CN111647688A - Freeze-dried PCR reagent for detecting COVID-19, FluA and FluB viruses and preparation method thereof - Google Patents

Freeze-dried PCR reagent for detecting COVID-19, FluA and FluB viruses and preparation method thereof Download PDF

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CN111647688A
CN111647688A CN202010561062.5A CN202010561062A CN111647688A CN 111647688 A CN111647688 A CN 111647688A CN 202010561062 A CN202010561062 A CN 202010561062A CN 111647688 A CN111647688 A CN 111647688A
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胡康
胡林立
张成军
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Anhui Guotai Guorui Pharmaceutical Technology Co ltd
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Abstract

The invention discloses a freeze-dried PCR reagent for detecting COVID-19, FluA and FluB viruses and a preparation method thereof, the freeze-drying PCR reagent designs three different upstream primers, downstream primers and TaqMan probes according to specific gene sequences of the COVID-19 virus, the FluA virus and the FluB virus, so that the freeze-drying PCR reagent can be used for patients with fever symptoms, simultaneously detecting three pathogens of COVID-19, FluA and FluB, judging whether the pathogen capable of causing fever symptoms is one or more of COVID-19, FluA and FluB, the reagent contains an uracil-DNA glycosylase anti-pollution system, dUTP replaces dTTP to be used as a PCR raw material, during PCR amplification, uracil-DNA glycosylase can digest the PCR product containing U in the PCR product, thereby preventing the aerosol pollution of the PCR product and ensuring the detection effect of the PCR reagent of the freeze-dried preparation.

Description

Freeze-dried PCR reagent for detecting COVID-19, FluA and FluB viruses and preparation method thereof
Technical Field
The invention belongs to the technical field of virus detection, and particularly relates to a freeze-dried PCR reagent for detecting COVID-19, FluA and FluB viruses and a preparation method thereof.
Background
The world health organization named the new coronavirus as COVID-19, patients infected with COVID-19 had fever, cough, fatigue, weakness as main symptoms, which were similar to those of influenza A virus (FluA) and influenza B virus (FluB) infections, and furthermore, COVID-19 was more contagious than SARS virus (SARS virus) which was outbreak in 2003 and was likely to have long-term and seasonal outbreaks similar to FluA.
The existing nucleic acid detection products mainly comprise a single virus detection product of COVID-19, can be used for screening the human population on a large scale with low cost, but cannot detect the fever symptoms caused by FluA and FluB pathogens, so that the application range of the kit is reduced; the existing nucleic acid detection product is basically in a liquid form, a liquid reagent needs to be subjected to cold chain transportation in the transportation process, the cost of the reagent in the transportation process is increased due to the cold chain transportation, the storage cost is greatly increased due to the need of refrigeration in the storage process, and the risk of reagent failure is high in the storage and transportation process; the existing nucleic acid detection product is easy to be polluted by PCR product aerosol when detecting the PCR product.
Disclosure of Invention
The invention aims to provide a freeze-dried PCR reagent for detecting COVID-19, FluA and FluB viruses and a preparation method thereof.
The technical problems to be solved by the invention are as follows:
the existing nucleic acid detection products mainly comprise a single virus detection product of COVID-19, can be used for screening the human population on a large scale with low cost, but cannot detect the fever symptoms caused by FluA and FluB pathogens, so that the application range of the kit is reduced;
the existing nucleic acid detection product is basically in a liquid form, a liquid reagent needs to be subjected to cold chain transportation in the transportation process, the cost of the reagent in the transportation process is increased due to the cold chain transportation, the storage cost is greatly increased due to the need of refrigeration in the storage process, and the risk of reagent failure is high in the storage and transportation process;
the existing nucleic acid detection product is easy to be polluted by the aerosol of the PCR product when detecting the PCR product.
