CN112029901A - Reagent for improving specificity of nucleic acid amplification reaction, nucleic acid amplification reaction solution and kit - Google Patents

Reagent for improving specificity of nucleic acid amplification reaction, nucleic acid amplification reaction solution and kit Download PDF

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CN112029901A
CN112029901A CN202010703846.7A CN202010703846A CN112029901A CN 112029901 A CN112029901 A CN 112029901A CN 202010703846 A CN202010703846 A CN 202010703846A CN 112029901 A CN112029901 A CN 112029901A
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王云龙
李玉林
王国强
王继创
王敏
程蕾
孙新城
王运从
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HENAN BIOENGINEERING RESEARCH CENTER
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Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to a reagent for improving specificity of a nucleic acid amplification reaction, a nucleic acid amplification reaction solution and a kit. The reagent for improving the specificity of the nucleic acid amplification reaction comprises glycine and trehalose, wherein the final concentration of the glycine is 1% -10%, the final concentration of the trehalose is 2% -10%, and the final concentration of the trehalose and the final concentration of the glycine are not equal to 10% at the same time. The glycine and the trehalose in the reagent for improving the specificity of the nucleic acid amplification reaction can play a role in synergistically improving the specificity of the nucleic acid amplification reaction. Compared with glycerol with high concentration (50%) commonly used in the field, the method can effectively avoid non-specific amplification under the condition of equivalent sensitivity.

Description

Reagent for improving specificity of nucleic acid amplification reaction, nucleic acid amplification reaction solution and kit
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a reagent for improving specificity of a nucleic acid amplification reaction, a nucleic acid amplification reaction solution and a kit.
Background
Nucleic acid amplification reactions are commonly used detection methods for the detection of pathogenic microorganisms such as viruses, bacteria, and the like. A plurality of enzymes are needed in the nucleic acid amplification reaction process, and the activity and stability of the enzymes can influence the nucleic acid amplification result, thereby influencing the detection result. Conventional enzyme-containing amplification buffers generally improve the activity and stability of the enzyme through the use of glycerol, but high concentrations of glycerol increase non-specific amplification of the enzyme, causing a bias in the detection results.
Coronaviruses are a large group of viruses belonging to the single-stranded plus-strand RNA virus, the order Nidovirales (Nidovirales) Coronaviridae (Coronaviridae) the orthocoronaviridae (Orthocoronavirinae), and are classified into the four genera α, β, γ, and. There are 6 types of coronavirus known to infect humans: 229E, NL63 from genus alpha; beta genus OC43, HKU1, MERS-CoV, SARS-CoV. The novel coronavirus belongs to a novel coronavirus of beta genus, and has envelope, round or elliptical particle, usually polymorphism, and diameter of 60-140 nm. The gene characteristics of the mutant are obviously different from those of SARSr-CoV and MERSR-CoV. The virus classification Committee was named SARS-CoV-2 in year 2020, 2, month 11. Meanwhile, the world health organization issued on the same day "clinical treatment guidelines for severe acute respiratory infections caused by suspected novel coronavirus infections". Guideline section 4 (collection of clinical specimens for laboratory diagnosis): it is clear that detection of nCoV nucleic acids by RT-PCR and periodic sampling of hospitalized patients diagnosed with nCoV infection until two consecutive negative results (both negative if URT and LRT samples are collected at the same time) are obtained, which can be regarded as clinical recovery.
In the diagnosis of COVID-19, the fluorescent quantitative PCR detection technique is determined as the gold standard. The fluorescent quantitative PCR technology gradually replaces the traditional PCR technology with the advantages of high sensitivity, high speed, quantifiability, pollution resistance, strong specificity and the like. The developed fluorescent probe makes this technique highly specific, and it utilizes the 3 '→ 5' exonuclease activity of Taq enzyme to cleave the probe and generate a fluorescent signal. Since the probe is specifically bound to the template, when the probe is intact, the fluorescent energy emitted by the reporter is absorbed by the quencher, and no signal is detected. As the PCR proceeds, Taq enzyme encounters the probe bound to the template during the chain extension process, the 3 '→ 5' exonuclease activity thereof cleaves the probe, and the reporter group is far away from the quencher group, thereby generating a fluorescent signal. Therefore, the fluorescent signal has a synchronous exponential growth process as the target fragment every PCR cycle. The intensity of the signal represents the copy number of the template DNA. The fluorescent quantitative technology is designed based on the principle and provides good technical support for the diagnosis, large-scale screening, dynamic analysis of curative effect, new drug development and the like of SARS-CoV-2 infection.
