Probe, freeze-drying reagent, lateral flow chromatography test strip, kit and method for detecting RNase activity

Abstract

The invention provides a probe, a freeze-drying reagent, a lateral flow chromatography test strip, a kit and a method for detecting RNase activity, and relates to the technical field of biology. According to the present invention, based on the specific antigen-antibody reaction and the immunochromatography technology, the RNA probe with the 5'end labeled with digoxin and the 3 'end labeled with biotin is selected as the substrate, and under the condition that the sample to be detected contains RNase and is mixed with the substrate, the RNase can specifically recognize and cut the phosphodiester bond on the RNA probe so as to fragment the RNA; then, the test strip is inserted into the reacted reagent, if the sample does not contain RNase, the RNA probe is not subjected to enzyme digestion and can be fixed on the quality control line and combined with streptavidin, the quality control line shows a red strip, and the detection line does not develop color; if RNase exists in the sample, the RNA probe is fixed on the detection line after being subjected to enzyme digestion and is combined with the secondary antibody, and a red strip appears on the detection line.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a probe, a lyophilized reagent, a lateral flow chromatography test strip, a kit and a method for detecting RNase activity. Background Art

[0002] Ribonuclease (RNase) can be classified according to substrate specificity and source. In terms of substrate specificity, there are RNase A family and RNase H family. RNase A can act specifically on single-stranded RNA and prefers to cleave the phosphodiester bond at the pyrimidine nucleotide;

[0003] The RNase H family hydrolyzes RNA hybridized with DNA. From the source, it includes endogenous RNases in cells, such as RNaseL, and exogenous RNases, such as RNase I of Escherichia coli. In molecular biology experiments, many processes involving RNA manipulation require attention to RNase residues, among which the enzymes that routinely need to detect residual enzymes are mainly RNase A. Because RNase A is widely present in the environment and has strong activity, it is very easy to degrade RNA, which has a huge impact on subsequent experiments such as RNA extraction and reverse transcription. Its main method of action is through an acid-base catalytic mechanism. The acid-base amino acid residues in the active center cooperate with each other to promote the hydrolysis of RNA substrates, and can also recognize specific nucleotide sequences or structures for cutting.

[0004] In the field of molecular biology, RNase activity or residue detection is of great significance. First of all, for RNA extraction, it is the key to ensure quality. It can determine whether the reagents, consumables and experimental environment used in the extraction process are contaminated by RNase. Only by eliminating the contamination can high-quality and complete RNA be obtained to ensure the smooth progress of subsequent experiments such as reverse transcription reactions and gene expression analysis. Secondly, in various RNA-related enzymatic reactions, it can ensure that the experimental results are accurate, reliable and repeatable, and avoid reaction failure or result deviation due to RNase contamination, which is especially important when dealing with trace RNA samples. In addition, in the production of RNA-based biological products, such as mRNA vaccine production, strict detection of RNase activity or residue is a necessary link to ensure product quality and stability, and prevent RNase from destroying RNA and affecting product efficacy and safety. There are currently a variety of detection technologies, each with its own characteristics and limitations.

[0005] The detection method based on electrophoresis is to incubate RNA with the sample to be tested and then perform electrophoresis to observe the changes in RNA bands to determine the RNase situation. Its advantages are that the operation is relatively simple, the cost is not high, and the integrity of the RNA can be intuitively seen. However, the disadvantages are also quite significant. The sensitivity is low, a small amount of RNase activity or trace residues are difficult to detect, false negative results are prone to occur, and it is difficult to achieve accurate quantification. It can only roughly determine whether the RNA is degraded. At the same time, the detection takes a long time. It takes a lot of time from sample processing to obtaining results, which is not conducive to rapid detection.

[0006] Radioactive labeling detection method, first radioactively label the RNA substrate, then incubate with the sample, and detect the radioactive fragments to determine the RNase activity. The advantage of this method is that it is highly sensitive and can accurately track the RNA degradation process. However, it has radioactive hazards, poses a threat to the health of experimenters and environmental safety, waste disposal is complicated and extremely costly, and the labeled substrate and detection instruments are expensive. The operation is also very complicated, requiring professional training, the process is cumbersome, and it is easy to make mistakes, affecting the reliability of the results.

[0007] The fluorescence detection method uses fluorescently labeled RNA substrates to detect RNase activity based on changes in fluorescence signals. Its advantages are high sensitivity, rapid reaction, and relative safety. However, the specificity is poor, and non-RNase factors such as pH changes and chemical interference may lead to false positives, so experimental conditions need to be strictly controlled. The background interference is large, and endogenous fluorescent substances in biological samples will affect the accuracy of the results, requiring complex corrections. In addition, there are problems with the stability of fluorescent labels, and label shedding or quenching will affect the reliability of detection.

