CRSPR-cas13a driven RNA rapid detection method based on double-enzyme signal amplification strategy

A detection method and enzyme signal technology, which is applied in the field of rapid RNA detection based on a dual-enzyme signal amplification strategy, can solve problems such as difficulty in reaching the detection range of nucleic acid sequence concentration, limited RNA content in biological fluids, and complex components in biological fluids. Sensitivity, fast separation speed, and simple instrument requirements

Pending Publication Date: 2021-03-16

台州市中心医院

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Problems solved by technology

[0004] The purpose of the present invention is to provide a CRSPR-cas13a-driven RNA rapid detection method based on a dual-enzyme signal amplification strategy to solve the problem of relatively complex biological fluid components and ...

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Abstract

The invention discloses a CRSPR-cas13a driven RNA rapid detection method based on double-enzyme signal amplification strategy in the technical field of biological detection. The RNA rapid detection method based on the double-enzyme signal amplification strategy comprises the following steps: collecting a sample; treating a sample; assembling a magnetic separation probe; amplification and separation of primary enzymatic signal specifically triggered by a target sequence; amplification of secondary enzymatic signal and sending the signal; sending the detection signal of the target sequence. According to the present invention, the nucleic acid detection sequence is controllable, a suitable leader sequence can be designed according to the actual requirement so as to control the identified target sequence, which has extremely high specificity, extremely high sensitivity and good color development effect, and the detection results can be judged by naked eyes without relying on analytical instruments, the separation speed is high, the requirement of instrument is simple, and can be used to treat multiple groups of samples simultaneously, so that the manpower and the cost are saved.

Application Domain

Microbiological testing/measurement

Technology Topic

Collection sampleEnzyme +8

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[0032] Next, the technical solutions in the embodiments of the present invention will be apparent from the embodiment of the present invention, and it is clearly described, and it is understood that the described embodiments are merely embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, there are all other embodiments obtained without making creative labor without making creative labor premises.

[0033] The present invention provides a RNA rapid detection method based on a double enzyme signal amplification strategy driven by CRSPR-CAS13A, and the nucleic acid detection sequence can be controlled, and the appropriate guidance sequence can be used to control the identified target sequence according to actual needs. The specificity and extremely high sensitivity, with a good color effect, can determine the analytical instrument in the case of qualitative case, the naked eye can determine the test results, the separation speed is fast, and the requirements of the instrument are simple, can process more simultaneously Group samples saved manpower and cost, please refer to figure 1 ,

[0034] The RNA rapid detection method based on the double enzyme signal amplification strategy includes the following steps:

[0035] S1: Sample Collection: Animal or human blood sample collection, animal or human urine sample collection, animal or human saliva sample collection, including the following steps:

[0036] A1: Animal or human cell culture medium collection: Cell culture medium DMEM, add volume fraction of 10% calf serum (FCS), cell culture dish, transfer to cell culture dishes, saturated at 37 ° C, 5% CO2 The water vapor carbon dioxide incubator was cultured for 24 hours, allowing the cells to mix 50% -60%, then cultured for 24-48 hours without serum DMEM culture solution, recovering the serum DMEM cell culture medium, stored at 4 ° C for a short period of time, long-term Plus-80 ° C save;

[0037] A2: Animal or human liquid sample collection: animal or human intravenous blood, add anticoagulant, centrifugation to remove cellular ingredients in the blood, to obtain plasma; or take blood to take precautions, obtained blood sample-80 ° C Preservation Or use immediately, collect the urine of the animal or human to the urine, to get the urine sample, try to avoid contamination of urine samples, immediately use or store it at -80 ° C;

[0038] A3: Cell collection: adhesive and tissue cells were obtained by 0.2% trypsin digestion for 1 minute, 1000 rpm is centrifuged for 5 minutes. If not immediately use, it should be frozen into a negative eighty or liquid nitrogen, non-adjective The cells are directly centrifuged. If they are not used immediately, they should be frozen to a negative eighty refrigerator or liquid nitrogen;

[0039] S2: Treatment of the sample: Includes the pre-processing of the sample and the acquisition of RNA in the sample, including the following steps:

[0040] B1: Pre-treatment of the sample: Blood samples need to be removed from 1500 rpm for 30 minutes, and the plasma components, urine and cell culture fluids generally need to concentrate after concentration of plasma components, urine and cell culture fluids.

