Application of haloacetic acid in enrichment of small molecule nucleic acid in biological sample

Abstract

The invention discloses application of haloacetic acid in enrichment of small molecule nucleic acid in a biological sample, and belongs to the technical field of molecular biology. According to the application, the haloacetic acid is utilized to enrich the micromolecular nucleic acid in the liquid biological sample, so that not only can the influence of interferents be eliminated, but also the enriched micromolecular nucleic acid sample can be stably stored at room temperature, and the micromolecular nucleic acid sample can be directly subjected to amplification detection without being extracted, so that the application value is very important.

Description

Technical Field

[0001] This application relates to the field of molecular biology technology, and in particular to the application of haloacetic acid in enriching small nucleic acids in biological samples. Background Technology

[0002] MicroRNAs (miRNAs) are a class of endogenous non-coding small RNA molecules, approximately 18-25 nucleotides in length, that play important regulatory roles within cells, participating in various biological processes such as cell proliferation, differentiation, apoptosis, and metabolism. Recent studies have discovered the presence of free miRNAs in plasma, and their expression profiles alter during the development and progression of various diseases, including tumors and cardiovascular diseases. Therefore, plasma-derived free miRNAs hold promise as novel biomarkers for disease diagnosis, prognostic assessment, and treatment monitoring.

[0003] Currently, the commonly used methods for extracting free miRNA mainly include silica adsorption column extraction and silica magnetic bead capture. The recovery efficiency of these two conventional nucleic acid extraction techniques is related to the length of the nucleic acid, and they can usually recover targets longer than 50 nt relatively well. However, the length of miRNA is generally no more than 25 nt, so its recovery rate is generally not high. In order to maximize the miRNA recovery rate, extraction kits usually limit the sample loading volume. For example, the Qiagen miRNeasy Serum / PlasmaAdvanced Kit only allows a maximum of 0.6 mL of plasma; and the Promega Maxwell® RSC miRNA from Plasmaand Serum Kits only allows a maximum of 0.5 mL of plasma. With a limited total reagent volume, the miRNA precipitation ability can be enhanced by achieving a higher ratio of lysis buffer and binding buffer, such as ultra-high concentrations of ethanol or isopropanol. However, in such ultra-high concentration ethanol or isopropanol reagent environments, molecular impurities in the sample are easily adsorbed onto the solid phase along with the miRNA, resulting in reduced purity and ultimately interfering with the sample detection stability.

[0004] Furthermore, when the sample volume is less than 1 mL, low-expression free miRNAs in plasma and serum are difficult to detect; while using large-volume samples directly can introduce excessive impurities, inhibiting the detection reaction and leading to detection failure. Therefore, it is necessary to develop a free miRNA enrichment technology for plasma / serum samples to concentrate large-volume samples for use with miRNA extraction kits. Simultaneously, this technology should ensure that most interfering substances are not enriched, significantly reducing the inhibitory interference of impurities in the initial sample on miRNA detection. Summary of the Invention

[0005] To address at least one of the aforementioned technical problems, the inventors discovered that when treating liquid biological samples such as plasma / serum with haloacetic acids such as trichloroacetic acid, small nucleic acids such as free miRNA can be enriched, and the influence of interfering substances in the initial sample on subsequent small nucleic acid detection can be reduced. Furthermore, the small nucleic acids enriched by haloacetic acids such as trichloroacetic acid can be stably stored at room temperature (20~40℃) for up to 7 days and can be directly used for RT-PCR without extraction, thus completing this invention.

[0006] The first aspect of this application provides the use of haloacetic acid in the preparation of precipitants for precipitating small molecule nucleic acids in liquid biological samples.

[0007] As previously stated, the inventors of this application accidentally discovered during research and development, and through extensive experimental verification, that trichloroacetic acid and other haloacetic acids can enrich small-molecule nucleic acids in biological samples. Haloacetic acids are a class of organic compounds formed by replacing one or more hydrogen atoms on the alkyl portion of acetic acid (particularly the α-carbon atom, i.e., the carbon atom directly bonded to the carboxyl group) with halogen atoms (fluorine, chlorine, bromine, iodine). In some embodiments of this application, the haloacetic acids include, but are not limited to, chloroacetic acid, dichloroacetic acid, trichloroacetic acid, trifluoroacetic acid, and tribromoacetic acid.