[0008] Colorimetric method detects by the color change after RNase degrades the chromophore-containing substrate. It is simple to operate, low-cost, and suitable for large-scale screening. However, it has low sensitivity and accuracy, is insensitive to low-activity RNase or trace residues, and may miss detection. At the same time, it is easily affected by the environment and sample matrix, making it difficult to accurately quantify RNase activity, and can only roughly determine the activity range.

[0009] In summary, the prior art lacks a simple, accurate, efficient and low-cost method for detecting RNase activity. Summary of the invention

[0010] The purpose of the present application is to provide a probe, a lyophilized reagent, a lateral flow chromatography test strip, a kit and a method for detecting RNase activity, aiming to solve the problems existing in the existing RNase activity detection methods.

[0011] To achieve the above objectives, the present application provides a probe for detecting RNase activity, wherein the probe is a single-stranded RNA of 20-60 nucleotides, the 5' end of the probe is labeled with digoxigenin, and the 3' end of the probe is labeled with biotin.

[0012] In some embodiments, the probe is used at a concentration of 0.5-1 pmol / reaction; the total reaction volume of each test reaction in the present application is 20 μL.

[0013] Preferably, the probe is used at a concentration of 1 pmol / reaction.

[0014] The present application also provides a lyophilized reagent for detecting RNase activity, comprising the above-mentioned probe for detecting RNase activity.

[0015] In some embodiments, 10× RNA reaction buffer and 2× lyophilization protectant are also included;

[0016] The 10× RNA reaction buffer comprises Tris-HCl, MgCl2, CaCl2 and Tween-20;

[0017] The 2× lyophilization protective agent includes trehalose and casein;

[0018] Preferably, the 10× RNA reaction buffer comprises 500 mM Tris-HCl, 100 mM MgCl2, 5 mM CaCl2 and 1% by volume Tween-20;

[0019] Preferably, the pH value of the 10× RNA reaction buffer is 7.5;

[0020] Preferably, the 2× lyophilization protective agent comprises 4%-8% by mass of trehalose and 2%-4% by mass of casein;

[0021] Preferably, the 2× lyophilization protectant comprises 6% by mass of trehalose and 2% by mass of casein.

[0022] The present application also provides a method for preparing the above-mentioned lyophilized reagent for detecting RNase activity, comprising:

[0023] The probe, the 10× RNA reaction buffer, the 2× freeze-drying protective agent and enzyme-free water are mixed and then freeze-dried to obtain the freeze-dried reagent;

[0024] Preferably, the freeze-drying process includes: pre-freezing, sublimation drying and analytical drying;

[0025] The pre-freezing temperature is -60°C to -50°C, and the maintenance time is 120 minutes;

[0026] The sublimation drying temperature is -30°C, the heating time from the pre-freezing temperature to the sublimation drying temperature is 30 minutes, and the maintenance time is 600 minutes;

[0027] The analytical drying temperature is 20° C. to 30° C., the heating time from the sublimation drying temperature to the analytical drying temperature is 60 minutes, and the maintaining time is 180 minutes.

[0028] The present application also provides a lateral flow chromatography test strip for detecting RNase activity, comprising a bottom plate, a sample pad, a conjugate pad, a nitrocellulose membrane, and a water-absorbing pad;

[0029] The nitrocellulose membrane is fixed with a quality control line and a detection line, the quality control line is coated with streptavidin, and the detection line is coated with rabbit anti-mouse IgG antibody; the conjugate pad is fixed with mouse anti-digoxigenin antibody.

[0030] In some embodiments, the mouse anti-digoxigenin antibody is labeled by covalent coupling to red latex microspheres to form a mouse anti-digoxigenin antibody microsphere complex, and the microsphere complex is bound to the conjugate pad;

[0031] Preferably, the particle size of the red latex microspheres is 200 nm;

[0032] Preferably, the concentration of the streptavidin is 1 mg / mL; the concentration of the rabbit anti-mouse IgG antibody is 1 mg / mL.

[0033] The present application also provides a kit for detecting RNase activity, comprising the above-mentioned probe for detecting RNase activity, or comprising the above-mentioned lyophilized reagent for detecting RNase activity, and comprising the above-mentioned lateral flow chromatography test strip for detecting RNase activity.

