only needs a 41 ℃ high condition in the reaction field, a water bath can meet the requirement of the isothermal reaction, does not need complex temperature-raising and lowering technologies, and the like, and has the advantages of being widely applicable to the NASBA detection technology, simple and convenient operation, low cost, simple and convenient detection technology of tumor diseases, and the like.
Currently, the detection of NASBA amplification products mainly comprises direct detection, SBA electrochemiluminescence technology, oligonucleotide detection and SBA molecular beacon detection. The direct detection is that the detection is carried out by adopting an electrophoresis gel mode, and belongs to a primary detection reaction product; the SBA electrochemiluminescence technology has high sensitivity but needs long time. The detection sensitivity of the SBA molecular beacon is slightly lower than that of the SBA electrochemical luminescence technology, but the required time is short. The most commonly used method for the on-line detection of the NASBA amplification product is a molecular beacon method, but the NASBA amplification reaction is jointly acted by three enzymes, the concentration of the three enzymes is relatively high, the attack on nucleic acid in a system is strong, and the molecular beacon in the system is easy to cause nonspecific reaction along with the extension of reaction time, so that false positive signals are generated, the result misjudgment is caused, and certain difficulty is brought to subsequent diagnosis.
Disclosure of Invention
In view of the above, the present invention provides a highly specific probe for real-time detection of NASBA product, which employs adjacent probes instead of molecular beacons, thereby effectively avoiding the above problems and improving the specificity of on-line detection of NASBA amplification product.
In order to achieve the purpose of the invention, the invention adopts the following technical scheme.
A highly specific probe which is composed of two adjacent nucleotide sequences, wherein the 1 st to 4 th bases at the 3' ends of the two adjacent nucleotide sequences are both ribonucleotide bases, and the rest bases are both deoxyribonucleotide bases.
The high-specificity Probe for NASBA product detection provided by the invention consists of two adjacent nucleotide sequences which are respectively named as a Probe Probe F and a Probe Probe R, wherein the two adjacent nucleotide sequences consist of ribonucleotides and deoxyribonucleotides, the 1 st to 4 th bases at the 3 'end of the nucleotide sequence are ribonucleotide bases, and the 5' end of the nucleotide sequence is deoxyribonucleotide bases.
The chain length of two adjacent nucleotide sequences in the high specificity probe is 8-18 bases. In some embodiments, the two contiguous nucleotide sequences in the high specificity probe have a chain length of 10-13 bases.
The two adjacent nucleotide sequences in the high specificity probe are combined with the NASBA amplification product through base complementary pairing. The Probe Probe-F binds to the upstream of the single-stranded RNA product, and the Probe Probe-R binds to the downstream of the single-stranded RNA product.
The distance between two adjacent nucleotide sequences in the high specificity probe of the invention and the two strands after the combination of the NASBA amplification product is 0-8 bases.
The 3 'end of the upstream nucleic acid sequence Probe-F in the two adjacent nucleotide sequences in the high specificity Probe is marked with a fluorescence reporter group, and the 5' end of the downstream nucleic acid sequence is marked with a fluorescence quencher group Probe-R. In the absence of the NASBA amplification product, the two adjacent nucleotide sequences are free in the system, emit fluorescence, and a strong fluorescent signal can be detected. When NASBA amplification products exist, after the two adjacent nucleotide sequences are combined with the NASBA amplification products, the fluorescence reporter group at the 3 'end of the Probe-F is very close to the fluorescence quenching group at the 5' end of the Probe-R, and fluorescence emitted by the fluorescence molecule is absorbed by the quenching molecule and dissipated in the form of heat energy, so that the fluorescence signal in the system is reduced, and real-time detection shows that the data is an inverted S-shaped curve.
Wherein, the fluorescence reporter group marked at the 3' end of the Probe-F can be FAM, TET, HEX, JOE, CY3, CY5, ROX, Texas Red and the like.
The 5' end of the Probe-R is marked with a fluorescence quenching group which can be TAMRA, BHQ1, BHQ2, CY5 and the like.
the high-specificity probe still has good specificity after being amplified for 6 hours in a negative control test, and has no non-specific amplification, which indicates that the high-specificity probe has strong specificity in the real-time detection of NASBA products. Therefore, the invention also provides the application of the high-specificity probe in the real-time online detection of the NASBA amplification product.
The invention also provides a real-time online detection method of the NASBA amplification product, which comprises the steps of mixing the constant-temperature amplification buffer solution containing the high-specificity probe, the constant-temperature amplification enzyme solution and the RNA template into a reaction system, placing the reaction system in a real-time fluorescence quantitative PCR instrument, and reacting for 1 hour at 41 ℃.
