CN111621595A - Novel coronavirus qRT-PCR one-step kit reaction solution and kit and method thereof - Google Patents

Novel coronavirus qRT-PCR one-step kit reaction solution and kit and method thereof Download PDF

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CN111621595A
CN111621595A CN202010316736.5A CN202010316736A CN111621595A CN 111621595 A CN111621595 A CN 111621595A CN 202010316736 A CN202010316736 A CN 202010316736A CN 111621595 A CN111621595 A CN 111621595A
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pcr
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禹文峰
张远波
梁贵友
黄智�
高睿
王碧
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Guizhou Medical University
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Abstract

The invention discloses a novel coronavirus qRT-PCR one-step method kit reaction solution, a kit and a method thereof, wherein the method comprises the following steps: PT-PCR reaction Solution, enzyme mixed Solution, specific primers and probes, wherein the formula of the Solution is as follows: (NH)4)2SO420-40 mM Tween 201.8E‑6～3.6E‑6Sodium nitride 4.5E‑2～9E‑20.05 to 0.1mM of glycerol, 0.07 to 0.14mM of dimethyl sulfoxide, and 1.5E of bovine serum albumin‑7～3E‑7(ii) a Comprises a kit containing known ORF1ab gene or N gene and a Solution formula; a detection method comprising the kit. The invention can effectively reduce the false negative of 2019-nCoV novel coronavirus detection by the qRT-PCR detection technology based on ORF1ab gene or N gene targetThe diagnosis accuracy of the kit is improved, and the infection hidden danger, the safety risk and the resource consumption are reduced.

Description

Novel coronavirus qRT-PCR one-step kit reaction solution and kit and method thereof
Technical Field
The invention relates to the field of biotechnology application, in particular to a novel coronavirus qRT-PCR one-step kit reaction solution, a kit and a method thereof.
Background
In the clinical practice process of the novel coronavirus, some initial-diagnosis patient samples with weak symptoms and low viral load, patient samples with high suppressive substances in the samples, patient samples to be treated for recheck and discharge, and the like cannot be diagnosed clearly, so that great potential infection hazards, safety risks and resource consumption are caused.
Therefore, in the detection and quantitative analysis of samples by using the known qRT-PCR technology and using ORF1ab gene or N gene for detection, in order to reduce and avoid false negative or false positive of the detection result and improve the accuracy of quantitative analysis, how to design a kit for detecting more sensitively based on the known target polynucleotide sequence becomes a key basic technical link.
Disclosure of Invention
In order to solve the technical problems, the invention provides a novel reaction solution of a coronavirus qRT-PCR one-step kit, a kit and a method thereof, so as to reduce and avoid false negative or false positive of a detection result by enhancing and improving a reaction system of the kit.
The technical scheme of the invention is as follows: a novel coronavirus qRT-PCR one-step method kit reaction solution comprises: PT-PCR reaction Solution, enzyme mixed Solution, specific primer and probe; the PT-PCR reaction solution comprises the following components in parts by weight: 5-40 mM DTT, 100-200 mM KCl, MgSO43-6 mM, Tris-HCl 20-40 mM; the enzyme mixed solution comprises the following components in parts by weight: 5-8mM of hot start Taq enzyme, 3-4mM of MMLV reverse transcriptase, 10-15mM of RNA inhibitor and 10mM of dNTP, and the amount is made up to 4uL by enzyme preservation solution; the formula of the Solution comprises the following components in parts by weight: (NH)4)2SO420-40 mM Tween 201.8E-6～3.6E-6Sodium nitride 4.5E-2～9E-20.05 to 0.1mM of glycerol, 0.07 to 0.14mM of dimethyl sulfoxide, and 1.5E of bovine serum albumin-7～3E-7。
Furthermore, the Solution formula also comprises an enhancer with 2.5 percent of volume ratio, and the preparation method of the enhancer comprises the following steps: mixing 10mM Tris-HCl, 1-1.5 mM trioctylphosphine oxide, 0.5-0.7 mM phenanthroline and 0.05-0.2 mM formamide, and shaking up to obtain the enhancer.
The invention also discloses a kit based on the novel coronavirus qRT-PCR one-step method kit reaction solution, which is characterized by comprising the kit reaction solution of claim 1 or 2.
Further, the reaction system in the kit comprises the following specific components in parts by weight: 0.8uL RT-PCR reaction Solution, 4uL enzyme mixed Solution, 2.0uL 10 × Solution, 0.25uL forward primer, 0.25uL reverse primer, 0.2uL probe, 0.4uL2019-nCoV novel coronavirus template, and sterile water is added to 20 uL; wherein, the content of the forward primer, the reverse primer and the probe is half of that of the corresponding 2019-nCoV novel coronavirus gene and the internal reference gene RPP 30.