The purpose of the invention can be realized by the following technical scheme:
the freeze-dried PCR reagent for detecting COVID-19, FluA and FluB viruses comprises 1-20 units of TaqDNA polymerase, 20-200 units of reverse transcriptase, 0.1-1 unit of uracil-DNA glycosylase, 1-40 units of ribonuclease inhibitor, 1-20pmol of upstream primer, 1-20pmol of downstream primer, 0.5-10pmol of TaqMan probe, 0.5-100 nmol of dATP2.5-100nmol of dCTP2.5-100nmol of dGTP2.5-100nmol, 2.5-100nmol of dUTP2, 0.01-0.2 μmol of magnesium ion, 0.25-2.5 μmol of Tris0.25-5 μmol of potassium ion and 2-15% (m/v) of freeze-dried protective agent;
the preparation steps of the freeze-dried PCR reagent are as follows:
step S1: mixing the reagents, and adjusting the pH value of the mixed solution to 8.3-8.9 after uniformly mixing to prepare a PCR reagent;
step S2: adding the PCR reagent prepared in the step S1 into a PCR tube, putting the PCR tube into a freeze dryer under the condition of not covering the PCR tube, carrying out freeze-drying reaction, and then packaging the freeze-dried PCR reagent in a nitrogen environment;
step S3: extracting nucleic acid from a sample to be detected to obtain a nucleic acid solution;
step S4: adding the freeze-dried PCR reagent prepared in the step S2 into the nucleic acid solution prepared in the step S3 to prepare a mixed solution;
step S5: the mixture solution prepared in step S4 is subjected to PCR amplification.
Further, the TaqDNA polymerase is 5 units, the reverse transcriptase is 50 units, the uracil-DNA glycosylase is0.25 unit, the ribonuclease inhibitor is 10 units, the upstream primer is 5pmol, the downstream primer is 5pmol, the TaqMan probe is 2.5pmol, the dATP is 5nmol, the dCTP is 5nmol, the dGTP is 5nmol, the dUTP is 10nmol, the magnesium ion is 0.05 μmol, the Tris is1.25 μmol, the potassium ion is 2.25 μmol, and the lyoprotectant is 7.2%.
Further, the reverse transcriptase is one or a mixture of two of AMV reverse transcriptase and M-Mulv reverse transcriptase in any proportion, the upstream primer is an upstream primer of COVID-19, FluA, FluB virus and human GAPDH gene, the downstream primer is a downstream primer of COVID-19, FluA, FluB virus and human GAPDH gene, the TaqMan probe is a TaqMan probe of COVID-19, FluA, FluB virus and human GAPDH gene, and the freeze-drying protective agent is one or a mixture of more of trehalose, sucrose, glucose, fucose, sorbitol, mannitol, dextran and polyethylene glycol in any proportion.
Furthermore, the upstream primer sequence of the COVID-19 virus is as follows:
5’-AGAATGGAGAACGCAGTGGG-3’,
the sequence of the downstream primer of the COVID-19 virus is as follows:
5’-TGAGAGCGGTGAACCAAGAC-3’,
the TaqMan probe sequence of the COVID-19 virus is as follows:
5’-FAM-CGCGATCAAAACAACGTCGGCC-BHQ13’,
the upstream primer sequence of the FluA virus is as follows:
5’-GTTGGTRATGAAACGRAAACGGG-3’,
the sequence of the downstream primer of the FluA virus is as follows:
5’-CCGAATYCTTTTGGTCGCTGT-3’,
the TaqMan probe sequence of the FluA virus is as follows:
5’-VIC-CTCTAGCATACTTACTGACAGC-MGB-3’,
the upstream primer sequence of FluB virus is as follows:
5’-CACAAATGCAACCAGACCTGC-3’,
the sequence of the downstream primer of the FluB virus is as follows:
5’-GGGGAGAGAAAATTCTCCTGCAT-3’,
the TaqMan probe sequence of the FluB virus is as follows:
5’-Cy5-TTAGACAGRATAGCTGCTGGCA-MGB-3’,
the upstream primer sequence of human GAPDH gene is as follows:
5’-ACTTAGAGAAGGGGTGGGCT-3’,
the sequence of the downstream primer of human GAPDH gene is as follows:
5’-ACGCTTGTACACTCAGCATCA-3’,
the TaqMan probe sequence of the human GAPDH gene is as follows:
5’-ROX-ccctgtccagttaatttctgacctttactcctgC-BHQ2-3’,
the 5 'end of the probe is marked with a fluorescent group, the 3' end of the probe is marked with a quenching group, and the colors of the fluorescent groups marked by the four probes can be adjusted.