[0008] In this application, the liquid biological sample is selected from body fluid samples, tissue processing fluid samples, cell suspension samples, exosome suspension samples, and environmental liquid samples such as water. The body fluid samples include, but are not limited to, serum, plasma, urine, and cerebrospinal fluid. The tissue processing fluid samples refer to liquid samples obtained after homogenization, lysis, or other treatments of fresh / frozen / paraffin-embedded tissue samples. The cells include, but are not limited to, cultured cell lines, primary cells, flow cytometry-sorted cells, and circulating tumor cells.

[0009] In some embodiments of this application, the liquid biological sample is a plasma or serum sample.

[0010] In some specific embodiments of this application, the plasma or serum sample contains interfering substances, meaning the liquid biological sample is a plasma or serum sample containing interfering substances. In some specific embodiments of this application, the interfering substances include anticoagulants added to the blood sample after acquisition, as well as common blood components such as glucose and uric acid, and drugs that may be present in the blood, such as antibiotics. In some specific embodiments of this application, the interfering substances include, but are not limited to, K2EDTA, lithium heparin, glucose, uric acid, ibuprofen, aspirin, and tetracycline.

[0011] In this application, the small nucleic acid molecules include miRNA, siRNA, tsRNA, piRNA, cfDNA, and ctDNA. Among them, microRNAs (miRNAs) are approximately 20-24 nucleotides long and participate in gene expression regulation. They are abundant and stable in serum, plasma, and exosomes, making them popular candidates for disease biomarkers.

[0012] Small interfering RNA (siRNA): approximately 20-25 nucleotides long, it can mediate the degradation of target mRNA.

[0013] tsRNA (tRF and tiRNA): Derived from tRNA, usually between 17 and 36 nucleotides in length, and associated with a variety of physiological and pathological processes.

[0014] PIWI interacting RNA (piRNA): mainly interacts with PIWI protein, is about 24-31 nucleotides in length, is highly expressed in germ cells, and its main function is to silence transposons.

[0015] Circulating cell-free DNA (cfDNA / ctDNA): DNA fragments released into the bloodstream during apoptosis or necrosis. Those originating from tumors are called ctDNA, which is commonly used for non-invasive cancer diagnosis and monitoring.

[0016] A second aspect of this application provides a precipitant for precipitating small molecule nucleic acids in liquid biological samples, said precipitant comprising haloacetic acid, with or without other components.

[0017] In some specific embodiments of this application, haloacetic acid is directly used as a precipitant. In other specific embodiments of this application, haloacetic acid is prepared into an aqueous solution and used as a precipitant.

[0018] A third aspect of this application provides a kit for enriching small molecule nucleic acids in liquid biological samples, including the precipitant described in the second aspect of this application.

[0019] In some embodiments of this application, after adding the precipitant to the liquid biological sample, separation yields a precipitate containing the small molecule nucleic acid. Prior to separation, a step of settling and / or centrifugation is included to allow substances, including at least the small molecule nucleic acid, to precipitate at the bottom of the solution, thus obtaining a precipitate containing the small molecule nucleic acid.

[0020] In some embodiments of this application, the precipitate needs to be resuspended to facilitate further detection of the small molecule nucleic acid. Therefore, the kit further includes a resuspension solution for resuspending the precipitate containing the small molecule nucleic acid obtained after treating the liquid biological sample with the precipitant.

[0021] In this application, the inventors experimented with different resuspensions and found that different resuspensions had a significant impact on subsequent detection results. For RT-PCR detection, the resuspension was an alkaline solution.

[0022] In some embodiments of this application, the resuspension solution is a KOH solution or a NaOH solution.