Wherein the concentration of the high-specific probe in the isothermal amplification buffer is preferably 0.01-1 mu M. In some embodiments, the high specific probe concentration is 0.1 μ M.
Preferably, the volume ratio of the isothermal amplification buffer solution to the isothermal amplification enzyme solution to the RNA template in the reaction system is 7:1: 2.
In some embodiments, the isothermal amplification buffer has the following composition: the solvent of the constant temperature amplification buffer solution is water, and the solutes and the concentrations are as follows: 200mM Tris-HCl (pH8.0), 0.5. mu.M forward primer, 0.5. mu.M reverse primer, 0.1. mu.M forward Probe Probe-F, 0.1. mu.M reverse Probe Probe-R, 50mM DTT, 10mM dNTP, 10mM rNTP, 80mM MgCl2, 450mM KCl, 15% volume percent DMSO, 1M sorbitol, 20mM tetramethylammonium chloride.
In some embodiments, the solvent of the isothermal amplification enzyme solution is water, and the solutes and concentrations are as follows: AMV reverse transcriptase 1U/. mu.L, T7RNA polymerase 5U/. mu.L, ribonuclease H0.5U/. mu.L, pyrophosphatase 0.5U/. mu.L, RNase inhibitor 5U/. mu.L, BSA 0.5. mu.g/. mu.L.
According to the technical scheme, the invention provides the high-specificity probe for detecting the NASBA product in real time and the method for detecting the NASBA product. The high-specificity probe consists of two adjacent nucleotide sequences, wherein the 1 st to 4 th bases at the 3' end of the two adjacent nucleotide sequences are ribonucleotide bases, and the rest bases are deoxyribonucleotide bases. The high-specificity probe is applied to the detection of the NASBA amplification product, has high sensitivity and strong specificity, shortens the detection time, and ensures that the NASBA detection technology can be better applied to the nucleic acid detection of pathogenic microorganisms.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 illustrates example 2 adjacent probes detecting NSABA amplification products; wherein, a is a detection amplification curve of universal detection primers InfA-TY-PrimerF and InfA-TY-PrimerR for influenza A and probes InfA-TY-PrimerF and InfA-TY-PrimerR, a line 1 is a detection result of a negative control group, and a line 2 is a detection result of amplified influenza A MP gene RNA; FIG. b shows the detection amplification curves of influenza A subtype H1 primers InfA-H1-PrimerF, InfA-H1-PrimerR and probes InfA-H1-ProbeF, InfA-H1-ProbeR, line 1 shows the detection results of a negative control group, and line 2 shows the detection results of amplified influenza A subtype H1 HA gene RNA; FIG. c is an influenza A H3 subtype primer InfA-H3-PrimerF, InfA-H3-PrimerR, and probe InfA-H3-ProbeF, InfA-H3-ProbeR detection amplification curve, line 1 is a negative control group detection result, and line 2 is a detection result of amplifying influenza A H3 subtype HA gene RNA; FIG. d is a graph showing the detection amplification curves for the universal influenza B detection primers InfB-TY-PrimerF and InfB-TY-PrimerR and the probes InfB-TY-PrimerF and InfB-TY-PrimerR, line 1 is the detection result of the negative control group, and line 2 is the detection result of the amplified influenza B MP gene RNA;
FIG. 2 shows the comparison of amplification of adjacent probes at different concentrations in example 3; wherein line 1 is the amplification detection result when the probe concentration is 1. mu.M, line 2 is the amplification detection result when the probe concentration is 0.1. mu.M, and line 3 is the detection result when the probe concentration is 0.01. mu.M;
FIG. 3 illustrates example 3 proximity probe specific analysis; wherein, line 1 is the detection result of detecting influenza A MP gene RNA amplification for 2h by adopting an adjacent probe, line 2 is the detection result of negative control amplification for two hours, and line 3 is the detection result of negative 2h by adopting a common molecular beacon.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
For a further understanding of the invention, reference will now be made in detail to the following examples. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
EXAMPLE 1 Adjacent probes for detection of NASBA amplification products of influenza Virus
The invention designs four sets of primers and adjacent probes for detecting NASBA amplification products of influenza viruses, wherein the primers for detecting genes of influenza A viruses are InfA-TY-PrimerF and InfA-TY-PrimerR, the corresponding adjacent probes are InfA-TY-ProbeF and InfA-TY-ProbeR, the primers for detecting genes of H1 subtype of influenza A viruses are InfA-H1-PrimerF and InfA-H1-PrimerR, the corresponding adjacent probes are InfA-H1-ProbeF and InfA-H1-ProbeR, the primers for detecting genes of H3 subtype of influenza A viruses are InfA-H3-PrimerF and InfA-H3-PrimerR, the corresponding adjacent probes are InfA-H3-ProbeF and InfA-H3-ProbeR, and the primers for detecting genes of influenza B viruses are InfA-TY-PrimerB and InfA-H3-PrimerR, the corresponding adjacent probes are InfB-TY-ProbeF, InfB-TY-ProbeR. The NASBA primers used for each virus detection are shown in Table 1, and the adjacent probe sequences are shown in Table 2.