Further, the final reaction volume of the PCR reagent described in step S1 is 25. mu.L or 50. mu.L, and when the final reaction volume is 50ul, the amount of the components other than the lyoprotectant is doubled.
Further, the freeze-drying reaction procedure of the step S2 is-45 ℃ and 2 h; -45 ℃, 20Pa, 2 h; -45 ℃, 10Pa, 16 h; 25 ℃, 10Pa, 2 h.
Further, the amount ratio of the lyophilized PCR reagents and the nucleic acid solution in step S4 is 25 μ L: 1-25 μ L or 50 μ L: 1 to 50. mu.L, and when the nucleic acid solution is less than 25. mu.L, the volume is made up to 25. mu.L with RNase-free water, and when the nucleic acid solution is less than 50. mu.L, the volume is made up to 50. mu.L with RNase-free water.
Further, the PCR amplification procedure described in step S5 is: 50 ℃, 5-15min, 94 ℃, 5 min; fluorescence was detected at 95 deg.C, 5 seconds, 60 deg.C, 30 seconds, 45 cycles, FAM/HEX/ROX/CY5 channel.
Further, the preparation method of the freeze-dried PCR reagent for detecting the COVID-19, FluA and FluB viruses comprises the following specific steps:
step S1: mixing the reagents, and adjusting the pH value of the mixed solution to 8.3-8.9 after uniformly mixing to prepare a PCR reagent;
step S2: adding the PCR reagent prepared in the step S1 into a PCR tube, putting the PCR tube into a freeze dryer under the condition of not covering the PCR tube, carrying out freeze-drying reaction, and then packaging the freeze-dried PCR reagent in a nitrogen environment;
step S3: extracting nucleic acid from a sample to be detected to obtain a nucleic acid solution;
step S4: adding the freeze-dried PCR reagent prepared in the step S2 into the nucleic acid solution prepared in the step S3 to prepare a mixed solution;
step S5: the mixture solution prepared in step S4 is subjected to PCR amplification.
The invention has the beneficial effects that: the invention designs three different upstream primers, downstream primers and TaqMan probes according to specific gene sequences of COVID-19 virus, FluA virus and FluB virus, so that a freeze-dried PCR reagent can simultaneously detect three pathogens of COVID-19, FluA and FluB aiming at a patient with fever symptom, judge whether the pathogen capable of causing the fever symptom is one or more of COVID-19, FluA and FluB, and reduce the misdiagnosis of the patient with fever symptom caused by non-COVID-19 virus into novel coronary pneumonia, the PCR reagent of the freeze-dried preparation prepared by the invention does not need cold chain transportation in the storage and transportation process, greatly reduces the cost of reagent transportation, does not have the risk of reagent failure in the transportation process, and the PCR reagent of the freeze-dried preparation contains a uracil-DNA glycosylase anti-pollution system and replaces dUTP as a PCR raw material, during PCR amplification, uracil-DNA glycosylase can digest a PCR product containing U in the PCR product, so that aerosol pollution of the PCR product is prevented, and the detection effect of the freeze-dried PCR reagent is ensured.
Drawings
FIG. 1 reagent status after lyophilization;
FIG. 2 multiplex PCR assay of inactivated COVID-19 samples;
FIG. 3 multiplex PCR detection of inactivated FluA samples;
FIG. 4 multiplex PCR detection of inactivated FluB samples.