[0023] The fourth aspect of this application provides a method for enriching small molecule nucleic acids in a liquid biological sample, comprising the step of adding haloacetic acid to the liquid biological sample, and after adding haloacetic acid, separating a precipitate containing the small molecule nucleic acid.

[0024] In some embodiments of this application, the final concentration of the haloacetic acid in the mixture after adding the haloacetic acid is 2-5% w / v. Those skilled in the art may choose to first prepare the haloacetic acid into a haloacetic acid solution, and then add it to the liquid biological sample in a certain proportion.

[0025] In some specific embodiments of this application, the concentration of the haloacetic acid solution is 20-50% w / v. The haloacetic acid solution is added at a ratio of liquid biological sample to haloacetic acid solution of 9:1.

[0026] Furthermore, to facilitate the analysis of the small molecule nucleic acid, the method further includes the step of resuspending the precipitate using a resuspension solution to obtain a resuspension containing the small molecule nucleic acid.

[0027] The volume of resuspension added is adjusted according to subsequent processing / analysis. If the subsequent step includes extraction of the small nucleic acid, the amount of resuspension added should not exceed the upper limit acceptable by the corresponding kit. Typically, the acceptable volume range for small nucleic acid extraction kits is 0.1 mL to 0.5 mL, and correspondingly, the volume of resuspension added should also be 0.1 mL to 0.5 mL. However, if the concentration of the small nucleic acid in the liquid biological sample is high, the amount of resuspension can be increased, and a portion of the resuspension can be used for subsequent processing / analysis.

[0028] In some preferred embodiments of this application, the precipitate is stored at 20-40°C and resuspended using the resuspension solution within 7 days.

[0029] In this application, the inventors also unexpectedly discovered that after enriching the small molecule nucleic acid with the haloacetic acid, the precipitate containing the small molecule nucleic acid can be stored at room temperature (20~40℃) for 7 days, and if the temperature is lower, it can be stored for a longer period of time.

[0030] The fifth aspect of this application provides a method for detecting small nucleic acid molecules in liquid biological samples based on RT-PCR, comprising the following steps: A heavy suspension containing the small molecule nucleic acid is obtained using the method described in the fourth aspect of this application; Choose one of the following processes to obtain an analyte containing the small molecule nucleic acid: (i) The resuspended sample is treated with a small molecule nucleic acid extraction kit to obtain the analyte containing the small molecule nucleic acid. (ii) Depending on the volume of resuspension added, the sample is diluted or undiluted and then lysed to obtain the analyte containing the small molecule nucleic acid; Using the analyte as a template, RT-PCR detection was performed.

[0031] In this application, the inventors also discovered that by enriching small-molecule nucleic acids in the liquid biological sample using haloacetic acid and resuspending them in a resuspension solution, further extraction can be performed using a conventional small-molecule nucleic acid extraction kit. Alternatively, nucleic acid extraction and purification can be omitted, and the sample can be directly lysed for RT-PCR detection. In other words, the resuspension containing the small-molecule nucleic acids obtained using the method for enriching small-molecule nucleic acids in liquid biological samples described in this application can be directly amplified without extraction.

[0032] In some embodiments of this application, the pyrolysis is selected from one of the following processing methods: (i) Incubate at 94~98℃ for 5~15 min; (ii) After adding the protease, incubate at 50-65℃ for 10-30 min, and then incubate at 94-98℃ for 5-15 min.

[0033] The sixth aspect of this application provides the use of haloacetic acid in the preparation of nuclease inhibitors.

[0034] As previously mentioned, haloacetic acids are a class of organic compounds formed by replacing one or more hydrogen atoms on the alkyl portion of acetic acid (particularly the α-carbon atom, i.e., the carbon atom directly bonded to the carboxyl group) with halogen atoms (fluorine, chlorine, bromine, iodine). In some embodiments of this application, the haloacetic acids include, but are not limited to, chloroacetic acid, dichloroacetic acid, trichloroacetic acid, trifluoroacetic acid, and tribromoacetic acid.

