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Detection test kit for virus inactivation, capture and real-time fluorescence isothermal amplification and application thereof

A technology for capturing reagents and kits, used in biochemical equipment and methods, microbial determination/inspection, microorganisms, etc.

Active Publication Date: 2020-06-05
苏州白垩纪生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In order to solve the problems of low sensitivity of the existing 2019-nCoV detection method, long detection time (collection, storage, transportation, inactivation, extraction and detection), easy to cause contamination of amplicons and cause false positive or false negative results of the experiment, In a first aspect, the present invention provides a virus sample inactivation and lysis kit, which includes the following reagents for preparing an inactivation lysis solution: ammonium salt, guanidine isothiocyanate, lithium chloride, ethylenediamine Tetraacetic acid, ethylene glycol bis(2-aminoethyl ether) tetraacetic acid, surfactant, ascorbic acid, dithiothreitol, and buffer

Method used

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  • Detection test kit for virus inactivation, capture and real-time fluorescence isothermal amplification and application thereof
  • Detection test kit for virus inactivation, capture and real-time fluorescence isothermal amplification and application thereof
  • Detection test kit for virus inactivation, capture and real-time fluorescence isothermal amplification and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0125] Preparation of inactivated lysate:

[0126] Use deionized water to prepare inactivated lysate according to the following components and concentrations: 0.2M ammonium salt, 2M guanidine isothiocyanate, 0.5M lithium chloride, 20mM ethylenediaminetetraacetic acid, 50mM ethylene glycol bis(2-amino ethyl ether) tetraacetic acid, 0.2% TritonX-100, 0.5% lithium dodecyl sulfate, 50 mM ascorbic acid, 0.5% dithiothreitol, 100 mM HEPES buffer, pH 5.5.

[0127] Add pseudoviruses of known concentration to 200 μL of the above-mentioned inactivated lysate and PBS solution, shake at room temperature for 20 minutes, and use Invitrogen TM Dynabeads supplied TM SILANE Viral NA Kit is verified, and Invitrogen is not added to those who have used the above inactivated lysate TM The lysis reagent in the kit does not use the lysate and lysis steps of the kit, directly extracts and detects with conventional RT-PCR, the results show that there is no significant difference between the two, whic...

Embodiment 2

[0130] Embodiment 2: the synthesis of intermediate probe sequence

[0131] Entrust Sangon Bioengineering (Shanghai) Co., Ltd. to synthesize SEQ ID NO.3 to 11 and the following intermediate probe sequences:

[0132] 5'biotin Oligo(dT) 15 3'

[0133] 5'biotin-C6-Oligo(dT) 25 VN 3'

[0134] 5'NH 2 -C6-Oligo(dT) 30 3'

Embodiment 3

[0135] Example 3: Preparation of capture magnetic beads Capture MB-1

[0136] 10 mg Dynabeads MyOne Streptavidin T1 (from Invitrogen) was washed twice with PBS, resuspended in 500 μL 5X SSC buffer, and 10 μmol 5’biotin Oligo(dT) was added 15 The 3' intermediate probe was reacted at room temperature for 30 minutes, then magnetically separated, and the remaining probe content was detected in the supernatant. The magnetic beads-intermediate probe was washed 5 times with 2X SSCbuffer, and finally stored in PBS buffer containing 0.1% preservatives and 0.05% Tween20. The content of the probe on the above-mentioned magnetic beads was 935 pmol / mg. Add 800 pmol of SEQ ID NO.3 and 800 pmol of SEQ ID NO.4 to the above-mentioned 10 mg magnetic bead-intermediate probe buffer, react at room temperature for 30 minutes, magnetically separate, and detect that the remaining probe content in the supernatant is zero, that is All capture probes have been bound to the surface of the above-mention...

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Abstract

The invention provides a virus sample inactivation cracking test kit. The virus sample inactivation cracking test kit comprises ammonium salt, guanidine isothiocyanate, lithium chloride, ethylenediamine tetraacetic acid and the like. The invention also provides a target nucleic acid magnetic capture reagent and a target nucleic acid capture test kit. The target nucleic acid magnetic capture reagent and the target nucleic acid capture test kit comprise magnetic polymer microspheres, an intermediate probe, a capture probe and the like. The invention also provides a primer pair and a real-time fluorescent nucleic acid isothermal amplification detection test kit for amplifying and detecting target nucleic acid, and a test kit used for virus inactivation, capture and real-time fluorescent isothermal amplification detection. The inactivation cracking test kit disclosed by the invention can realize quick inactivation at a room temperature and reduce RNA degradation, and is high in sensitivity; the target nucleic acid magnetic capture reagent has stronger specificity, and can obtain templates with good quality and large quantity; and a real-time fluorescent nucleic acid isothermal amplification technology enables reverse transcription and amplification to be carried out together, so the reaction time is shortened, and the sensitivity of a detection reagent is improved.

Description

technical field [0001] The present invention relates to the technical field of biological detection of viruses, in particular to a virus inactivation kit, a virus nucleic acid capture kit, a real-time fluorescent constant temperature amplification detection kit and applications thereof, and in particular to an integration of collection, inactivation and lysis, specific Viruses, especially 2019 novel coronavirus (2019-nCoV) pretreatment and detection related kits and their applications, which combine magnetic capture of sexual targets and real-time fluorescent nucleic acid constant temperature amplification detection technology. Background technique [0002] In the Spring Festival of 2020, the sudden outbreak of novel coronavirus pneumonia continued to spread, and the current situation is still severe. Novel coronavirus (2019Novel Coronavirus, 2019-nCoV) is an RNA virus, its genetic material is ribonucleic acid, and its specific nucleic acid sequence is a marker to distinguis...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6806C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6806C12Q1/6844C12Q2527/125C12Q2523/308C12Q2565/519C12Q2563/107C12Q2521/107Y02A50/30
Inventor 何宗顺曲峰
Owner 苏州白垩纪生物科技有限公司
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