Furthermore, the sequences of the primer and the probe of the 2019-nCoV novel coronavirus gene and the reference gene RPP30 are respectively as follows:
1)2019-nCoV novel coronavirus gene (ORF1ab gene or N gene)
A forward primer: 1 or 4 SEQ ID NO;
reverse primer: 2 or 5 SEQ ID NO;
and (3) probe: 3 or 6 SEQ ID NO;
2) internal reference gene RPP30
A forward primer: 7 in SEQ ID NO;
reverse primer: 8 in SEQ ID NO;
and (3) probe: SEQ ID NO 9.
Furthermore, 5 'end of the probe shown as SEQ ID NO. 3, SEQ ID NO. 6 and SEQ ID NO. 9 is marked with a fluorescent group FAM, 3' end of the probe shown as SEQ ID NO. 3 is marked with a quenching group BHQ-1, 3 'end of the probe shown as SEQ ID NO. 6 is marked with a quenching group TAMRA, 3' end of the probe shown as SEQ ID NO. 9 is marked with a quenching group TAMRA/BHQ-1, and meanwhile, in order to reduce interference of a reaction system, the synthesized primer and probe are purified by HPLC.
The invention also discloses a kit using method based on the novel coronavirus qRT-PCR one-step method kit reaction solution, which comprises the following steps:
s1: extracting RNA of the virus from a detection sample as a template, configuring an amplification reaction system by using the kit prepared in claim 4, and performing real-time fluorescence PCR amplification to obtain an amplification curve;
s2: and analyzing and judging the amplification curve to obtain a conclusion whether the 2019-nCoV novel coronavirus exists in the detection sample.
Further, the reaction parameters and amplification procedures of the amplification reaction system are as follows:
[1]42℃30min
[2]95℃5min
[3]94℃30sec
[4]60℃1min
[5] goto 3 and 4, 40-45cycles, collecting FAM channel fluorescence signal in step 4
[6]End。
The detection principle of the kit provided by the invention is as follows:
adopting qRT-PCR, carrying out reverse transcription on novel coronavirus nucleic acid (RNA) into DNA before carrying out PCR reaction, and carrying out specific primers and Taqman probes contained in a PCR reaction system;
when the probe is complete, the fluorescent signal emitted by the reporter group is absorbed by the quenching group; if a target sequence exists in a PCR reaction system, a probe is combined with a template during the PCR reaction, DNA polymerase enzyme cuts and degrades the probe along the template by utilizing the activity of exonuclease of the enzyme, a reporter group is separated from a quenching group to emit fluorescence, and one fluorescent molecule is generated for each amplified DNA chain;
the ABI7500 fluorescent PCR amplification instrument sold by Saimei Fei company monitors that the cycle number (Ct value) of fluorescence reaching a preset threshold value is related to the concentration of virus nucleic acid, the higher the concentration of the virus nucleic acid is, the smaller the Ct value is, a real-time amplification curve is automatically drawn by a PCR instrument software system, and the instant result judgment is realized; thereby achieving the purpose of fast, real-time and quantitative detection of the novel coronavirus.
The invention has the beneficial effects that:
(1) the Solution formula can effectively reduce the condition that false negative occurs to 2019-nCoV novel coronavirus detection by the qRT-PCR detection technology based on ORF1ab gene or N gene target, thereby improving the diagnostic accuracy of the kit and reducing the infection hidden danger, the safety risk and the resource consumption.
(2) According to the invention, an enhancer formula is added on the basis of the disclosed Solution formula, and the condition that the 2019-nCoV novel coronavirus detection is false negative by the known qRT-PCR detection technology based on ORF1ab gene or N gene target is further reduced through the effect of the enhancer formula, so that the detection reliability is improved.
(3) The kit and the detection method can be used for qualitatively detecting the new coronavirus nucleic acid in a nose/throat swab/whole blood/serum sample, have high detection sensitivity and strong specificity, can effectively avoid false negative or false positive in coronavirus detection, and have the advantages of simple and convenient operation and high detection reliability.
Drawings
FIG. 1 is a diagram of a fluorescence quantitative PCR specificity experiment of a Solution type I formulation according to an embodiment of the present invention,
FIG. 2 is a diagram of a fluorescence quantitative PCR specificity experiment of a Solution type II formulation according to an embodiment of the present invention,
wherein, 1: 2019-nCoV novel coronaviruses; 2: SARS coronavirus; 3: influenza virus type a; 4: influenza virus type B; 5: influenza virus type C virus; 6: coronavirus 229E; 7: metapneumovirus HMP.