[0035] In this application, the term "nuclease inhibitor" is interpreted broadly. It can refer to a substance that directly acts on the nuclease to reduce or eliminate its activity, or it can refer to a substance that reduces or removes the contact between the nuclease and nucleic acid molecules, thereby weakening or eliminating the effect of the nuclease on nucleic acid molecules.

[0036] In some embodiments of this application, haloacetic acid is used as a nuclease inhibitor, or as an active ingredient or one of the active ingredients of a nuclease inhibitor.

[0037] In some embodiments of this application, haloacetic acid is used as or to prepare nuclease inhibitors, which can be used to preserve small molecule nucleic acids in liquid biological samples at room temperature, and can be preserved for at least 7 days at room temperature.

[0038] In this application, as previously stated, the small molecule nucleic acids include miRNA, siRNA, tsRNA, piRNA, cfDNA, and ctDNA.

[0039] The seventh aspect of this application provides the application of haloacetic acid in the preparation of a kit for extraction-free direct amplification of liquid biological samples, wherein the extraction-free direct amplification refers to: after adding the haloacetic acid to the liquid biological sample, a precipitate containing small molecule nucleic acids is obtained; without nucleic acid extraction and purification steps, the precipitate is resuspended, diluted, and lysed before being directly loaded onto the sample for PCR amplification to complete the detection of small molecule nucleic acids in the liquid biological sample.

[0040] The eighth aspect of this application provides a kit for extraction-free direct amplification of liquid biological samples, the kit comprising haloacetic acid, and further comprising resuspension, lysis reagent and / or RT-PCR amplification reagent.

[0041] In this application, after precipitation with haloacetic acid and resuspension with the resuspending solution, there is no need for further extraction and purification operations. Otherwise, the sample state and nucleic acid concentration will be changed, resulting in adverse results such as significant changes in the detection results.

[0042] It is particularly important to emphasize that the kit for direct amplification of liquid biological samples without extraction does not include nucleic acid extraction reagents and is not intended for use with nucleic acid extraction reagents. Furthermore, the instructions for use with this kit may state that it should not be used with nucleic acid extraction reagents or something similar. Alternatively, the instructions may include usage steps that do not include an extraction step.

[0043] Compared with the prior art, this application has the following advantages: This application utilizes haloacetic acid to enrich small molecule nucleic acids in liquid biological samples. This not only eliminates the influence of interfering substances but also allows the enriched small molecule nucleic acid samples to be stably stored at room temperature. Furthermore, it enables direct amplification and detection without extraction, which has significant application value.

[0044] It should be understood that the description in this section is not intended to identify key or essential features of the embodiments of this application, nor is it intended to limit the scope of this application. Other features of this application will become readily apparent from the following description. Attached Figure Description

[0045] The above and other objects, features, and advantages of exemplary embodiments of this application will become readily apparent from the following detailed description taken in conjunction with the accompanying drawings. Several embodiments of this application are illustrated in the drawings by way of example and not limitation, in which: Figure 1 This document illustrates a schematic diagram of the miRNA enrichment and extraction process in plasma / serum samples according to Example 1 of this application. Figure 2 The effect of different precipitants on miRNA enrichment in Example 1 of this application is shown; Figure 3 This illustrates the effect of different resuspensions on miRNA enrichment in Example 2 of this application; Figure 4 The effect of precipitant and resuspension concentrations on miRNA enrichment is shown in Example 3 of this application; Figure 5 The anti-interference performance of enrichment combination 6 in embodiment 4 of this application is shown; Figure 6 The target detection results of plasma samples of different volumes enriched or unenriched in Example 5 of this application are shown; Figure 7 This document shows a schematic diagram of the miRNA enrichment and extraction process in plasma / serum samples in Example 6 of this application. After obtaining the precipitate, it is stored at room temperature. Figure 8 The preservation effect of the miRNA sample treated with the precipitant in Example 6 of this application at room temperature is shown; Figure 9 This demonstrates the effect of storing the miRNA sample treated with the precipitant in a blood collection tube at room temperature in Example 6 of this application; Figure 10 The effects of different treatments in Example 7 of this application on the inhibition of RNase activity in plasma samples are shown. Figure 11 This document illustrates a flowchart of the direct amplification and detection process for miRNAs enriched in plasma / serum samples in Example 8 of this application. Figure 12 The effect of different dilution factors and lysis conditions on the detection results of direct amplification of miRNA without extraction is shown in Example 8 of this application. Detailed Implementation