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely below, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all 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
A freeze-dried PCR reagent for detecting COVID-19, FluA and FluB viruses comprises 5 units of TaqDNA polymerase, 50 units of reverse transcriptase, 0.25 unit of uracil-DNA glycosylase, 10 units of ribonuclease inhibitor, 5pmol of upstream primer, 5pmol of downstream primer, 2.5pmol of TaqMan probe, 5nmol of dATP, 5nmol of dCTP, 5nmol of dGTP, 10nmol of dUTP, 0.05 μmol of magnesium ion, 1.25 μmol of Tris, 2.25 μmol of potassium ion and 7.2% (m/v) of freeze-dried protective agent;
the upstream primers of the COVID-19, FluA, FluB virus and human GAPDH gene, the downstream primers of the COVID-19, FluA, FluB virus and human GAPDH gene, and the TaqMan probe of the COVID-19, FluA, FluB virus and human GAPDH gene;
the preparation steps of the freeze-dried PCR reagent are as follows:
step S1: mixing the reagents, and after uniformly mixing, adjusting the pH value of the mixed solution to 8.7 to prepare a PCR reagent with the final reaction volume of 25 mu L;
step S2: adding the PCR reagent prepared in the step S1 into a PCR tube, putting the PCR tube into a freeze dryer without a cover, and carrying out freeze-drying reaction for 2 hours at the temperature of minus 45 ℃; -45 ℃, 20Pa, 2 h; -45 ℃, 10Pa, 16 h; carrying out freeze-drying reaction at 25 ℃ under the conditions of 10Pa and 2h, and packaging the freeze-dried PCR reagent in a nitrogen environment after the freeze-drying reaction is finished;
step S3: extracting nucleic acid from a sample to be detected to obtain a nucleic acid solution;
step S4: adding the freeze-dried PCR reagent prepared in the step S2 into a nucleic acid solution with the volume of 25 mu L to prepare a mixed solution;
step S5: performing PCR amplification on the mixed solution prepared in the step S4 at 50 ℃ for 5-15min and 94 ℃ for 5 min; PCR amplification was performed at 95 deg.C, 5 seconds, 60 deg.C, 30 seconds, 45 cycles, and fluorescence detection of the FAM/HEX/ROX/CY5 channel.
Example 2
Detection of inactivated COVID-19 virus positive samples:
collecting nasopharyngeal swabs of patients with positive COVID-19 virus, heating and inactivating the swab samples at 56 ℃ in a biological safety laboratory, extracting nucleic acid after inactivation, and detecting the nucleic acid solution of COVID-19 by using the freeze-dried PCR reagent prepared in the example 1, wherein the detection result is shown in figure 2;
as is clear from FIG. 2, the results of detection of the COVID-19 virus and the human GAPDH gene were positive, and the results of detection of the FluA virus and the FluB virus were negative.
Example 3
Detection of inactivated FluA virus positive samples:
collecting nasopharyngeal swabs of patients with FluA virus positive, heating and inactivating the swab samples at 56 ℃ in a biological safety laboratory, extracting nucleic acid after inactivation, and detecting the nucleic acid solution of COVID-19 by using the freeze-dried PCR reagent prepared in the example 1, wherein the detection result is shown in figure 3;
as is clear from FIG. 3, the results of detection of FluA virus and human GAPDH gene were positive, and those of COVID-19 virus and FluB virus were negative.
Example 4
Detection of inactivated FluB positive samples:
collecting nasopharyngeal swabs from patients with FluB virus positive, heating and inactivating the swab samples at 56 ℃ in a biological safety laboratory, extracting nucleic acid after inactivation, and detecting the nucleic acid solution of COVID-19 by using the freeze-dried PCR reagent prepared in example 1, wherein the detection result is shown in FIG. 4;
as is clear from FIG. 4, the results of detection of FluB virus and human GAPDH gene were positive, and those of COVID-19 virus and FluA virus were negative.
Example 5
After the lyophilized PCR reagents prepared in example 1 were stored in a thermostat at 50 ℃, a refrigerator at-20 ℃ and a normal temperature cabinet at 25 ℃ for one month, pseudoviruses containing specific gene sequences of COVID-19 were added to three different lyophilized PCR reagents, and three samples were prepared for each of the lyophilized PCR reagents and tested, the test results are shown in table 1 below;
TABLE 1 storage stability test PCR amplification Ct value statistical Table (ND: not detected)
Figure BDA0002546073180000081
Figure BDA0002546073180000091
As can be seen from Table 1 above, the performance of the lyophilized PCR reagents in samples 1-3 stored at 50 ℃ did not decrease.