[0034] The present application also provides a method for detecting RNase activity, comprising:

[0035] Adding RNase or a sample to be detected into the above-mentioned freeze-dried reagent solution for detecting RNase activity to react and obtain a detection solution;

[0036] The lateral flow chromatography test strip for detecting RNase activity is inserted into the test solution, and the RNase activity or whether the sample to be tested contains RNase is determined based on the quality control line and the detection line.

[0037] In some embodiments, the total reaction volume of the reaction is 20 μL, and if it is less than 20 μL, it is supplemented with enzyme-free water;

[0038] Preferably, the reaction temperature is 37°C and the reaction time is 30 min;

[0039] Preferably, the detection solution is supplemented to 60 μL by adding 40 μL of pure water diluent.

[0040] Compared with the prior art, the beneficial effects of this application include:

[0041] The method for detecting RNase activity provided by the present application relies on specific antigen-antibody reaction and immunochromatography technology, selects an RNA probe labeled with digoxin at the 5' end and biotin at the 3' end as a substrate, and carries out an enzymatic reaction under suitable conditions when the sample to be tested contains RNase and is mixed with the substrate. During this process, RNase will specifically recognize and cut the phosphodiester bond on the RNA probe, causing RNA fragmentation. The test strip is then inserted into the reagent after the reaction. If the sample does not contain RNase, the RNA probe will not be subjected to enzyme cleavage, and will be fixed on the quality control line and combined with streptavidin. At this time, the quality control line will show a red strip, and the test line of the test strip will not show color; if RNase is present in the sample, the RNA probe will be fixed on the test line after enzyme cleavage and combined with the second antibody, then a red strip will appear on the test line. When all RNA probes in the sample are completely cleaved, the quality control line will not show color.

[0042] The present application uses an immunoassay strip to identify whether the probe has been enzymatically degraded. Compared with traditional fluorescent probes, it can detect samples that are fluorescent in themselves and has a wider range of applications.

[0043] This application uses test strips to interpret the results, which has low sample requirements, simple operation, short interpretation time, accurate results, high sensitivity, low price, no need for large equipment, and can achieve rapid on-site testing.

[0044] The reaction reagent of the present application is a freeze-dried reagent, which is simpler to operate than a liquid reagent and does not require a reagent preparation step. Only the sample reagent needs to be added. At the same time, the freeze-dried reagent can improve the storage stability of the RNA probe. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope of the present application.

[0046] Figure 1 A schematic diagram of the process for detecting RNase activity of the present application;

[0047] Figure 2 This is a diagram of the probe screening results of this application;

[0048] Figure 3 This is a graph showing the probe concentration optimization results for this application;

[0049] Figure 4 This is a graph showing the probe concentration determination results for this application;

[0050] Figure 5This is a graph showing the screening results of the 10× RNA reaction buffer of this application;

[0051] Figure 6 This is a diagram of the screening results of the lyoprotectant of this application;

[0052] Figure 7 This is a graph showing the optimization results of the freeze-drying protective agent casein concentration of the present application;

[0053] Figure 8 This is a graph showing the optimization results of the freeze-drying protective agent trehalose concentration in this application;

[0054] Fig. 9 This is a diagram showing the screening results of the freeze-dried protective agent formula of this application;

[0055] Fig.10 This is a detection limit result diagram of the kit for detecting RNase activity of the present application;

[0056] Fig.11 This is a graph showing the reproducibility of the kit for detecting RNase activity of the present application;

[0057] Fig.12 This is a graph showing the stability results of the kit for detecting RNase activity of the present application. DETAILED DESCRIPTION

[0058] As used herein:

[0059] "Prepared from" is synonymous with "comprising." As used herein, the terms "comprising," "including," "having," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0060] The conjunction "consisting of excludes any unspecified element, step, or component. If used in a claim, this phrase renders the claim closed-ended so that it does not include materials other than those described, except for conventional impurities associated therewith. When the phrase "consisting of" appears in a clause of the body of a claim rather than immediately following the subject matter, it limits only the elements described in that clause; other elements are not excluded from the claim as a whole.

[0061] When an amount, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted as including ranges "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described in this article, unless otherwise stated, the range is intended to include its end values ​​and all integers and fractions within the range.

[0062] The embodiments of the present application will be described in detail below in conjunction with specific examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present application and should not be considered as limiting the scope of the present application. If specific conditions are not specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0063] (1) The main reagents used in the examples of this application are shown in Table 1 below.

[0064] Table 1 Reagent information

[0065]

[0066]

[0067] (2) The main materials used in the examples of this application are shown in Table 2 below.

