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Freeze-dried PCR (polymerase chain reaction) reagent for detecting COVID-19, FluA and FluB viruses and preparation method thereof

A COVID-19 and virus detection technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of reduced scope of application of kits, aerosol pollution, and increased storage costs, etc., to achieve Prevent aerosol pollution, ensure detection effect, and reduce cost

Pending Publication Date: 2020-09-11
ANHUI GUOTAI GUORUI MEDICAL TREATMENT TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Most of the existing nucleic acid detection products are based on COVID-19 single virus detection products, which can carry out large-scale COVID-19 screening on the population at a low cost, but cannot detect fever symptoms caused by FluA and FluB pathogens. Reduces the scope of application of the kit; the existing nucleic acid detection products are basically liquid dosage forms, and liquid dosage form reagents need to be transported in the cold chain during transportation. Cold chain transportation increases the cost of reagents in the transportation process, and they need to be refrigerated during storage The storage cost is greatly increased, and the risk of reagent failure during storage and transportation is high; existing nucleic acid detection products are easily polluted by PCR product aerosols when detecting PCR products

Method used

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  • Freeze-dried PCR (polymerase chain reaction) reagent for detecting COVID-19, FluA and FluB viruses and preparation method thereof
  • Freeze-dried PCR (polymerase chain reaction) reagent for detecting COVID-19, FluA and FluB viruses and preparation method thereof
  • Freeze-dried PCR (polymerase chain reaction) reagent for detecting COVID-19, FluA and FluB viruses and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Freeze-dried PCR reagents for COVID-19, FluA, FluB virus detection, including 5 units of TaqDNA polymerase, 50 units of reverse transcriptase, 0.25 units of uracil-DNA glycosylase, 10 units of ribonuclease inhibitor, upstream Primer 5pmol, downstream primer 5pmol, TaqMan probe 2.5pmol, dATP 5nmol, dCTP5nmol, dGTP5nmol, dUTP10nmol, magnesium ion 0.05μmol, Tris1.25 μmol, potassium ion 2.25μmol, lyoprotectant 7.2% (m / v);

[0062] The upstream primers of the COVID-19, FluA, FluB virus and human GAPDH gene, the downstream primers are the downstream primers of COVID-19, FluA, FluB virus and human GAPDH gene, and the TaqMan probes are COVID-19, FluA, FluB virus and TaqMan probes for the human GAPDH gene;

[0063] The preparation steps of this lyophilized PCR reagent are as follows:

[0064] Step S1: Mix the above reagents, and after mixing evenly, adjust the pH value of the mixture to 8.7 to prepare a PCR reagent with a final reaction volume of 25 μL;

[0065] Step S2: Add t...

Embodiment 2

[0070] Tests for Inactivated COVID-19 Virus Positive Samples:

[0071] Collect nasopharyngeal swabs from COVID-19 virus-positive patients, heat and inactivate the swab samples at 56°C in a biosafety laboratory, extract nucleic acid from them after inactivation, and use the COVID-19 nucleic acid solution for implementation The freeze-dried PCR reagent that example 1 makes detects, and the detection result is as follows: figure 2 shown;

[0072] Depend on figure 2 It can be seen that the detection results of COVID-19 virus and human GAPDH gene are positive, and the detection results of FluA virus and FluB virus are negative.

Embodiment 3

[0074] Detection of inactivated FluA virus positive samples:

[0075] Collect nasopharyngeal swabs from FluA virus-positive patients, heat and inactivate the swab samples at 56°C in a biosafety laboratory, and extract nucleic acid after inactivation, and use the nucleic acid solution of COVID-19 using Example 1 The prepared freeze-dried PCR reagent was tested, and the test results were as follows: image 3 shown;

[0076] Depend on image 3 It can be seen that the detection results of FluA virus and human GAPDH gene are positive, and the detection results of COVID-19 virus and FluB virus are negative.

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Abstract

The invention discloses a freeze-dried PCR (polymerase chain reaction) reagent for detecting COVID-19, FluA and FluB viruses and a preparation method thereof. The freeze-dried PCR reagent is characterized in that three different upstream primers, downstream primers and TaqMan probes are designed according to the specific gene sequences of the COVID-19, FluA and FluB viruses, so that the freeze-dried PCR reagent can simultaneously detect three pathogens of COVID-19, FluA and FluB for patients with fever symptoms, and judge whether the pathogens capable of causing the fever symptoms are one or more of COVID-19, FluA and FluB. The reagent contains a uracil-DNA glycosylase anti-pollution system, and dUTP is used as a PCR raw material instead of dTTP; and during PCR amplification, the uracil-DNA glycosylase can digest the U-containing PCR product in the PCR product, thereby preventing the PCR product from aerosol pollution and ensuring the detection effect of the freeze-dried PCR reagent.

Description

technical field [0001] The invention belongs to the technical field of virus detection, in particular to a freeze-dried PCR reagent for COVID-19, FluA and FluB virus detection and a preparation method thereof. Background technique [0002] The World Health Organization named the new coronavirus COVID-19. Patients with COVID-19 infection have fever, cough, fatigue, and weakness as the main symptoms, which are similar to those of influenza A virus (FluA) and influenza B virus (FluB) infection symptoms. In addition, unlike the SARS virus (atypical pneumonia virus) that broke out in 2003, COVID-19 is more contagious and has the potential to be long-lasting and seasonal, similar to FluA. [0003] Most of the existing nucleic acid detection products are based on COVID-19 single virus detection products, which can carry out large-scale COVID-19 screening on the population at a low cost, but cannot detect fever symptoms caused by FluA and FluB pathogens. Reduces the scope of applic...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/701C12Q1/686C12Q2521/531C12Q2561/101C12Q2563/107
Inventor 胡康胡林立张成军
Owner ANHUI GUOTAI GUORUI MEDICAL TREATMENT TECH CO LTD
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