Disclosure of Invention
The glycine and the trehalose in the reagent for improving the specificity of the nucleic acid amplification reaction can play a role in synergistically improving the specificity of the nucleic acid amplification reaction. Compared with glycerol with high concentration (50%) commonly used in the field, the method can effectively avoid non-specific amplification under the condition of equivalent sensitivity.
The second object of the present invention is to provide a nucleic acid amplification reaction solution.
The invention also aims to provide a kit.
The reagent for improving the specificity of the nucleic acid amplification reaction adopts the following technical scheme: a reagent for improving specificity and sensitivity of a nucleic acid amplification reaction, which comprises glycine and trehalose, wherein the final concentration of the glycine is 1-10% (mass percent), and the final concentration of the trehalose is 2-10% (mass percent); and the final concentration of trehalose and the final concentration of glycine cannot be equal to 10% at the same time. Compared with glycerol with high concentration (50%) commonly used in the field, the reagent for improving the specificity of the nucleic acid amplification reaction can effectively avoid non-specific amplification under the condition of equivalent sensitivity.
As a further preferable technical scheme, the final concentration of glycine in the reagent is 1% -10%, and the final concentration of trehalose is 2% -5%; and when the final concentration of glycine is 10%, the final concentration of trehalose is 2%. The reagent can not only improve the specificity of nucleic acid amplification reaction, but also make the sensitivity better.
As a further preferable technical scheme, the final concentration of the glycine is 1% -5%, and the final concentration of the trehalose is 2% -5%. The reagent can not only improve the specificity of nucleic acid amplification reaction, but also make the sensitivity better.
As a further preferred technical scheme, the reagent comprises glycine with a final concentration of 2% -5% and trehalose with a final concentration of 5%. The reagent can not only improve the specificity of nucleic acid amplification reaction, but also optimize the sensitivity.
The nucleic acid amplification reaction solution adopts the following technical scheme: a nucleic acid amplification reaction solution comprising a reagent as described in any of the above and any one or a combination of UNG enzyme, reverse transcriptase and hot start Taq enzyme.
As a further preferred technical scheme, the nucleic acid amplification reaction solution also comprises at least one of Tris, KCl, MgCl2, DTT, EDTA, Tween-20 and RNase inhibitor; preferably, the final concentration of Tris is 8.0 and 10mM, the final concentration of KCl is 40mM, the final concentration of MgCl2 is 5mM, the final concentration of DTT is 1mM, the final concentration of EDTA is 0.5mM, the final concentration of Tween-20 is 0.5%, the final concentration of RNase inhibitor is 10U/. mu.L, the final concentration of UNG enzyme is 1U/. mu.L, the final concentration of reverse transcriptase is 100U/. mu.L, and the final concentration of hot start Taq enzyme is 2U/. mu.L.
The kit adopts the following technical scheme: a kit comprising, as components, the reagent according to any one of the above and/or the nucleic acid amplification reaction solution according to any one of the above.