[0068] Table 2 Material information

[0069]

[0070] (3) The main experimental instruments used in the examples of this application are shown in Table 3 below.

[0071] Table 3 Experimental instrument information

[0072] Instrument Name Instrument Model Manufacturer Mini Metal Bath G100517 Bioengineering Film Spraying Instrument HM3030 Shanghai Gold Label Strip cutting machine ZQ2002 Shanghai Gold Label Freeze Dryer Pilot2-4M Bo Yi Kang

[0073] The probes used in the examples of the present invention were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0074] Example 1 Method for detecting RNase activity

[0075] See also Figure 1 The method for detecting RNase activity of the present application comprises the following steps:

[0076] 1. Add RNase or sample to the reaction tube containing the freeze-dried reagent of the probe for detecting RNase activity of the present application. The total reaction volume is 20 μL. If it is less than 20 μL, add enzyme-free water to make up.

[0077] 2. After vortexing and mixing, spin for a few seconds and place at 37℃ for 30 minutes. The reaction time can be extended appropriately.

[0078] 3. After the reaction is completed, add 40 μL of pure water to the reaction product to make up to 60 μL and mix well. Insert the sample pad end of the test strip for detecting RNase activity of the present application into 60 μL of liquid, and the liquid level should not be higher than the marked line.

[0079] 4. Record the test results in the interpretation area within 5 minutes after starting chromatography.

[0080] When there is a band in the detection area (T), it means that there is RNase in the sample, and the enzyme activity can be roughly judged based on the depth of the band.

[0081] Example 2 Design and screening of probes for detecting RNase activity

[0082] First, the following probes were designed and synthesized, and five probes of different lengths were designed by screening random AUCG sequences and UG special sequences of different lengths.

[0083] (1) SEQ ID NO. 1: DIG 5'-AUACAGCUUUGGUGCCACCUGCG UGAAGAAGUGUCCCCGU-3'Biotin, full length 40 bp;

[0084] (2) SEQ ID NO. 2: DIG 5'-AUACAGCUUUGGUGCCACCU-3'Biotin, full length 20 bp;

[0085] (3) SEQ ID NO.3: DIG 5'-CCCCAAAGAGAGCUCAUGGCCCA ACCACACCGUAACCGGAGUAUCAGCAUCAUGCUCCCA-3'Biotin, full length 60bp;

[0086] (4) SEQ ID NO.4: DIG 5'-CCCCAAAGAGAGCUCAUGGC-3'Biotin, full length 20 bp;

[0087] (5) SEQ ID NO.5: DIG 5'-UGUGUGUGUGUGUGUGUGG-3'Biotin, full length 20bp.

[0088] Then, according to the detection scheme described in Example 1, 1 pmol or more of the probe was added to each test, and 10 -1 / 10 -2 / 10 -3 / 10 -4 / 10 -5 / 10 -6 / 10 -7 mg / reaction RNase detection situation, and enzyme-free water was used as a negative control. The detection was carried out according to the detection method steps of Example 1.

[0089] The results are as follows Figure 2 As shown, all five probes can detect 10 -6 mg / reaction RNase, and there were no false positives. -6 When the detection line (T) of SEQ ID NO.5 was the darkest and the quality control line (C) was the lightest, SEQ ID NO.5 was selected as the preferred detection probe of the present application and used as the probe material for subsequent experiments.

[0090] Example 3 Probe Concentration Determination

[0091] SEQ ID NO.5 was used as the test probe, and the following probe dosages were used, including: 1 / 0.5 / 0.25 / 0.125 / 0.0625 / 0.031 pmol / reaction, to test the lowest concentration of the test strip to detect false positives. The results are as follows Figure 3 As shown, a minimum of 0.5 pmol / reaction can be added without false positives, so the probe concentration is tentatively set at 0.5 pmol / reaction and 1 pmol / reaction.

[0092] The probe concentration was further determined. At probe concentrations of 0.5 pmol / reaction and 1 pmol / reaction, different concentration gradients of RNase were tested (the concentration gradients were 1 pg / μL, 0.5 pg / μL, 0.25 pg / μL and 0.125 pg / μL, and enzyme-free water was used as a negative control). The reaction system was prepared with 2 μL of 10× RNA reaction buffer, 1 μL of probe, 1 μL of RNase and 16 μL of enzyme-free water, and the test was performed according to the detection method steps of Example 1.

[0093] The results are as follows Figure 4 As shown, both groups can detect up to 0.125pg / reaction, but 0.5pmol / reaction is prone to false positives after 5 minutes. To ensure that no false positives occur, the probe amount is set at 1pmol / reaction.