FIG. 3 is a graph of the fluorescence quantitative PCR sensitivity test of Solution type I formulation according to an embodiment of the present invention,
FIG. 4 is a graph of the fluorescence quantitative PCR sensitivity test of Solution type II formulation according to an embodiment of the present invention,
wherein, 1: 1 × 100copies/μL；2：1×101copies/μL；3：1×102copies/μL；4： 1×103copies/μL；5：1×104copies/μL；6：1×105copies/μL；7：1×106copies/μL；8： 1×107copies/μL；9：1×108copies/μL。
FIG. 5 is a standard curve of fluorescent quantitative PCR for Solution type I formulation according to an embodiment of the present invention.
FIG. 6 is a standard curve of fluorescent quantitative PCR for Solution type II formulation according to an embodiment of the present invention.
Detailed Description
The invention relates to a novel coronavirus qRT-PCR one-step method kit reaction Solution, a kit and a method thereof, which are aRT-PCR detection kits based on known ORF1ab genes or N genes and carry a reaction system formed by a Solution formula and the like.
Example 1(Solution formulation)
Solution type I: according to (NH)4)2SO435mM, Tween 203.2E-6Sodium nitride 7.5E-20.07mM of glycerol, 0.12mM of dimethyl sulfoxide, and 2.3E of bovine serum albumin-7Preparing Solution I type, and adjusting the pH value to 6.5-8.5.
Solution type II: according to (NH)4)2SO435mM, Tween 203.2E-6Sodium nitride 7.5E-20.07mM of glycerol, 0.12mM of dimethyl sulfoxide, and 2.3E of bovine serum albumin-7Preparing Solution II type with 2.5% volume ratio of the reinforcing agent, and adjusting the pH value to 6.5-8.5;
the preparation method of the reinforcing agent comprises the following steps: mixing 10mM Tris-HCl, 1.2mM trioctylphosphine oxide, 0.6mM phenanthroline and 0.15mM formamide, and shaking up to obtain the enhancer.
Example 2 (primer and Probe)
Selecting known ORF1ab gene or N gene, and designing primer and probe of RPP30 reference gene, wherein the sequence is synthesized by Takara Baozhen (Dalian) company;
the 5 'end of the probe shown in SEQ ID NO. 3, SEQ ID NO. 6 and SEQ ID NO. 9 is marked with a fluorescent group FAM, the 3' end of the probe shown in SEQ ID NO. 3 is marked with a quenching group BHQ-1, the 3 'end of the probe shown in SEQ ID NO. 6 is marked with a quenching group TAMRA, the 3' end of the probe shown in SEQ ID NO. 9 is marked with a quenching group TAMRA/BHQ-1, and meanwhile, in order to reduce the interference of a reaction system, the synthesized primer and probe are purified by HPLC;
all primer and probe sequences are shown in table 1:
TABLE 1 primer sequences and Probe sequences
Figure RE-GDA0002602589610000061
Example 3 (New coronavirus kit and reaction System)
Reaction systems of the kits are respectively configured according to Solution type I and Solution type II prepared in example 1, which are specifically as follows:
target one (ORF1ab)
1) The specific components of the reaction system based on Solution type I are shown in the following table 2:
TABLE 2 Solution I type ORF1ab gene kit reaction system
Figure RE-GDA0002602589610000062
Figure RE-GDA0002602589610000071
2) The specific components of the reaction system based on Solution II are shown in the following table 3:
TABLE 3 Solution II type ORF1ab gene kit reaction system
Figure RE-GDA0002602589610000072
Wherein the PT-PCR reaction solution comprises the following components in parts by weight: DTT 30mM, KCl 170mM, MgSO45mM、Tris-HCl 30mM；
The enzyme mixed solution comprises the following components in parts by weight: hot start Taq enzyme 6mM, MMLV reverse transcriptase 4mM, RNA inhibitor 13mM, dNTP 10mM, and make up to 4uL through enzyme preservation solution;
the ORF1ab gene forward primer, ORF1ab gene reverse primer, ORF1ab gene probe, RPP30 reference gene forward primer, RPP30 reference gene reverse primer and RPP30 reference gene probe are respectively shown as SEQ ID NO 1, 2, 3, 7, 8 and 9.