As a further preferable technical scheme, the kit further comprises a primer group for detecting the novel coronavirus, wherein the primer group comprises a first primer pair and a first probe for detecting ORF1a/b gene, a second primer pair and a second probe for detecting N gene, a third primer pair and a third probe for detecting E gene and/or a fourth primer pair and a fourth probe for detecting human beta-Globin gene; the nucleotide sequence of the F primer of the first primer pair is shown as SEQ ID No.1, the nucleotide sequence of the R primer of the first primer pair is shown as SEQ ID No.2, and the nucleotide sequence of the first probe is shown as SEQ ID No. 3; the nucleotide sequence of the F primer of the second primer pair is shown as SEQ ID No.4, the nucleotide sequence of the R primer of the second primer pair is shown as SEQ ID No.5, and the nucleotide sequence of the second probe is shown as SEQ ID No. 6; the nucleotide sequence of the F primer of the third primer pair is shown as SEQ ID No.7, the nucleotide sequence of the R primer of the third primer pair is shown as SEQ ID No.8, and the nucleotide sequence of the third probe is shown as SEQ ID No. 9; the nucleotide sequence of the F primer of the fourth primer pair is shown as SEQ ID No.10, the nucleotide sequence of the R primer of the fourth primer pair is shown as SEQ ID No.11, and the nucleotide sequence of the fourth probe is shown as SEQ ID No. 12. Wherein, the length of the amplification product of SARS-CoV-2ORF1a/b gene is 480-550 bP, and the length of the amplification product of N gene, E gene and reference gene is 150-300 bP. The primer group is suitable for the fluorescent quantitative PCR detection technology of SARS-CoV-2 virus, and can detect the novel coronavirus quickly, sensitively, accurately and quantitatively. The technology selects three groups of primer probes (cell membrane protein gene, E, open reading frame 1ab, ORF1ab, nucleocapsid protein gene, N) of SARS-CoV-2 coronavirus, realizes the detection of the novel coronavirus through different fluorescent markers and multiple fluorescent composite PCR technology, provides basis for early screening and auxiliary diagnosis of the novel coronavirus, and can monitor the curative effect of clinical treatment.
The detection system of the kit uses three fluorophores marked with different colors to independently detect in different wavelength regions, and realizes the simultaneous detection and resolution of three target sequences of the E gene (FAM), the 2019-nCoV N gene (CY5) and the ORF1ab (ROX) by a single tube. The internal standard uses the human beta-Globin gene (beta-Globin gene), which is labeled HEX/VIC. The UNG-dUTP anti-contamination system can effectively prevent possible contamination of amplification products. The negative and positive quality control products contain internal standard systems, and can monitor the whole process of sample extraction and amplification. The kit is suitable for in vitro qualitative detection of suspected cases of novel coronavirus infection, ORF1ab, E gene and N gene of novel coronavirus SARS-CoV-2 in pharynx swab, sputum, alveolar lavage fluid and other samples of patients suspected of aggregating venereal disease cases and other patients needing novel coronavirus infection diagnosis or differential diagnosis.
As a further preferred technical scheme, the components of the kit also comprise another amplification reaction solution A, and the amplification reaction solution A comprises Tris, KCl, (NH4)2SO4DTT, MgCl2, dATP, dGTP, dUTP, oligdt and the primer set; preferably, the final concentration of Tris pH 8.0 is 10mM, the final concentration of KCl is 40mM, and the (NH4)2SO415mM, 1mM of DTT, 5mM of MgCl2, 1mM of dATP, 1mM of dGTP, 1mM of dUTP, 1mM of dCTP, 0.1. mu.M of oligdt, and 0.1. mu.M of each primer in the primer set.
As a further preferred technical scheme, the components of the kit also comprise a positive quality control substance and/or a negative quality control substance; when the kit is used for nucleic acid amplification reaction, the amplification procedure is 37 ℃ for 1 min; at 42 ℃ for 20 min; 95 ℃ for 5 min; 10s at 95 ℃ and 30s at 58 ℃ for 40 cycles.
Reference interval of the kit of the invention: (1) the experiment should be set with negative quality control material and positive quality control material. The determination results of the negative quality control material and the positive quality control material meet the requirements. If the results of the negative quality control product and the positive quality control product do not meet the requirements, the test results of the whole batch are invalid.
(2) Negative quality control product: the negative quality control product is detected to be negative, and meanwhile, the internal standard Ct is less than or equal to 35, so that the negative quality control product result is effective; if the negative quality control product is detected to be negative and the Ct of the internal standard is more than 35, the detection result of the negative quality control product is invalid.