[0094] Example 4 Optimization of 10× RNA Reaction Buffer

[0095] First, in this experimental example, two 10× RNA reaction buffers were prepared according to Table 4, and then 2 μL of 10× RNA reaction buffer was added to each 20 μL reaction system, and 1 pmol / reaction SEQ ID NO.5 was used as a test probe to test RNases of different concentration gradients (1 pg / reaction, 0.5 pg / reaction, 0.25 pg / reaction and 0.1 pg / reaction, and enzyme-free water was used as a negative control).

[0096] Table 5 Compositions of two 10× DNA reaction buffers

[0097]

[0098] The results are as follows Figure 5 As shown in the figure, Buffer ① can detect to 0.1pg / reaction, and Buffer ② can detect to 0.25pg / reaction. Buffer ① is better than Buffer ②. Therefore, Buffer ① is selected as the final 10× RNA reaction buffer.

[0099] Example 5 Optimization of 2× Lyoprotectant

[0100] First, the lyoprotectant components were screened.

[0101] To each 20 μL reaction system, 10 μL of four 2× lyophilized protectants (2% casein by mass, 8% sucrose by mass, 8% trehalose by mass and 8% mannitol by mass, each prepared with enzyme-free water) were added, 2 μL of 10× RNA reaction buffer was added, 1 pmol / reaction SEQ ID NO.5 was used as the test probe, and different concentration gradients of RNase (1 pg / reaction, 0.5 pg / reaction, 0.25 pg / reaction, 0.125 pg / reaction) were tested, and enzyme-free water was used as a negative control.

[0102] The results are as follows Figure 6 As shown, the final concentration of 1% by mass casein and 4% by mass trehalose has the best effect.

[0103] Further, trehalose and casein were selected and their added concentrations were screened.

[0104] A 2× casein solution was prepared with water, and the concentration was set to 2% and 4% by mass. 10 μL of the above 2× casein solution was added to every 20 μL reaction system, and then 2 μL of 10× RNA reaction buffer was added. 1 pmol / reaction SEQ ID NO.5 was used as the test probe, and different concentration gradients of RNase (1 pg / reaction, 0.5 pg / reaction, 0.25 pg / reaction, 0.1 pg / reaction) were tested respectively, and enzyme-free water was used as a negative control.

[0105] The screening results of casein at different concentrations are as follows Figure 7 As shown, the detection sensitivity is consistent. From the 0.5 pg / reaction group, the C line of the final concentration of 1% casein is weaker. Therefore, the use of casein with a final concentration of 1% by mass is more effective, so 2% by mass casein is preferably used as the 2× freeze-drying protectant.

[0106] 2× trehalose solution was prepared with water with concentrations of 4%, 6% and 8% by mass, 10 μL of the above 2× trehalose solution was added to every 20 μL reaction system, and then 2 μL of 10× RNA reaction buffer was added. 1 pmol SEQ ID NO.5 was used as the test probe, and different concentration gradients of RNase (1 pg / reaction, 0.5 pg / reaction, 0.25 pg / reaction, 0.1 pg / reaction) were tested, and enzyme-free water was used as a negative control.

[0107] like Figure 8 As shown, the effect of trehalose at a final concentration of 3% by mass is better than that of trehalose at 2% by mass and 4% by mass, the T line of 0.1 pg / reaction is deeper, and the C line of 0.25 pg / reaction is shallower. Therefore, trehalose at a final concentration of 6% by mass is preferably used as a 2× lyoprotectant.

[0108] Furthermore, the combination formula of the lyophilized protective agent was optimized, and three 2× lyophilized protective agents were prepared with water: 1.6% mass percentage of trehalose, 2.2% mass percentage of casein, 3.6% mass percentage of trehalose and 2% mass percentage of casein, and 10 μL of the above 2× lyophilized protective agent was added to each 20 μL reaction system, and then 2 μL of 10× RNA reaction buffer was added, 1 pmol / reaction SEQ ID NO.5 was used as the test probe, and water was added to 20 μL, and the reagents were lyophilized according to the lyophilization procedure in Table 5, and the detection effect after lyophilization was compared. After the reagents were lyophilized with the three lyophilized protective agents, different concentration gradients of RNase (1 pg / reaction, 0.5 pg / reaction, 0.25 pg / reaction, 0.1 pg / reaction) were tested, and enzyme-free water was used as a negative control.