TABLE 1 NSABA primer sequences for influenza virus specific detection
TABLE 2 NSABA Adjacent Probe sequences for influenza Virus-specific detection
Index (I) Probe-F(5’-3’) Probe-R(5’-3’)
Influenza A virus CCTTGTGCUAUG GGTGTTCAUCAC
Influenza a virus subtype H1 GCCAGTTGUAUG ACTCATATACAAC
Influenza a virus subtype H3 CTCAACAGGUG TATGCGACAGUCC
Influenza B virus TGCTCGAACCAUU GATTCTUACA
The primers and the probes can be used for specifically detecting influenza A virus, influenza A virus H1 subtype, influenza A virus H3 subtype and influenza B virus.
Example 2 use of proximity probes for NASBA product detection
First, preparation of RNA template
Plasmids for preparing the RNA template are provided by Boo biological group, Inc., and comprise a recombinant plasmid pUC-InfA-TY containing an influenza A virus gene, a recombinant plasmid pUC-InfA-H1 containing an influenza A virus H1 subtype HA gene, a recombinant plasmid pUC-InfA-H3 containing an influenza A virus H3 subtype HA gene, and a recombinant plasmid pUC-InfB-TY containing an influenza B virus gene. The RNA template was prepared as follows.
1. Enzyme digestion: each recombinant plasmid was digested with EcoRI endonuclease at 37 ℃ for 2 h.
2. Transcription: a reaction system with a total volume of 50. mu.L was prepared from 5. mu.L of 5 × Transcription Optimized Buffer (Promega), 2U T7RNA polymerase, 10mM DTT (Promega), 10U recombinant RNase inhibitor (Promega), 2mM rNTP, and 5. mu.L of the enzyme-cleaved product, and transcribed at 37 ℃ for 4 hours after shaking uniformly.
3. Digestion: after completion of transcription, 1. mu.L of DNA digesting enzyme (rDNA II, 5U/. mu.L) was added to the system, centrifuged with shaking, and incubated at 37 ℃ for 20 min.
4. And (3) purification: the RNA transcript was purified using the RNA purification kit product from Macherey-Nagel, catalog No. 740948, as follows:
a) Preparing RA1-C 2 H 5 OH mixed solution, namely RA1: C 2 H 5 OH, in a ratio of 1:1 (volume ratio), adding 600 mu L of RA1-C 2 H 5 OH mixed solution, namely 300 mu L of RA1+300 mu L C 2 H 5 OH solution, into 100 mu L of transcription product, wherein the volume of the mixed solution is calculated according to the number of tubes of the transcription product, and supplementing the product to 100 mu L by using water if the product is less than 100 mu L (namely adding 50 mu L of Tiangen RNase-free H 2 O into each tube of product).
b) The previously prepared RA1-C 2 H 5 OH mixed solution is divided into two tubes of 600. mu.L each, and 100. mu.L of the product is transferred into the corresponding centrifugal tube of 700. mu.L in total, and the tubes are fully shaken and centrifuged.
c) Two adsorption columns are prepared and marked correspondingly, 700 mu L of product mixed liquor is transferred into the adsorption columns (the maximum capacity of the adsorption columns is 700 mu L), the adsorption columns are centrifuged at 1.2 ten thousand rpm for 2min, and the lower layer solution is poured out.
d) Adding 700 μ L of RA3 into adsorption column, standing for 1min to make it fully disperse at the bottom of the adsorption column, centrifuging at 1.2 ten thousand rpm for 2min, and pouring off the lower solution.
e) mu.L of RA3 was added to the adsorption column, and the column was left for 2min, centrifuged at 1.2 ten thousand rpm for 2min, and the lower layer solution was decanted off.
f) mu.L of RA3 was added to the adsorption column, and the column was left for 2min, centrifuged at 1.2 ten thousand rpm for 2min, and the lower layer solution was decanted off.
g) After opening the lid of the adsorption column for 3min, the column was left idle at 1.2 ten thousand rpm for 2min, the lower layer solution was decanted, and the procedure was repeated once.