[0046] Unless otherwise stated, implied from the context, or as is customary in the art, all parts and percentages in this application are based on weight, and all testing and characterization methods used are concurrent with the filing date of this application. Where applicable, any patent, patent application, or disclosure relating to this application is incorporated herein by reference in its entirety, and its equivalent patent families are also incorporated herein by reference, particularly the definitions of relevant terms in the art disclosed in such documents. If any definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition provided in this application shall prevail.

[0047] The numerical ranges used in this application are approximate values ​​and therefore may include values ​​outside the range unless otherwise stated. The numerical range includes all values ​​from the lower limit to the upper limit, increasing by one unit, provided that there is an interval of at least two units between any lower and any higher value. For ranges containing values ​​less than 1 or fractions greater than 1 (e.g., 1.1, 1.5, etc.), one unit is appropriately considered as 0.0001, 0.001, 0.01, or 0.1. For ranges containing single digits less than 10 (e.g., 1 to 5), one unit is generally considered as 0.1. These are merely specific examples of what is intended to be expressed, and all possible combinations of values ​​between the listed lowest and highest values ​​are considered to be clearly described in this application.

[0048] The terms “comprising,” “including,” “having,” and their derivatives do not exclude the presence of any other components, steps, or processes, regardless of whether such other components, steps, or processes are disclosed in this application. To eliminate any doubt, unless expressly stated otherwise, all compositions using the terms “comprising,” “including,” or “having” in this application may contain any additional additives, excipients, or compounds. Conversely, except for those necessary for operational performance, the term “substantially constitutes…” excludes any other components, steps, or processes described below with respect to that term. The term “consisting of…” does not include any components, steps, or processes not specifically described or listed. Unless expressly stated otherwise, the term “or” refers to the individual members listed or any combination thereof.

[0049] To make the technical problems, technical solutions and beneficial effects solved by this application clearer, the following detailed description is provided in conjunction with embodiments.

[0050] The following examples are used to illustrate preferred embodiments of this application. Those skilled in the art will understand that the techniques disclosed in the examples represent technologies discovered by the inventors that can be used to implement this application, and therefore can be considered preferred embodiments of this application. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of this application.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains, and all materials cited herein and referenced by them are incorporated herein by reference.

[0052] Those skilled in the art will recognize, or can learn through routine experimentation, many equivalents of the specific embodiments of the invention described herein. These equivalents will be included in the claims.

[0053] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are standard laboratory instruments and equipment. The samples used in the following examples are plasma samples obtained through legal channels, and the plasma / serum miRNA extraction kit used is the Qiagen miRNeasy Serum / Plasma Advanced Kit; the operating procedure follows the kit's instruction manual. Unless otherwise specified, other experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0054] Example 1: Effect of precipitant on enrichment effect of free miRNA in plasma / serum The inventive concept of this invention is as follows: Free miRNA in plasma / serum samples is precipitated using a precipitant, the precipitate containing miRNA is further resuspended using a resuspension solution, and finally, miRNA is extracted. Figure 1 As shown.

[0055] In this embodiment, the inventors designed various precipitants, as follows: Precipitant 1: 4M ammonium sulfate Precipitator 2: 10% w / v PEG8000 Precipitant 3: 5% w / v potassium dodecyl sulfate Precipitant 4: 50% w / v trichloroacetic acid (TCA) It should be noted that, since the solutes in the above-mentioned precipitants are of different types, their solubilities also vary. Therefore, the concentrations of the precipitants mentioned above are the commonly used concentrations or higher concentrations (close to their saturation concentrations) of each reagent. For example, the saturation concentration of ammonium sulfate is approximately 4M, so 4M ammonium sulfate was chosen as precipitant 1. After determining the optimal precipitant, the inventors further explored the optimal concentration.