[0109] The results are as follows Fig. 9 As shown, the sensitivity of the three lyophilized protectants after freeze-drying is consistent, and all can be detected to 0.1pg / reaction. From the perspective of the depth of the band, the effect of lyophilized protectant No. ③ is the best. Therefore, 6% by mass of trehalose and 2% by mass of casein are determined as the components of the final 2× lyophilized protectant.

[0110] Example 6 Kit for detecting RNase activity

[0111] 1. Preparation of lyophilized reagents

[0112] (1) Reaction Buffer Preparation: First, prepare 10× RNA reaction buffer, which includes 500 mM Tris-HCl, 100 mM MgCl2, 5 mM CaCl2 and 1% by volume Tween-20, and adjust the pH value to 7.5.

[0113] (2) Preparation of lyophilization protective agent: Then, 2× lyophilization protective agent was prepared with water, which included 6% by weight of trehalose and 2% by weight of casein.

[0114] (3) Lyophilization system composition: Prepare the lyophilized liquid reagent by adding 1 μL of RNA probe (1 μM), 2 μL of 10× RNA reaction buffer, 10 μL of 2× lyoprotectant, and 7 μL of enzyme-free water per tube, and dispense into eight tubes and place on a metal ice plate.

[0115] (4) Freeze-drying program: Before preparing the freeze-dried reagent, turn on the freeze dryer and precool it to -55°C. After the reagent is prepared, place the metal ice plate containing the reagent on the freeze dryer plate layer, close the box, and set the freeze-drying program as shown in Table 5 below.

[0116] Table 5 Freeze-drying procedure

[0117]

[0118] (5) After freeze-drying, turn on the laboratory dehumidifier. When the humidity drops below 20%, start packaging the freeze-dried reagents. After the eight-tube tubes are covered, they are divided into aluminum foil bags, and desiccant is added. Vacuum, seal, and store at -20°C.

[0119] 2. Preparation of lateral flow chromatography test strips for detecting RNase activity

[0120] The chromatographic test strip includes a base plate, a nitrocellulose membrane, a conjugated pad, a sample pad, a blotting paper and a protective sticker (optional). Among them, a quality control line and a detection line are fixed on the nitrocellulose membrane, the quality control line is coated with streptavidin (1 mg / mL), and the detection line is coated with rabbit anti-mouse IgG antibody (1 mg / mL). The conjugated pad is soaked in a conjugated pad treatment solution and dried. Mouse anti-digoxigenin antibody is covalently coupled to red latex microspheres to form a mouse anti-digoxigenin antibody microsphere complex, and the red microspheres are 200 nm in diameter. The sample pad is soaked in a sample pad treatment solution and dried. Nitrocellulose membrane, conjugated pad, sample pad, blotting paper and a protective sticker (optional) are sequentially adhered to the base plate to obtain a lateral flow chromatography test strip for detecting RNase activity.

[0121] The preparation method of the above test strip is as follows:

[0122] 1. Activation of latex microspheres and labeling of conjugates

[0123] (1) Prepare labeling activation buffer (1 L): 50 mM MES buffer, pH = 5.5-6.5.

[0124] (2) Prepare labeling coupling buffer (1 L): 50 mM MES buffer, pH = 6.5-7.5.

[0125] (3) Prepare labeling storage buffer (1 L): 50 mM boric acid-borax buffer, 20 g sucrose, 10 g casein, 1 mL Proclin-300, pH = 7.0-8.0.

[0126] (4) Add 0.9 mL of pure water into the activation container.

[0127] (5) Add 100 μL of microspheres to the activation container, mix well, and ultracentrifuge at 20,000 rpm / min in an ultracentrifuge for 10 minutes, and discard the supernatant.

[0128] (6) Add 1 mL of labeling activation buffer to the precipitate, disperse by ultrasonication, and stir until completely mixed.

[0129] (7) Add 0.5 mg NHS to the activation container and stir until completely mixed. Immediately add 0.5 mg EDC and stir until completely mixed. Stir and react for 10-20 minutes. After the reaction is completed, ultracentrifuge at 20,000 rpm / min in an ultracentrifuge for 10 minutes and discard the supernatant.

[0130] (8) Add 1 mL of labeling coupling buffer to the precipitate, disperse by ultrasonication, and stir until completely mixed.

[0131] (9) Add 0.4 mg of mouse anti-digoxin antibody to the activated microsphere solution, stir until completely mixed, and disperse by ultrasound for 1-2 minutes. Then react while stirring for 1-3 hours.

[0132] (10) After the coupling reaction time is over, add 100 μL of 10% BSA solution to the above solution, mix thoroughly and sonicate for 1 minute, then react with stirring for more than 2 hours.