h) After the completion of the air separation, transferring the adsorption column into a corresponding centrifuge tube, opening the cover of the adsorption column, standing for 15min to completely volatilize ethanol, adding 60 μ L of RNase-H 2 O (carried in the kit) into the adsorption column, eluting, standing for 2min, and centrifuging at 1.2 ten thousand rpm for 2 min.
i) The template obtained by centrifugation was sucked back into the adsorption column, left to stand for 2min, centrifuged at 1.2 ten thousand rpm for 2min, and the procedure was repeated twice.
j) Discard the adsorption column, take out 2 μ L of template in the centrifuge tube, measure its concentration with Nano drop, and record its concentration and 260/280, 260/230 ratio, dilute the RNA template concentration to 10 10 copies/μ L according to the measured concentration.
Two, adjacent probe for detection of NASBA amplification product
1. the RNA template prepared above was diluted to 10 3 copies/. mu.L and used as a template for NASBA amplification.
2. The NSABA amplification system consists of constant-temperature amplification buffer solution and constant-temperature amplification enzyme solution, wherein the solvent of the constant-temperature amplification buffer solution is water, and the solute and the concentration are 200mM Tris-HCL (pH8.0), 0.5 mu M upstream primer, 0.5 mu M downstream primer, 0.1 mu M upstream probe, 0.1 mu M downstream probe, 50mM DTT, 10mM dNTP, 10mM rNTP, 80mM MgCl 2, 450mM KCl, 15% volume percentage DMSO, 1M sorbitol and 20mM tetramethylammonium chloride.
The solvent of the constant temperature amplification enzyme solution is water, and the solutes and the concentrations are as follows: AMV reverse transcriptase 1U/. mu.L, T7RNA polymerase 5U/. mu.L, ribonuclease H0.5U/. mu.L, pyrophosphatase 0.5U/. mu.L, RNase inhibitor 5U/. mu.L, BSA 0.5. mu.g/. mu.L. Preparing corresponding enzyme solution according to the concentration.
3. Preparation of amplification System
Mixing 7 μ L of constant temperature amplification buffer (see step 2), 1 μ L of constant temperature amplification enzyme solution (see step 2), and 2 μ L of corresponding template diluent to obtain 10 μ L of reaction solution, and placing in a PCR tube, and performing vortex oscillation uniformly. And (3) placing the PCR tube in a 7500 real-time fluorescent quantitative PCR instrument, setting the temperature to 41 ℃ for reaction for 1 hour, and simultaneously completing real-time online detection.
4. Interpretation of results
As shown in FIG. 1, the amplification curve is an inverted S-shaped curve when the system is amplified, and a straight line when the system is not amplified. From the results, it can be seen that NSABA amplification has good specificity.
Example 3 Adjacent Probe characterization
Concentration of adjacent probes
1. Preparation of isothermal amplification system with different adjacent probe concentrations
Isothermal amplification buffer B1 200mM Tris-HCl (pH8.0), 0.5. mu.M InfA-TY-PrimerF, 0.5. mu.M InfA-TY-PrimerR, 0.01. mu.M InfA-TY-ProbeF, 0.01. mu.M InfA-TY-ProbeR, 50mM DTT, 10mM dNTP, 10mM rNTP, 80mM MgCl 2, 450mM KCl, 15% volume percent DMSO, 1M sorbitol, 20mM tetramethylammonium chloride.
Isothermal amplification buffer B2 200mM Tris-HCl (pH8.0), 0.5. mu.M InfA-TY-PrimerF, 0.5. mu.M InfA-TY-PrimerR, 0.1. mu.M InfA-TY-ProbeF, 0.1. mu.M InfA-TY-ProbeR, 50mM DTT, 10mM dNTP, 10mM rNTP, 80mM MgCl 2, 450mM KCl, 15% volume percent DMSO, 1M sorbitol, 20mM tetramethylammonium chloride.
Isothermal amplification buffer B3 200mM Tris-HCl (pH8.0), 0.5. mu.M InfA-TY-PrimerF, 0.5. mu.M InfA-TY-PrimerR, 1. mu.M InfA-TY-ProbeF, 1. mu.M InfA-TY-ProbeR, 50mM DTT, 10mM dNTP, 10mM rNTP, 80mM MgCl 2, 450mM KCl, 15% volume percent DMSO, 1M sorbitol, 20mM tetramethylammonium chloride.
The enzyme solution for isothermal amplification was prepared in the same manner as in example 2.