(3) Positive quality control product: the positive quality control products should be E gene positive, ORF positive and N gene positive, and the internal standard result can be disregarded.
The invention has the beneficial effects that: the glycine and the trehalose in the reagent for improving the specificity of the nucleic acid amplification reaction can play a role in synergistically improving the specificity of the nucleic acid amplification reaction. Compared with glycerol with high concentration (50%) commonly used in the field, the method can effectively avoid non-specific amplification under the condition of equivalent sensitivity.
The trehalose and the glycine in the reagent for improving the specificity of the nucleic acid amplification reaction have a synergistic effect, so that the sensitivity of the nucleic acid amplification reaction can be improved to a certain extent.
The kit of the invention can determine various detection indexes for detecting SARS-CoV-2 virus by detecting samples such as throat swab, sputum, alveolar lavage fluid and the like.
As can be seen from the following experiments, the kit of the present invention can achieve better sensitivity when glycine and trehalose are used in the range of 2-5%, and optimally both glycine and trehalose are 5%.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments, and it should be understood 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.
Example 1
The composition reagent comprises: the nCoV amplification reaction solution A, nCoV is used for amplifying the reaction solution B, a negative quality control product and a positive quality control product sample.
The reagent provided by the invention comprises the following steps:
1. nucleic acid extraction
The nucleic acid extraction kit (magnetic bead method) was purchased from bioscience, Huayi Mei, Suzhou, according to the instructions.
2. Amplification reagent preparation
Taking out the 2019-nCoV amplification reaction liquid A and the 2019-nCoV amplification reaction liquid B, and melting and uniformly mixing at room temperature. And taking reaction liquid A (the number of samples to be detected is plus 2) multiplied by 10 mu l, taking reaction liquid B (the number of samples to be detected is plus 2) multiplied by 10 mu l, adding the reaction liquid A and the reaction liquid B into a 1.5ml centrifuge tube, mixing uniformly, centrifuging, subpackaging the mixture into PCR reaction tubes according to 20 mu l per hole, and transferring the mixture to a sample preparation area for later use after the subpackaging is finished.
2.1 the primer sets used were:
first primer pair and first probe (ORF1 ab):
F：CCCCCTGTGGGTTTTACACTTAA(SEQ ID No.1)
R：ACGATTGTGCATCAGCTGA(SEQ ID No.2)
a first probe: 5 '-ROX-CCGTCTGCGGTATGTGGAAAGGTTATGG-3' BHQ1(SEQ ID No.3)
Second primer set and second probe (E gene):
F：ACAGGTACGTTAATAGTTAATAGCGT(SEQ ID No.4)
R：ATATTGCAGCAGTACGCACACA(SEQ ID No.5)
a second probe: 5 '6-FAM-ACACTAGCCATCCTTACTGCGCTTCG-3' BHQ1(SEQ ID No.6)
Third primer set and third probe (N gene):
F：TGAGTGAGCTGCACTGTGAC(SEQ ID No.7)
R：CCCAAGCTTCAGACATTTTGCTCTCAAGCTG(SEQ ID No.8)
a third probe: 5 '-CY 5-AGCTGCACGTGGATCCTGAGA-3' BHQ1(SEQ ID No.9)
Fourth primer set and fourth Probe (. beta. -Globin Gene)
F：TGAGTGAGCTGCACTGTGAC(SEQ ID No.10)
R：ACATCAAGCGTCCCATAGACT(SEQ ID No.11)
A fourth probe: 5 '-VIC-AGCTGCACGTGGATCCTGAGA-3' BHQ1(SEQ ID No.12)
2.22019-nCOV amplification reaction solution A comprises the following components: tris 10mM pH 8.0, KCl 40mM, (NH4)2SO415mM, DTT 1mM, MgCl 25 mM, dATP 1mM, dGTP 1mM, dUTP 1mM, dCTP 1mM, oligdt 0.1. mu.M, concentration of each primer in the above primer set0.1. mu.M; the concentrations of the above components are final concentrations.