[0056] Nuclease-free water was used as the resuspension solution.

[0057] The above-mentioned precipitants and resuspensions were used to enrich free miRNAs in plasma samples. The specific steps are as follows: (1) Add precipitant to 200 μL of plasma sample, with a volume ratio of plasma sample: precipitant = 9:1, mix thoroughly to obtain a mixture.

[0058] (2) Centrifuge the above mixture at 4000×g for 3 min in a high-speed centrifuge, discard the supernatant, and collect the precipitate (containing free miRNA).

[0059] (3) Add the resuspended solution to the precipitate and shake thoroughly to mix.

[0060] The volume of resuspension solution added can be adjusted according to the sample volume requirements of subsequent steps (such as plasma / serum miRNA extraction kits), and the volume range is generally 0.1mL-0.5mL.

[0061] (4) Use a plasma / serum miRNA extraction kit to extract and purify miRNA to obtain purified miRNA.

[0062] The extracted and purified miRNAs were analyzed using RT-PCR, with miRNAs extracted directly from unenriched plasma samples serving as a control. Each plasma sample group underwent three parallel tests. Specifically, using the extracted miRNAs as templates, the target hsa-miR-21-5p was detected. The sequence of target hsa-miR-21-5p and its primer sequences are shown in Table 1. Table 1: Sequence and primer sequences (5'-3') of the target hsa-miR-21-5p

[0063] In the miRNA sequence, "U" is displayed as "t" in the sequence listing.

[0064] The RT reaction system is shown in Table 2: Table 2: RT reaction system

[0065] The RT reaction procedure is shown in Table 3: Table 3: RT Reaction Procedure

[0066] The qPCR reaction system is shown in Table 4: Table 4: qPCR reaction system

[0067] The qPCR reaction procedure is shown in Table 5: Table 5: qPCR reaction procedure

[0068] The RT-qPCR detection results of the enriched miRNAs using different precipitants are shown in Table 6 and... Figure 2 As shown.

[0069] Table 6: Effects of different precipitants on miRNA enrichment

[0070] As shown in Table 6, the miRNA detection results obtained by different precipitants varied. Among them, ammonium sulfate, PEG8000 and potassium dodecyl sulfate had poor recovery effects on free miRNA. In contrast, 50% w / v TCA achieved partial recovery of free miRNA, but its efficiency was low and there is still considerable room for optimization.

[0071] Example 2: Effects of different resuspensions on the enrichment of free miRNA Based on the results of Example 1, this example uses 50% w / v TCA as a precipitant and designs the following different resuspensions to further improve the miRNA enrichment and recovery efficiency: Resuspension 1: Nuclease-free water Resuspension 2: 1×PBS, pH 7.4 Resuspension 3: 50 mM Tris-HCl, pH 8.0 Resuspension 4: 100mM EDTA Resuspension 5: 0.1% Tween 20 Resuspension 6: 0.1% Triton X-100 Resuspension 7: 100mM glucose Resuspension 8: 100mM Trehalose Resuspension 9:1% w / v PEG 2000 Resuspension 10:10% v / v glycerol Resuspension 11:1% w / v BSA Resuspension 12: 50mM NaCl Resuspension 13: 50mM KCl Resuspension 14: 50mM NaOH Resuspension 15: 50mM KOH Resuspension 16: 50mM guanidine thiocyanate Resuspension 17: 50 mM dithiothreitol Resuspension 18: 50 mM Tris(2-carbonylethyl)phosphohydrochloride Similarly, the concentration of the resuspension is also the commonly used concentration or a relatively high concentration (close to its saturation concentration) of each solute.