[0041] B2: RNA Extraction: The use of commercial kits after treatment or by TRIZOL method;

[0042] S3: Assembly of magnetic separation probes: including a buffer solution, an amino modified signal probe and a carboxyl magnetic bead to activate and amino modified signal probes and carboxyl magnetic beads, including the following steps:

[0043] C1: Activated carboxyl magnetic beads: Electronic balance weigh 9.76 g 3, 29.22 g NaCl, 0.191GeDC, 0.115GNHS, 9.76GMES, 29.22 g NaCl dissolved in 900 ml of ultra-pure water. PH-6 with 3mol / L sodium hydroxide, and will volume Set to 1L, formulated into MES buffer, EDC, NHS, dissolved in 10 ml of centrifuge tube, separately 50 μl of the carboxyl magnetic beads, and remove the protective reagent, magnetic separation removal protective reagent, add 1 ml Me Cushion to the EP tube The liquid is mixed, and the 100μLEDC solution was added to the magnetic bead solution, and the room temperature was placed for half an hour, and 100 μlNHS solution was added to the magnetic bead, and the activation was allowed to activate at room temperature, and the EP tube was placed in a magnetic separator, allowed for 30 seconds, Remove the liquid to retain the carboxy magnetic bead to obtain a carboxyl bead after activation;

[0044] C2: Carboxy magnetic beads and signal probes Based: The activated carboxyl magnetic bead is dissolved in 1 mlpbs buffer, and the signal probe is added, and the temperature after 24 hours is shaken at room temperature, and the solution after the above reaction is placed in a magnetic separator, still 30s And remove the liquid portion to retain the magnetic bead, and the modified magnetic bead potential changes, and the probe can be combined to the magnetic bead surface by detection of fluorescence probe.

[0045] C3: Activation of Streptaviarin Ham Root Peroxidase: Electronic Balance Weigh 0.292GEDta, 29.22 g NaCl, 2.423gtris in 1L beaker, add water to 800ml, stir mixed under magnetic stirrer, resulting The solution was transferred to 1 L to 1 L to 1L and the pH was adjusted to 7.5 with 1 mol / L of HCl, and the obtained solution was filtered, and the reaction liquid was mixed, and the streptavidin hosporque hydrogen peroxidase was obtained. The mother liquor was placed in 1.5MLEP tube, and 375 μl of reaction solution was added to obtain activated form of streptocin;

[0046] C4: Streptaviarin conjugated horseradish peroxidase and probe binding: Electronic balance weigh 0.05 mNAcl, 20 mmtris-HCl, 7.51mMedta in the beaker, stir mixed under magnetic stirrer, 500 μl reaction Liquid I, the activated streptavidin homocaroxide, added to the magnetic bead mix, place the mixed liquid in the constant temperature shaker, constant temperature clocked bed 60R / s, room temperature for 30 minutes, and place the mixed liquid In the magnetic separator, stand for 30 seconds, remove the supernatant, retain the magnetic beads that bind to the probe, the magnetic beads, the modified magnetic beads, and the modified magnetic beads have horseradish peroxidase activity, which can make the substrate Generate color reactions;

[0047] S4: Target sequence specific triggering primary enzymatic signal amplification and separation: primary enzymatic signal amplification and separation of target sequences: the RNA obtained in the sample is added to 100 μL modified signal probe magnetic beads, The 10 nM purified LBucas 13a, 10 nm crRNA was synchronized synchronously, and mixed well, placed at room temperature for incubation, and the incubated sample was placed in a magnetic separator frame, stand, and collected a solution portion;

[0048] S5: The secondary enzymatic signal amplification and signal given: the primary reaction product prepared by step S4 is recovered, and the liquid portion 50 ul is recovered, and the 50 ul of the reaction substrate is added at the same time, and the reaction is 10-30 min. Pay attention to the change of color changes to avoid missed the optimal measurement time;

[0049] S6: The nucleic acid sequence detection signal is given to the following steps:

[0050] D1: Semi-quantitative analysis: Terrace after the end of the reaction system, 96-well plates, and measure the absorption value at 450 nm, compared to the control group and the positive group;

[0051] D2: Quantitative Analysis: Set the concentration gradient of the target sequence in step S3, followed by the above process, and the termination reaction system is placed into the enzyme gospeller, measure the absorption value at 450 nm, and the measured value Do linear regression, compare the target sequence concentration of the sample group;

[0052] D3: Qualitative Analysis: Contrast the terminated reaction system after termination, observing differences with the control group, if there is significant difference, indicating that the target sequence is present.

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High sensitivity
Good color rendering

CRSPR-cas13a driven RNA rapid detection method based on double-enzyme signal amplification strategy

AI-Extracted Technical Summary

Problems solved by technology

Abstract

Image

Examples

Example Embodiment

PUM

Description & Claims & Application Information

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