The foregoing is merely exemplary and illustrative of the principles of the present invention and various modifications, additions and substitutions may be made in the specific embodiments described by those skilled in the art without departing from the principles of the present invention or exceeding the scope of the claims set forth herein.

Claims (9)

1. A freeze-drying PCR reagent for detecting COVID-19, FluA and FluB viruses is characterized in that: comprises 1-20 units of Taq DNA polymerase, 20-200 units of reverse transcriptase, 0.1-1 unit of uracil-DNA glycosylase, 1-40 units of ribonuclease inhibitor, 1-20pmol of upstream primer, 1-20pmol of downstream primer, 0.5-10pmol of TaqMan probe, 2.5-100nmol of dATP, 2.5-100nmol of dCTP, 2.5-100nmol of dGTP, 2.5-100nmol of dUTP, 0.01-0.2 μmol of magnesium ion, 0.25-2.5 μmol of Tris, 0.25-5 μmol of potassium ion and 2-15% of freeze-drying protective agent of m/v;
the preparation steps of the freeze-dried PCR reagent are as follows:
step S1: mixing the reagents, and adjusting the pH value of the mixed solution to 8.3-8.9 after uniformly mixing to prepare a PCR reagent;
step S2: adding the PCR reagent prepared in the step S1 into a PCR tube, putting the PCR tube into a freeze dryer under the condition of not covering the PCR tube, carrying out freeze-drying reaction, and then packaging the freeze-dried PCR reagent in a nitrogen environment;
step S3: extracting nucleic acid from a sample to be detected to obtain a nucleic acid solution;
step S4: adding the freeze-dried PCR reagent prepared in the step S2 into the nucleic acid solution prepared in the step S3 to prepare a mixed solution;
step S5: the mixture solution prepared in step S4 is subjected to PCR amplification.
2. The lyophilized PCR reagent for COVID-19, FluA, FluB virus detection according to claim 1, wherein: the kit comprises 5 units of Taq DNA polymerase, 50 units of reverse transcriptase, 0.25 unit of uracil-DNA glycosylase, 10 units of ribonuclease inhibitor, 5pmol upstream primer, 5pmol downstream primer, 2.5pmol TaqMan probe, 5nmol dATP, 5nmol dCTP, 5nmol dGTP, 10nmol dUTP, 0.05 μmol magnesium ion, 1.25 μmol Tris, 2.25 μmol potassium ion and 7.2% freeze-drying protective agent m/v.
3. The lyophilized PCR reagent for COVID-19, FluA, FluB virus detection according to claim 2, wherein: the reverse transcriptase is one or two of AMV reverse transcriptase and M-Mulv reverse transcriptase which are mixed in any proportion, the upstream primer is the upstream primer of COVID-19, FluA, FluB virus and human GAPDH gene, the downstream primer is the downstream primer of COVID-19, FluA, FluB virus and human GAPDH gene, the TaqMan probe is the TaqMan probe of COVID-19, FluA, FluB virus and human GAPDH gene, and the freeze-drying protective agent is one or more of trehalose, sucrose, glucose, fucose, sorbitol, mannitol, dextran and polyethylene glycol which are mixed in any proportion.
