RNA virus inactivation preservation solution and application thereof

A technology of RNA virus and preservation solution, which is applied in the field of virus biological detection, can solve the problems of cumbersome virus decrosslinking process, inactivation of RNA virus, and operator hazards, so as to preserve the integrity of RNA, reduce the risk of infection, and save energy. the effect of time

Active Publication Date: 2020-07-03
苏州白垩纪生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

CN110684747A discloses a kind of use formaldehyde solution and paraformaldehyde solution to inactivate respiratory syncytial virus (RSV) and can stabilize RSV virus, but needs the processing time of about 12 hours, and the virus decrosslinking process after formaldehyde treatment is loaded down with trivial details
However, although Trizol preservation solution can preserve RNA samples well, and can also inactivate viruses by heating, it is harmful to operators due to the presence of toxic volatile gases
In addition, RNase inhibitors are used to preserve RNA samples, but the cost is very expensive and RNA viruses cannot be inactivated, so their application is greatly limited

Method used

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  • RNA virus inactivation preservation solution and application thereof
  • RNA virus inactivation preservation solution and application thereof
  • RNA virus inactivation preservation solution and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0065] (S1) The formula of RNA virus inactivation preservation solution: 2M ammonium chloride, 0.5M 1-ethyl-3-methylimidazolium bisulfate, 0.2M tartaric acid, 0.25M lithium chloride, 20mM ethylenediaminetetraacetic acid , 5 mM ethylene glycol bis(2-aminoethyl ether) tetraacetic acid, 1% (w / v) lithium dodecyl sulfate, 0.5% (w / v) Triton X-100, 0.2% (w / v) Tween 20, 0.05M sodium pyrophosphate, 1% (w / v) dithiothreitol, 50 mM HEPES buffer, pH5.

[0066] The contrast solution used VTM sterile virus transport solution preservation solution (preservation solution prepared by Hank’s solution) (from Shenzhen Huachenyang Technology Co., Ltd.) and Buffer AVL (lysate) in the Viral RNA Mini Kit, the swabs are from Italian COPAN disposable flocking swabs, the pseudovirus (SARS-CoV2) is the quality control substance containing the N gene fragment (Shanghai Yisheng Biotechnology Co., Ltd.), kit use Viral RNA Mini Kit for manual operation. The real-time PCR detection reagents were prepared ...

Embodiment 2

[0075]Using the recipe (S1) of the RNA virus inactivation preservation solution in Example 1 and the sample collection and processing methods in Example 1, the storage of the inactivation preservation solution in Example 1 at different temperatures was investigated. Among them, after air-drying the high-value samples on the surface of the swab, add 2 mL of the preservation solution (S1) of Example 1, vortex and shake for 10 minutes, take out the swab, and store the preservation solution at -20°C, 4°C, At 25°C and 56°C for a period of time, the control group was inactivated with the preservation solution of group B at 56°C (the control group is the method recommended by the Centers for Disease Control and Prevention (CDC) at this stage). 400 μL was taken out at each time point for extraction and RT-PCR test. From Table 3, we found that after treatment at 56°C for 30 minutes, the amount of templates detected in group A did not decrease, while the Ct of group B changed by more th...

Embodiment 3

[0081] Adopt the formula (S1) of embodiment 1 RNA virus inactivation preservation liquid and the mode of embodiment 1 sample collection processing to three groups of preservation liquids: embodiment 1 formula preservation liquid S1 (group A), VTM aseptic virus transport liquid preservation liquid ( Group B) and BufferAVL (group C) were investigated with different kits, and the kits were respectively using MagMAX TM Viral RNA Isolation Kit (magnetic bead virus RNA extraction kit), provided by Qiagen Viral RNA Mini (column method virus RNA extraction kit) and Trizol method extraction kit (solvent method RNA extraction kit). The results are shown in Table 4. We found that whether Group A was used in high-value samples or low-value samples, after using different kits for extraction, the RT-PCR test results showed that the difference in Ct value was within 1; The solution is suitable for the column method, but the compatibility with the magnetic bead method is reduced, and it can...

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Abstract

The present invention discloses an RNA virus inactivation preservation solution. The inactivation preservation solution comprises ammonium salt, 1-ethyl-3-methylimidazolium bisulfate, lithium chloride, ethylenediaminetetraacetic acid, an RNase inhibitor, a surfactant, a reducing agent, a buffer agent and water. The present invention also provides an RNA virus sample inactivation and preservation method and an RNA virus detection method, and the method is conducted by using the inactivation preservation solution. The preservation solution has low cost, inhibits RNase activity, preserves RNA integrity, realizes preservation and transportation of samples at room temperature, can also quickly inactivate pathogens at room temperature, reduces infection risks of medical personnel, and reduces false negatives in detection. At the same time, the inactivation preservation solution can realize a lysis function, does not contain chaotropic reagents and alcohols, is compatible with mainstream nucleic acid extraction kits on the market, greatly saves time of an entire process, and is particularly suitable for 2019 new coronavirus (COVID-19) inactivation and lysis steps after swab sampling.

Description

technical field [0001] The invention relates to the technical field of biological detection of viruses, in particular to a preservation solution for inactivating an RNA virus and its application. Background technique [0002] Regarding the new type of coronavirus (COVID-19, referred to as: new coronavirus) pneumonia, on the one hand, it is known that the epidemic prevention departments and hospitals regard the positive nucleic acid test of the new coronavirus as the gold standard for diagnosis; The PCR tests were all negative, and there was even a report that a case did not detect a positive result until the seventh test. Therefore, how to avoid the false negative rate of nucleic acid detection is the biggest dilemma encountered by current PCR detection. [0003] The "Diagnosis and Treatment Plan for Novel Coronavirus Infected Pneumonia" has two criteria for the diagnosis of infection cases: 1) positive real-time fluorescence RT-PCR detection of new coronavirus nucleic acid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/06C12N15/10C12Q1/70C12Q1/686
CPCC12N7/00C12N15/10C12Q1/68C12Q1/701Y02A50/30
Inventor 曲峰何宗顺
Owner 苏州白垩纪生物科技有限公司
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