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Pretreatment method, pretreatment solution, kit and use thereof for viral nucleic acid detection

A technology of pretreatment solution and viral nucleic acid, which is applied in microorganism-based methods, biochemical equipment and methods, and microbial determination/inspection, etc. Conducive to long-term storage and detection, improving detection efficiency and shortening detection time

Active Publication Date: 2020-05-26
SANSURE BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the current common virus pretreatment solutions, such as Hank's and guanidine salt-based pretreatment solutions, cannot be used well for subsequent one-step detection, making it difficult to be used in the application scenarios of rapid detection and screening of virus samples

Method used

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  • Pretreatment method, pretreatment solution, kit and use thereof for viral nucleic acid detection
  • Pretreatment method, pretreatment solution, kit and use thereof for viral nucleic acid detection
  • Pretreatment method, pretreatment solution, kit and use thereof for viral nucleic acid detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 The present invention is used for the pretreatment and rapid detection of respiratory syncytial virus (RSV) oropharyngeal swab samples

[0054] In order to evaluate the virus pretreatment solution in this invention, the virus pretreatment solution of this invention (the concentration of Tris-HCl is 100mM, the concentration of EDTA-2Na is 10mM, the concentration of sodium chloride is 0.9% (w / v), The concentration of RNasin was 20U / mL, and the concentration of Proclin 950 was 0.04% (v / v)) were compared with normal saline and commercial virus pretreatment solution. The method for comparison is pretreatment with a clinically diagnosed positive respiratory syncytial virus (RSV) throat swab sample diluted (1:9, v / v). hours / 24 hours / 48 hours and 72 hours for direct amplification of samples, and by comparing the detection efficiency of real-time fluorescent quantitative PCR (real-timeqPCR) under room temperature pretreatment conditions by Ct value, to evaluate the eff...

Embodiment 2

[0059] Example 2 The present invention is used for nucleic acid pretreatment and rapid detection after purification of 2019 novel coronavirus (2019-nCoV) samples

[0060] In order to evaluate the virus pretreatment solution in this invention, the virus pretreatment solution of this invention (the concentration of Tris-HCl is 100mM, the concentration of EDTA-2Na is 10mM, the concentration of sodium chloride is 0.9% (w / v), The concentration of RNasin was 20U / mL, and the concentration of Proclin 300 was 0.01% (v / v)) were compared with normal saline and commercial virus pretreatment solution. The method for comparison is to dilute (1:9, v / v) pretreatment with the nucleic acid of the 2019 novel coronavirus (2019-nCoV) that is clinically diagnosed as positive. hours / 24 hours / 48 hours and 72 hours for direct amplification of samples, and by comparing the detection efficiency of real-time fluorescent quantitative PCR (real-timeqPCR) under room temperature pretreatment conditions by Ct...

Embodiment 3

[0063] Example 3 The present invention is used for pretreatment and rapid detection of enterovirus versatility (EV) throat swab samples

[0064] In order to evaluate the virus pretreatment solution in this invention, the virus pretreatment solution of this invention (the concentration of Tris-HCl is 100mM, the concentration of EDTA-2Na is 10mM, the concentration of sodium chloride is 0.9% (w / v), The concentration of SDS was 0.1%, and the concentration of Proclin950 was 0.04% (v / v)) were compared with normal saline and commercial virus pretreatment solution. The method for comparison is pretreatment with the common enterovirus (EV) throat swab samples clinically diagnosed as positive (1:9, v / v). The pretreatment condition is room temperature 25°C. Samples were directly amplified at 0 hours / 24 hours / 48 hours and 72 hours, and the detection efficiency of real-time qPCR (real-time qPCR) under room temperature pretreatment conditions was compared by Ct value to evaluate the effect ...

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Abstract

The invention relates to the field of virus nucleic acid detection. Specifically, the present invention provides a pretreatment method for viral nucleic acid detection, the method comprising mixing a pretreatment solution containing a sample with a nucleic acid releasing agent and a qPCR amplification reagent, wherein the pretreatment solution includes: Tris-HCl, EDTA-2Na, sodium chloride, ribonuclease inhibitors and antibiotics; wherein the pH of the pretreatment solution is 6.5 to 8.0. The nucleic acid detection reaction solution prepared by the method of the present invention can directly perform qPCR, improve the detection efficiency, and shorten the detection time.

Description

technical field [0001] The invention relates to the field of nucleic acid detection of virus samples; in particular, it relates to a virus sample pretreatment method and a pretreatment solution for pretreatment of DNA / RNA viruses. Background technique [0002] There are two main ways of traditional virus pretreatment solution: the pretreatment solution based on Hank's matrix and the pretreatment solution based on guanidinium salt. Hank's solution is a common balanced salt solution (Balanced Salt Solution, BSS) for virus delivery. Affected by various factors, Hank's solution can only preserve respiratory viruses (such as influenza virus, new coronavirus, etc.) for a few hours. After a few hours, the shape of the virus will be affected, which will affect the nucleic acid detection efficiency of the virus. If If the time is prolonged, bacteria and fungi can easily breed, causing the pH value of Hank's solution to drop and accelerating virus degradation, so it is difficult to u...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6806C12Q1/70C12Q1/6851C12R1/93
CPCC12Q1/6806C12Q1/701C12Q1/706C12Q1/6851C12Q2527/127C12Q2527/125C12Q2527/119C12Q2531/113C12Q2527/137
Inventor 戴立忠纪博知范旭谭德勇邓中平刘佳
Owner SANSURE BIOTECH INC
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