2.32019-nCOV amplification reaction solution B comprises the following components: tris 10mM pH 8.0, KCl 40mM, MgCl 25 mM, DTT 1mM, EDTA 0.5mM, Tween-200.5% (volume%), glycine 5% (mass%), trehalose 5% (mass%), rnase inhibitor 10U/. mu. L, UNG enzyme 1U/. mu.l, reverse transcriptase 100U/. mu.l, hot start Taq enzyme 2U/. mu.l; the concentrations of the above components are final concentrations.
3. Addition of nucleic acids
3.1 taking out the extracted nucleic acid template for later use.
3.2 absorbing the nucleic acid template, adding the nucleic acid template into the PCR reaction tube with the split reaction mixed solution, covering the PCR reaction tube with a cover, uniformly mixing and centrifuging.
PCR amplification
4.1 the PCR reaction tube added with the reaction mixture and the template is placed into a fluorescent PCR instrument.
4.2 cycle parameter settings.
Figure RE-RE-GDA0002741928340000061
Note: fluorescence collection: pathogen nucleic acid: FAM, ROX, CY 5; internal standard: VIC (HEX).
5. Analysis of results
The real-time fluorescence PCR instrument simultaneously collects fluorescence signals in the PCR amplification process according to the set requirements. The multichannel fluorometer performs multi-signal fluorescence acquisition according to a preset setting. The kit is characterized in that an internal standard system is added into an amplification detection system and is used for monitoring the whole process of nucleic acid extraction and amplification detection.
Example 2 the influence of the trehalose and/or glycine concentration in the 2019-nCoV amplification reaction liquid B on the sensitivity and specificity of the nucleic acid amplification reaction was examined.
In the experiment, only the concentration of trehalose and/or glycine was different from that of example 1, and the remaining reagents and reaction conditions were the same.
The experimental results show that when glycine and trehalose are used in the range of 2-5%, better sensitivity can be obtained, and the optimal choice is that glycine and trehalose are both 5%.
The detailed experimental results are shown in table 1 below:
TABLE 1
Figure RE-RE-GDA0002741928340000081
Remarking: in the art, negative amplification generally did not occur before Ct40, so Ct38.7 and Ct37.6 in Table 1 above indicate that non-specific amplification occurred during the experiment.
Example 3
The data of the detection result is processed according to a hygiene statistical method, a standard statistical method is required to be adopted in the statistical analysis process and the result, and the analysis result can indicate the quality of the test kit and the specificity, sensitivity and accuracy in the clinical application process.
The collected clinical specimens were tested according to the instructions of the kit to be verified (the reagents contained therein were the same as in example 1), and the results of the two measurements were analyzed by using a reference reagent (the "novel coronavirus 2019-nCoV nucleic acid detection kit (fluorescent PCR method)" of shanghai berjie medical science and technology ltd).
1) Consistency of detection results of examination reagent and reference reagent
And summarizing the detection results of the assessment reagent and the reference reagent in a 2X 2 table form, and calculating the coincidence rate.
TABLE 1 consistency of assessment reagents with reference reagents
Figure RE-RE-GDA0002741928340000091
The calculation formula is as follows:
positive coincidence rate is a/(a + c) × 100%,
The negative coincidence rate is d/(b + d) x 100%,
the total coincidence rate is (a + d)/T multiplied by 100 percent,
95% confidence interval: lower limit of 100% × (Q1-Q2)/Q3 and upper limit of 100% × (Q1+ Q2)/Q3
Q1＝2(a+d)+1.96*2
Figure RE-RE-GDA0002741928340000092
Q3＝2(n+1.96)
Kappa：Kappa＝(PA-Pe)/(1-Pe)
PA＝(a+d)/(T)，Pe＝[(a+b)(a+c)+(c+d)(b+d)]/T)2
The Kappa coefficient is more than or equal to 0.75, the heights are consistent, and the two systems are considered to be equivalent; the Kappa coefficient is more than or equal to 0.4, which is considered to be consistent, but further related statistical analysis is needed; and if the Kappa coefficient is less than 0.4, the two systems are not consistent and are not equivalent.