Target two (N)
1) The specific components of the reaction system based on Solution type I are shown in the following table 4:
TABLE 4 Solution I type N gene kit reaction system
Figure RE-GDA0002602589610000081
2) The specific components of the reaction system based on Solution II are shown in the following table 5:
TABLE 5 Solution II type N gene kit reaction system
Figure RE-GDA0002602589610000082
Figure RE-GDA0002602589610000091
Wherein the PT-PCR reaction solution comprises the following components in parts by weight: DTT 30mM, KCl 170mM, MgSO45mM、Tris-HCl 30mM；
The enzyme mixed solution comprises the following components in parts by weight: hot start Taq enzyme 6mM, MMLV reverse transcriptase 4mM, RNA inhibitor 13mM, dNTP 10mM, and make up to 4uL through enzyme preservation solution;
the N gene forward primer, the N gene reverse primer, the N gene probe, the RPP30 reference gene forward primer, the RPP30 reference gene reverse primer and the RPP30 reference gene probe are respectively shown in SEQ ID NO. 4, 5, 6, 7, 8 and 9.
Example 4(qRT-PCR amplification procedure)
The qRT-PCR amplification program is constructed, and is specifically shown as follows:
[1]42℃30min
[2]95℃5min
[3]94℃30sec
[4]60℃1min
[5] goto 3 and 4, 40-45cycles, collecting FAM channel fluorescence signal in step 4
[6]End。
Example 5 (Solution formula-based 2019-nCoV novel coronavirus detection kit detection method)
1. Preparation of control samples:
1) a positive control sample, which is artificially synthesized with the concentration of 1 × 10 and takes pseudovirus containing the target amplification region as a positive control sample of the 2019-nCoV novel coronavirus6A pseudo virus recombined with 2019-nCoV novel coronavirus sequences of Copies/mL;
2) negative control samples: sterile normal saline is used as a negative control sample;
3) specific reference control samples: SARS coronavirus, influenza virus A, influenza virus B, influenza virus C, coronavirus 229E, and metapneumovirus HMP were used as specific reference controls.
2. Collecting a sample: the clinician collects samples of corresponding parts according to actual conditions, and the detectable samples comprise nasal/pharyngeal swabs/whole blood/serum; and (4) processing the collected sample according to the conventional requirements, sealing and storing the sample, and performing low-temperature inspection.
3. Detection method
1) RNA extraction and treatment: taking 100 mu L of each detection sample, extracting by a conventional RNA/DNA extraction method to obtain 50 mu L of nucleic acid extraction product, and diluting the nucleic acid extraction product by 10 times by using DEPC water to serve as a template;
2) and (3) PCR reaction: configuring a reaction system of the 2019-nCoV novel coronavirus kit according to example 3, taking two reaction systems of a target I (ORF1ab) as an example, respectively adding 5 mu L of each of a negative control, a specific reference control and a positive control, and performing real-time fluorescence PCR amplification by using a PCR instrument with the model number of ABI7500, which is manufactured by Saimerfi corporation, 2019-nCoV novel coronavirus primers and probes designed according to example 1 and an amplification program constructed according to example 4 to obtain an amplification curve;
3) and (4) analyzing results: analyzing the experimental result of the amplification curve, if the amplification curve has obvious exponential growth period, judging the amplification curve to be positive, otherwise, judging the amplification curve to be negative; the result shows that the test result of the positive control is positive, and the test results of the negative control and the specific reference control are negative; it was demonstrated that the kit comprising the primer and probe of the present invention has good specificity.
Example 6 (specificity experiment)
The specificity of each kit is detected by performing fluorescent quantitative PCR reaction by taking the DNA of SARS coronavirus, influenza virus A, influenza virus B, influenza virus C, coronavirus 229E, metapneumovirus HMP and 2019-nCoV novel coronavirus as templates and Solution type I and type II reaction systems of a target I (ORF1ab) as examples.
As shown in FIGS. 1 and 2, only the 2019-nCoV plasmid has obvious amplification, and no other samples have amplification, which shows that the specificity of the kit formed by the reaction systems formed by two solutions I and II is better, and the specificity of the reaction system formed by using the Solution II by contrasting the curves of I and II is relatively better.
Example 7 (sensitivity test)
Preparation 1 × 100-1×108The sensitivity test of the product is carried out by taking the Solution I type and II type reaction systems of a target I (ORF1ab) as an example, and carrying out fluorescent quantitative PCR reaction on the novel 2019-nCoV coronavirus positive standard plasmid of copies/mu L with 3 repetitions arranged in each gradient.
As shown in FIGS. 3 and 4, the kit of the reaction system composed of two solutions type I and type II can effectively detect 10copies of samples, when the sample concentration is more than 10 copies/. mu.L, obvious amplification can occur, and simultaneously, the detection sensitivity of the reaction system using the solutions type II is higher by comparing the curves of type I and type II.