Three NSABA amplification systems with different adjacent probe concentrations are prepared according to the preparation method of the amplification system in the embodiment 2, a PCR tube of the prepared system is placed in a 7500 real-time fluorescent quantitative PCR instrument, the reaction is carried out for 1 hour at 41 ℃, and the real-time online detection is simultaneously completed.
2. Analysis of results
As shown in FIG. 2, the results showed that the initial fluorescence value increased with the increase of the probe concentration, and the rate of change of fluorescence also increased to some extent, but the change was not significant. In order to obtain better experimental data, it is recommended to set the concentration of the adjacent probe to 0.1. mu.M, which can well react with the change of fluorescence during amplification, and the fluorescence value is moderate, and the fluorescence value is not beyond the detection range of the detector because the fluorescence value is too low or too high.
Two, adjacent probe specific analysis
1. Preparation of constant temperature amplification buffer solution
The preparation method of the isothermal amplification buffer adjacent to the probe is the same as the isothermal amplification buffer B2 in step one.
A conventional molecular beacon isothermal amplification system B4 was prepared by 200mM Tris-HCl (pH8.0), 0.5. mu.M InfA-TY-PrimerF, 0.5. mu.M InfA-TY-PrimerR, 0.1. mu.M InfA-TY-Pro, 50mM DTT, 10mM dNTP, 10mM rNTP, 80mM MgCl 2, 450mM KCl, 15% volume percent DMSO, 1M sorbitol, 20mM tetramethylammonium chloride.
The isothermal amplification system was prepared according to the method for preparing the amplification system described in example 2. And (3) placing the prepared system in a 7500 real-time fluorescence quantitative PCR instrument, setting the temperature to 41 ℃ for reaction for 1 hour, and simultaneously completing real-time online detection.
2. analysis of results
The results are shown in fig. 3, and show that the adjacent probe provided by the invention has better specificity of NSABA amplification. With the extension of the amplification time, false positive amplification signals can not be generated as long as no specific template exists in the system, while with the extension of the amplification time, weak false positive signals can be generated even if no specific template exists in the system by adopting the common molecular beacon.

Claims (6)

1. A high specificity probe is characterized by consisting of two adjacent nucleotide sequences, wherein the 1 st to 4 th bases at the 3' ends of the two adjacent nucleotide sequences are both ribonucleotide bases, and the rest bases are both deoxyribonucleotide bases; the two contiguous nucleotide sequences base-complementarily pair with the NASBA amplification product; the 3 'end of the upstream nucleic acid sequence in two adjacent nucleic acid sequences is marked with a fluorescent reporter group, and the 5' end of the downstream nucleic acid sequence is marked with a fluorescent quencher group; the two contiguous nucleotide sequences are each between 8-18 bases long; after the two adjacent nucleotide sequences are bound to the NSABA amplification product by base complementary pairing, the distance between the two strands is 0-8 bases.
2. The highly specific probe according to claim 1, wherein the fluorescent reporter group is FAM, TET, HEX, JOE, CY3, CY5, ROX or Texas Red; the fluorescence quenching group is TAMRA, BHQ1, BHQ2 or CY 5.
3. The use of the highly specific probe according to claim 1 or 2 for real-time on-line detection of NASBA amplification products.
4. A real-time on-line detection method of NASBA amplification product is characterized in that a constant temperature amplification buffer solution containing the high specificity probe of claim 1 or 2, a constant temperature amplification enzyme solution and an RNA template are mixed into a reaction system, and the reaction system is placed in a real-time fluorescence quantitative PCR instrument and reacts for 1 hour at 41 ℃; the concentration of the high-specificity probe is 0.01-1 mu M.
5. The real-time online detection method according to claim 4, wherein the volume ratio of the isothermal amplification buffer solution to the isothermal amplification enzyme solution to the RNA template in the reaction system is 7:1: 2.
6. The real-time on-line detection method of claim 4, wherein the isothermal amplification buffer comprises water as a solvent, 200mM pH8.0 Tris-HCl as a solute, 0.5. mu.M forward primer, 0.5. mu.M backward primer, 0.1. mu.M forward probe, 0.1. mu.M backward probe, 50mM DTT, 10mM dNTP, 10mM rNTP, 80mM MgCl 2, 450mM KCl, 15% volume percent DMSO, 1M sorbitol, and 20mM tetramethylammonium chloride, and water as a solvent, 1U/. mu.L as a solute, 5U/. mu.L as a T7RNA polymerase, 0.5U/. mu.L as a ribonuclease H, 0.5U/. mu.L as a pyrophosphatase, 5U/. mu.L as an RNase inhibitor, and 0.5. mu.g/. mu.L as BSA.
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