4. The lyophilized PCR reagent for COVID-19, FluA, FluB virus detection according to claim 3, wherein:
the upstream primer sequence of the COVID-19 virus is as follows:
5’-AGAATGGAGAACGCAGTGGG-3’,
the sequence of the downstream primer of the COVID-19 virus is as follows:
5’-TGAGAGCGGTGAACCAAGAC-3’,
the TaqMan probe sequence of the COVID-19 virus is as follows:
5’-FAM-CGCGATCAAAACAACGTCGGCC-BHQ13’,
the upstream primer sequence of the FluA virus is as follows:
5’-GTTGGTRATGAAACGRAAACGGG-3’,
the sequence of the downstream primer of the FluA virus is as follows:
5’-CCGAATYCTTTTGGTCGCTGT-3’,
the TaqMan probe sequence of the FluA virus is as follows:
5’-VIC-CTCTAGCATACTTACTGACAGC-MGB-3’,
the upstream primer sequence of FluB virus is as follows:
5’-CACAAATGCAACCAGACCTGC-3’,
the sequence of the downstream primer of the FluB virus is as follows:
5’-GGGGAGAGAAAATTCTCCTGCAT-3’,
the TaqMan probe sequence of the FluB virus is as follows:
5’-Cy5-TTAGACAGRATAGCTGCTGGCA-MGB-3’,
the upstream primer sequence of human GAPDH gene is as follows:
5’-ACTTAGAGAAGGGGTGGGCT-3’,
the sequence of the downstream primer of human GAPDH gene is as follows:
5’-ACGCTTGTACACTCAGCATCA-3’,
the TaqMan probe sequence of the human GAPDH gene is as follows:
5’-ROX-ccctgtccagttaatttctgacctttactcctgC-BHQ2-3’,
the 5 'end of the probe is marked with a fluorescent group, and the 3' end of the probe is marked with a quenching group.
5. The lyophilized PCR reagent for COVID-19, FluA, FluB virus detection according to claim 1, wherein: the pH value in step S1 is 8.7, the final reaction volume of the PCR reagent is 25. mu.L or 50. mu.L, and when the final reaction volume is 50ul, the amount of the components except the lyoprotectant is doubled.
6. The lyophilized PCR reagent for COVID-19, FluA, FluB virus detection according to claim 1, wherein: the freeze-drying reaction procedure of the step S2 is-45 ℃ and 2 h; -45 ℃, 20Pa, 2 h; -45 ℃, 10Pa, 16 h; 25 ℃, 10Pa, 2 h.
7. The lyophilized PCR reagent for COVID-19, FluA, FluB virus detection according to claim 1, wherein: the dosage ratio of the freeze-dried PCR reagent and the nucleic acid solution in the step S4 is 25 mu L: 1-25 μ L or 50 μ L: 1 to 50. mu.L, and when the nucleic acid solution is less than 25. mu.L, the volume is made up to 25. mu.L with RNase-free water, and when the nucleic acid solution is less than 50. mu.L, the volume is made up to 50. mu.L with RNase-free water.
8. The lyophilized PCR reagent for COVID-19, FluA, FluB virus detection according to claim 1, wherein: the PCR amplification procedure described in step S5 is: 50 ℃, 5-15min, 94 ℃, 5 min; fluorescence was detected at 95 deg.C, 5 seconds, 60 deg.C, 30 seconds, 45 cycles, FAM/HEX/ROX/CY5 channel.
9. The method for preparing the lyophilized PCR reagent for COVID-19, FluA and FluB virus detection according to claim 1, wherein the method comprises the following steps: the method comprises the following specific steps:
step S1: mixing the reagents, and adjusting the pH value of the mixed solution to 8.3-8.9 after uniformly mixing to prepare a PCR reagent;
step S2: adding the PCR reagent prepared in the step S1 into a PCR tube, putting the PCR tube into a freeze dryer under the condition of not covering the PCR tube, carrying out freeze-drying reaction, and then packaging the freeze-dried PCR reagent in a nitrogen environment;
step S3: extracting nucleic acid from a sample to be detected to obtain a nucleic acid solution;
step S4: adding the freeze-dried PCR reagent prepared in the step S2 into the nucleic acid solution prepared in the step S3 to prepare a mixed solution;
step S5: the mixture solution prepared in step S4 is subjected to PCR amplification.
CN202010561062.5A 2020-06-18 2020-06-18 Freeze-dried PCR reagent for detecting COVID-19, FluA and FluB viruses and preparation method thereof Pending CN111647688A (en)

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