[0133] (11) After the blocking reaction time is over, the above reaction solution is ultracentrifuged at 20,000 rpm / min in an ultracentrifuge for 10 minutes, and the supernatant is discarded. Then, 1 mL of the labeled storage buffer is added, ultrasonically dispersed, and stirred until completely mixed. Store in an environment of 2-8°C.

[0134] 2. Preparation of Sample Pad

[0135] The sample pad treatment solution includes (1L, pH = 7.0-8.0): 50mM phosphate buffer, 50g sucrose, 50g sodium chloride, 2g casein, 5mL Tween 20, 5mL Triton-100, 1mL Proclin-300. The sample pad is cut into 1.5cm×30cm specifications, soaked in the sample pad treatment solution for 1 minute, then taken out and spread on a drying net, and placed in a constant temperature drying oven for more than 12 hours (45°C).

[0136] 3. Preparation of the Conjugate Pad

[0137] The conjugate pad treatment solution includes (1L, pH = 7.0-8.0): 50mM phosphate buffer, 50g sucrose, 2g casein, 2g BSA, 1mL Proclin-300. Cut the blank conjugate pad into 30cm×1.1cm strips, soak them in the conjugate pad treatment solution for 1 minute, then take them out and spread them on a drying net, and put them in a constant temperature drying oven to dry for more than 12 hours (45°C).

[0138] The mouse anti-digoxin antibody latex solution prepared in step 1 was diluted 5 times with the labeling storage buffer, and then sprayed using a film drawing instrument with a spray volume of 5 μL / cm. After spraying, it was placed in a constant temperature drying oven for more than 12 hours (45° C.).

[0139] 4.NC membrane coating

[0140] Prepare coating buffer (1L, pH=7.0-8.0), including 50mM phosphate buffer and 20g sucrose. Dilute streptavidin to 1mg / mL with coating buffer, and dilute rabbit anti-mouse IgG antibody to 1mg / mL with coating buffer, then use a film stripper, set the film stripping volume to 1μL / cm for film stripping. After film stripping, place in a constant temperature drying oven and dry for more than 12 hours (45°C).

[0141] 5.Assembly and cutting of test strips

[0142] Tear off the film on the bottom plate, and paste the NC membrane, conjugate pad, sample pad, absorbent paper and protective film (optional), with 1-2mm overlap between adjacent parts. Use a cutter to cut the test paper into strips with a width of 3mm.

[0143] Example 7 Detection Limit of a Kit for Detecting RNase Activity

[0144] RNase A (10 mg / mL) was diluted with enzyme-free water to 1 pg / μL, 0.5 pg / μL, 0.25 pg / μL, and 0.1 pg / μL, and 1 μL of diluted RNase + 19 μL of water was prepared. 20 μL of each was added to the lyophilized reagent of Example 6, and the detection method of Example 1 was followed.

[0145] The results are as follows Fig.10 As shown, the freeze-dried detection kit can detect down to 0.1 pg / reaction with clear bands.

[0146] Example 8 Repeatability of a kit for detecting RNase activity

[0147] To illustrate the repeatability of the product, the same batch of freeze-dried reagents was taken and 0.25 pg / reaction was tested. RNase A (10 mg / mL) was diluted to 0.25 pg / μL with enzyme-free water, and 1 μL of diluted RNase + 19 μL of water was prepared. 20 μL was taken and added to the freeze-dried system, and the test was repeated 10 times. The detection method process in Example 1 was followed.

[0148] The results are as follows Fig.11 As shown, 0.25 pg / reaction can be detected 100%, with good repeatability.

[0149] Example 9 Stability of the kit for detecting RNase activity

[0150] The kits of the same batch were placed at 37°C and 42°C for 7 days, and different concentration gradients of RNase (1pg / reaction, 0.5pg / reaction, 0.25pg / reaction, 0.1pg / reaction) were tested respectively, and enzyme-free water was used as a negative control. RNase A (10mg / mL) was diluted with enzyme-free water to 1pg / μL, 0.5pg / μL, 0.25pg / μL, 0.1pg / μL, and 1μL of diluted RNase + 19μL of water was prepared, and 20μL was taken and added to the freeze-dried system, and each concentration was repeated 2 times, and the operation was performed according to the detection method flow of Example 1.

[0151] like Fig.12 As shown, the test kit was stable when placed at 37°C and 42°C for 7 days, with no false positives within 5 minutes and could detect up to 0.1 pg / reaction.