2) Consistency analysis of assessment reagents with clinical validation/exclusion results
The assessment reagent and the clinical confirmation/exclusion result are summarized in a 2X 2 table form, and the clinical sensitivity, the clinical specificity and the clinical total coincidence rate are calculated.
TABLE 2 agreement of assessment reagents with clinical reference standards
Figure RE-RE-GDA0002741928340000101
Calculating the formula:
clinical sensitivity ═ a/(a + c) × 100%
Clinical specificity ═ d/(b + d). times.100%
The total clinical coincidence rate is (a + d)/T multiplied by 100%
95% confidence interval: lower limit of 100% × (Q1-Q2)/Q3 and upper limit of 100% × (Q1+ Q2)/Q3
Q1＝2(a+d)+1.96*2
Figure RE-RE-GDA0002741928340000102
Q3＝2(n+1.96)
Kappa：Kappa＝(PA-Pe)/(1-Pe)
PA＝(a+d)/T)，Pe＝[(a+b)(a+c)+(c+d)(b+d)]/(T)2
The Kappa coefficient is more than or equal to 0.75, the heights are consistent, and the two systems are considered to be equivalent; the Kappa coefficient is more than or equal to 0.4, which is considered to be consistent, but further related statistical analysis is needed; and if the Kappa coefficient is less than 0.4, the two systems are not consistent and are not equivalent.
Consistency analysis of results of assessment reagent and reference reagent
In the clinical research, the total number of examination case specimens is 150, and the consistency of the examination reagent results and the reference reagent results is as follows:
TABLE 3 consistency of assessment reagents with reference reagents
Figure RE-RE-GDA0002741928340000103
Sensitivity: the percent of positive agreement (percent) is 62/62X 100 percent and 100 percent
Specificity: negative match rate (%) 88/88 × 100%: 100%
The accuracy is as follows: the total percent (%) of coincidence is 150/150X 100%: 100%
95% confidence interval: 0.9743-1.0256
Kappa＝(PA－Pe)/(1－Pe)＝1.00
The K values of the assessment reagent and the reference reagent are 1.00, which indicates that the consistency of the two reagents is good.
3.2.1.2 assessment reagent results analysis of compliance with clinical reference standards
The clinical study examined the total number of the case specimens of 150 cases, 63 cases were confirmed and 87 cases were excluded. The consistency of the detection result of the assessment reagent and the clinical reference standard is as follows:
TABLE 4 agreement of assessment reagent results with clinical reference standards
Figure RE-RE-GDA0002741928340000111
Clinical sensitivity (%) 62/63 × 100%, (98.41%)
Clinical specificity (%): 87/87 × 100%: 100%
The clinical total coincidence rate (%) is 149/150 × 100% >, 99.33%
Reference interval of 95%: 0.9783-1.0085
Kappa＝(PA-Pe)/(1-Pe)＝0.98
The K value of the examination reagent and the clinically confirmed diagnosis is 0.98, which shows that the consistency of the detection result of the examination reagent and the clinical reference standard is good.
The inconformity sample is a positive sample of early detection of a later clinical confirmed case, and the detection result of the examination reagent is negative.
The present invention is not limited to the above-described preferred embodiments, but rather, the present invention is to be construed broadly and cover all modifications, equivalents, and improvements falling within the spirit and scope of the present invention.
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agctgcacgt ggatcctgag a 21

Claims (10)

1. A reagent for improving specificity of nucleic acid amplification reaction is characterized in that the reagent comprises glycine and trehalose, wherein the final concentration of the glycine is 1% -10%, and the final concentration of the trehalose is 2% -10%; and the final concentration of trehalose and the final concentration of glycine cannot be equal to 10% at the same time.
2. The reagent for improving the specificity of a nucleic acid amplification reaction according to claim 1, wherein the final concentration of glycine is 1% to 10%, and the final concentration of trehalose is 2% to 5%; and when the final concentration of glycine is 10%, the final concentration of trehalose is 2%.