Establishing a standard curve as shown in figures 5 and 6 by taking the CT value as an ordinate and the copy number as an abscissa; the standard curves of the reaction system kit composed of Solution type I and II are respectively shown in FIGS. 5 and 6The equation is: 15.683x0.0428；R2＝0.9963。
Sequence listing
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Claims (7)

1. A novel coronavirus qRT-PCR one-step method kit reaction solution is characterized by comprising the following steps: PT-PCR reaction Solution, enzyme mixed Solution, specific primer and probe;
the PT-PCR reaction solution comprises the following components in parts by weight: 5-40 mM DTT, 100-200 mM KCl, MgSO43～6mM、Tris-HCl 20～40mM；
The enzyme mixed solution comprises the following components in parts by weight: 5-8mM of hot start Taq enzyme, 3-4mM of MMLV reverse transcriptase, 10-15mM of RNA inhibitor and 10mM of dNTP, and the amount is made up to 4uL by enzyme preservation solution;
the formula of the Solution comprises the following components in parts by weight: (NH)4)2SO420-40 mM Tween 201.8E-6～3.6E-6Sodium nitride 4.5E-2～9E-20.05 to 0.1mM of glycerol, 0.07 to 0.14mM of dimethyl sulfoxide, and 1.5E of bovine serum albumin-7～3E-7。
2. The reaction Solution of the qRT-PCR one-step kit for the novel coronavirus of claim 1, wherein the Solution formula further comprises an enhancer with a volume ratio of 2.5%, and the preparation method of the enhancer comprises: mixing 10mM Tris-HCl, 1-1.5 mM trioctylphosphine oxide, 0.5-0.7 mM phenanthroline and 0.05-0.2 mM formamide, and shaking up to obtain the enhancer.
3. A kit based on a novel coronavirus qRT-PCR one-step kit reaction solution, which is characterized by comprising the kit reaction solution of claim 1 or 2.
4. The kit based on the novel coronavirus qRT-PCR one-step kit reaction solution as claimed in claim 3, wherein the reaction system in the kit comprises the following specific components in parts by weight: 0.8uL RT-PCR reaction Solution, 4uL enzyme mixed Solution, 2uL 10 × Solution, 0.25uL forward primer, 0.25uL reverse primer, 0.2uL probe, 0.4uL2019-nCoV novel coronavirus template, and sterile water is added to 20 uL; wherein the content of the forward primer, the reverse primer and the probe is half of that of the corresponding 2019-nCoV novel coronavirus gene and reference gene RPP30, and the pH of the whole reaction system is controlled to be 6.5-8.5.
5. The kit based on the reaction solution of the novel coronavirus qRT-PCR one-step kit of claim 4, wherein the sequences of the primer and the probe of the 2019-nCoV novel coronavirus gene and the reference gene RPP30 are respectively as follows:
1)2019-nCoV novel coronavirus gene (ORF1ab gene or N gene)
A forward primer: 1 or 4 SEQ ID NO;
reverse primer: 2 or 5 SEQ ID NO;
and (3) probe: 3 or 6 SEQ ID NO;
2) internal reference gene RPP30
A forward primer: 7 in SEQ ID NO;
reverse primer: 8 in SEQ ID NO;
and (3) probe: SEQ ID NO 9.
6. A kit using method based on a novel coronavirus qRT-PCR one-step method kit reaction solution is characterized by comprising the following steps:
s1: extracting RNA of the virus from a detection sample as a template, configuring an amplification reaction system by using the kit prepared in claim 4, and performing real-time fluorescence PCR amplification to obtain an amplification curve;
s2: and analyzing and judging the amplification curve to obtain a conclusion whether the 2019-nCoV novel coronavirus exists in the detection sample.
7. The method for using the kit based on the novel coronavirus qRT-PCR one-step kit reaction solution as claimed in claim 6, wherein the reaction parameters and the amplification procedure of the amplification reaction system are as follows:
[1]42℃30min
[2]95℃5min
[3]94℃30sec
[4]60℃1min
[5] goto 3 and 4, 40-45cycles, collecting FAM channel fluorescence signal in step 4
[6]End。
CN202010316736.5A 2020-04-21 2020-04-21 Novel coronavirus qRT-PCR one-step kit reaction solution and kit and method thereof Pending CN111621595A (en)

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GB2593010B (en) * 2020-10-20 2022-03-30 Primer Design Ltd Composition and Method
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KR102532739B1 (en) * 2022-08-04 2023-05-17 경기도 Composition for pretreatment for detecting coronavirus and/or diagnosing coronavirus infection, and pretreatment method using same

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