[0152] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

[0153] In addition, those skilled in the art will appreciate that, although some embodiments herein include certain features included in other embodiments but not other features, the combination of features of different embodiments is meant to be within the scope of the present application and form different embodiments. For example, in the above claims, any one of the claimed embodiments may be used in any combination. The information disclosed in this background technology section is intended only to deepen the understanding of the overall background technology of the present application and should not be regarded as an admission or in any form of implication that the information constitutes prior art known to those skilled in the art.

Claims

1. A probe for detecting RNase activity, characterized in that: The probe is a single-stranded RNA of 20-60 nucleotides, the 5' end of the probe is labeled with digoxigenin, and the 3' end of the probe is labeled with biotin.

2. The probe for detecting RNase activity according to claim 1, characterized in that The probe is used at a concentration of 0.5-1 pmol / reaction; Preferably, the probe is used at a concentration of 1 pmol / reaction.

3. A freeze-dried reagent for detecting RNase activity, characterized in that: The invention comprises the probe for detecting RNase activity as described in claim 1 or 2.

4. The freeze-dried reagent for detecting RNase activity according to claim 3, characterized in that: Also included are 10× RNA reaction buffer and 2× lyoprotectant; The 10× RNA reaction buffer comprises Tris-HCl, MgCl2, CaCl2 and Tween-20; The 2× lyophilization protective agent includes trehalose and casein; Preferably, the 10× RNA reaction buffer comprises 500 mM Tris-HCl, 100 mM MgCl2, 5 mM CaCl2 and 1% by volume Tween-20; Preferably, the pH value of the 10× RNA reaction buffer is 7.5; Preferably, the 2× lyophilization protective agent comprises 4%-8% by mass of trehalose and 2%-4% by mass of casein; Preferably, the 2× lyophilization protectant comprises 6% by mass of trehalose and 2% by mass of casein.

5. A method for preparing a lyophilized reagent for detecting RNase activity according to claim 3 or 4, characterized in that: include: The probe, the 10× RNA reaction buffer, the 2× freeze-drying protective agent and enzyme-free water are mixed and then freeze-dried to obtain the freeze-dried reagent; Preferably, the freeze-drying process includes: pre-freezing, sublimation drying and analytical drying; The pre-freezing temperature is -60°C to -50°C, and the maintenance time is 120 minutes; The sublimation drying temperature is -30°C, the heating time from the pre-freezing temperature to the sublimation drying temperature is 30 minutes, and the maintenance time is 600 minutes; The analytical drying temperature is 20° C. to 30° C., the heating time from the sublimation drying temperature to the analytical drying temperature is 60 minutes, and the maintaining time is 180 minutes.

6. A lateral flow chromatography test strip for detecting RNase activity, characterized in that: Including base plate, sample pad, conjugate pad, nitrocellulose membrane, absorbent pad; The nitrocellulose membrane is fixed with a quality control line and a detection line, the quality control line is coated with streptavidin, and the detection line is coated with rabbit anti-mouse IgG antibody; the conjugate pad is fixed with mouse anti-digoxigenin antibody.

7. The lateral flow chromatography test strip for detecting RNase activity according to claim 6, characterized in that: The mouse anti-digoxigenin antibody is labeled to red latex microspheres by covalent coupling to form a mouse anti-digoxigenin antibody microsphere complex, and the microsphere complex is bound to the conjugate pad; Preferably, the particle size of the red latex microspheres is 200 nm; Preferably, the concentration of the streptavidin is 1 mg / mL; the concentration of the rabbit anti-mouse IgG antibody is 1 mg / mL.

8. A kit for detecting RNase activity, characterized in that: The invention comprises the probe for detecting RNase activity according to claim 1 or 2, or the freeze-dried reagent for detecting RNase activity according to claim 3 or 4, and the lateral flow chromatography test strip for detecting RNase activity according to claim 6 or 7.

9. A method for detecting RNase activity, characterized in that include: Adding RNase or a sample to be detected into the freeze-dried reagent solution for detecting RNase activity according to claim 3 or 4 to react and obtain a detection solution; The lateral flow chromatography test strip for detecting RNase activity according to claim 6 or 7 is inserted into the detection solution, and the enzyme activity of RNase or whether the sample to be detected contains RNase is determined based on the strips of the quality control line and the detection line.

10. The method for detecting RNase activity according to claim 9, characterized in that: The total reaction volume of the reaction was 20 μL, and if it was less than 20 μL, it was supplemented with enzyme-free water; Preferably, the reaction temperature is 37°C and the reaction time is 30 min; Preferably, the detection solution is supplemented to 60 μL by adding 40 μL of pure water diluent.

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