3. The reagent for improving the specificity of a nucleic acid amplification reaction according to claim 2, wherein the final concentration of glycine is 1% to 5%, and the final concentration of trehalose is 2% to 5%; preferably, the final concentration of glycine is 2% to 5%.
4. The reagent for improving the specificity of a nucleic acid amplification reaction according to claim 1, wherein the reagent comprises glycine at a final concentration of 2% to 5% and trehalose at a final concentration of 5%.
5. A nucleic acid amplification reaction solution comprising the reagent according to any one of claims 1 to 4 and one or more of UNG enzyme, reverse transcriptase and Hot Start Taq enzyme in combination.
6. The nucleic acid amplification reaction solution of claim 5, further comprising at least one of Tris, KCl, MgCl2, DTT, EDTA, Tween-20, and RNase inhibitor; preferably, the final concentration of Tris is 8.0 and 10mM, the final concentration of KCl is 40mM, the final concentration of MgCl2 is 5mM, the final concentration of DTT is 1mM, the final concentration of EDTA is 0.5mM, the final concentration of Tween-20 is 0.5%, the final concentration of RNase inhibitor is 10U/. mu.L, the final concentration of UNG enzyme is 1U/. mu.L, the final concentration of reverse transcriptase is 100U/. mu.L, and the final concentration of hot start Taq enzyme is 2U/. mu.L.
7. A kit comprising the reagent according to any one of claims 1 to 4 and/or the nucleic acid amplification reaction solution according to any one of claims 5 to 6 as a component.
8. The kit according to claim 7, further comprising a primer set for detecting a novel coronavirus, wherein the primer set comprises a first primer pair and a first probe for detecting ORF1a/b gene, a second primer pair and a second probe for detecting N gene, a third primer pair and a third probe for detecting E gene, and/or a fourth primer pair and a fourth probe for detecting human β -Globin gene; the nucleotide sequence of the F primer of the first primer pair is shown as SEQ ID No.1, the nucleotide sequence of the R primer of the first primer pair is shown as SEQ ID No.2, and the nucleotide sequence of the first probe is shown as SEQ ID No. 3; the nucleotide sequence of the F primer of the second primer pair is shown as SEQ ID No.4, the nucleotide sequence of the R primer of the second primer pair is shown as SEQ ID No.5, and the nucleotide sequence of the second probe is shown as SEQ ID No. 6; the nucleotide sequence of the F primer of the third primer pair is shown as SEQ ID No.7, the nucleotide sequence of the R primer of the third primer pair is shown as SEQ ID No.8, and the nucleotide sequence of the third probe is shown as SEQ ID No. 9; the nucleotide sequence of the F primer of the fourth primer pair is shown as SEQ ID No.10, the nucleotide sequence of the R primer of the fourth primer pair is shown as SEQ ID No.11, and the nucleotide sequence of the fourth probe is shown as SEQ ID No. 12.
9. The kit according to claim 7 or 8, wherein the components of the kit further comprise another amplification reaction solution A, the amplification reaction solution A comprising Tris, KCl, (NH4)2SO4、DTT、MgCl2、dATP、dGTP, dUTP, oligdt and the primer set; preferably, the Tris has a final pH of 8.0 of 10mM, the KCl has a final concentration of 40mM, and the (NH4)2SO415mM, 1mM of DTT, 5mM of MgCl2, 1mM of dATP, 1mM of dGTP, 1mM of dUTP, 1mM of dCTP, 0.1. mu.M of oligdt, and 0.1. mu.M of each primer in the primer set.
10. The kit of claim 9, wherein the components of the kit further comprise a positive quality control material and/or a negative quality control material; when the kit is used for nucleic acid amplification reaction, the amplification procedure is 37 ℃ for 1 min; at 42 ℃ for 20 min; 95 ℃ for 5 min; 10s at 95 ℃ and 30s at 58 ℃ for 40 cycles.
CN202010703846.7A 2020-07-21 2020-07-21 Reagent for improving specificity of nucleic acid amplification reaction, nucleic acid amplification reaction solution and kit Pending CN112029901A (en)

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