[0072] The miRNA enrichment, extraction, and detection methods were the same as in Example 1. Similarly, miRNA extracted directly from unenriched plasma samples was used as a control, and the detection target was also hsa-miR-21-5p. The mean Ct value of three parallel detections in the control group was selected as the Ct reference value for calculating the recovery rate. The detection results are shown in Table 7 and... Figure 3 As shown: Table 7: Effects of different resuspensions on miRNA enrichment

[0073] From Table 7 and Figure 3 It can be seen that using 50mM NaOH or 50mM KOH as the resuspension solution resulted in the highest miRNA enrichment and recovery rate.

[0074] Example 3: Determination of the optimal concentration range of TCA precipitant and NaOH resuspension To further enhance the miRNA enrichment effect, in this embodiment, the inventors set different concentrations of TCA precipitant and NaOH resuspension to obtain the optimal combined concentration.

[0075] The miRNA enrichment, extraction, and detection methods were the same as in Example 1. In the control group without enrichment, miRNA was extracted directly from plasma samples. The detection target was also hsa-miR-21-5p. Detection results are shown in Table 8. Figure 4 As shown: Table 8: Effects of precipitant and resuspension concentrations on miRNA enrichment

[0076] From Table 8 and Figure 4 It can be seen that the optimal recovery rate of free miRNA enrichment is achieved when the concentration of the precipitant TCA is in the range of 20%-50% w / v and the concentration of the resuspension NaOH is in the range of 10-200mM.

[0077] Example 4: Anti-interference ability of miRNA enrichment reagent combination Based on the results of Examples 1-3, this example uses enrichment combination 6 from Example 3, which uses 50% w / v TCA as the precipitant and 50 mM NaOH as the resuspension for enrichment. The precipitant and resuspension are packaged separately and can be assembled into a kit for use.

[0078] In this embodiment, enrichment combination 6 is further used to enrich free miRNA in plasma samples containing different interfering agents in order to test the anti-interference ability of enrichment combination 6.

[0079] First, the plasma samples were divided into different groups, each with 200 μL. Interfering substances were added directly to each group of plasma samples to the concentrations corresponding to Table 9 (each interfering substance corresponds to two gradients: low concentration and high concentration).

[0080] The miRNA enrichment, extraction, and detection methods were the same as in Example 1. In the control group without enrichment, miRNA was extracted directly from plasma samples. The detection target was also hsa-miR-21-5p. Detection results are shown in Table 9. Figure 5 As shown.

[0081] Table 9: Anti-interference performance of enrichment combination 6

[0082] From Table 9 and Figure 5 It can be seen that when using conventional methods without enrichment, the low concentration of interfering substances does not affect the final free miRNA detection results thanks to the purification effect of the plasma / serum miRNA extraction kit. However, when the concentration of interfering substances increases, the purification capability of the extraction kit is insufficient to eliminate the influence of the interfering substances, ultimately causing a significant change in the free miRNA detection results. In contrast, after using enrichment combination 6 for free miRNA enrichment, the problem of high concentrations of interfering substances in the initial sample inhibiting free miRNA PCR is solved.

[0083] This embodiment also demonstrates that enrichment combination 6 is applicable to the detection of free miRNA in plasma / serum samples containing interfering substances. Through enrichment, the influence of interfering substances can be eliminated without affecting the recovery rate of free miRNA.

[0084] Example 5: Enrichment of free miRNAs in large-volume plasma samples using a combination of enrichment reagents. In existing technologies, the maximum volume of plasma / serum samples suitable for plasma / serum miRNA extraction kits is 0.6 mL. However, for free miRNAs with low abundance, such a small sample volume is insufficient to extract miRNAs that meet the detection limit. Therefore, in this embodiment, the inventors attempted to enrich miRNAs in plasma / serum samples of different volumes in order to obtain information on more types of miRNAs.

[0085] Specifically, the inventors divided plasma samples into groups of 0.2 mL, 0.6 mL, 2 mL, 6 mL, and 10 mL. For each group, free miRNA was enriched using enrichment combination 6 from Example 3.

[0086] The miRNA enrichment, extraction, and detection methods are the same as in Example 1. In the control group without enrichment, miRNA was extracted directly from plasma samples. The detection targets included, in addition to the highly expressed hsa-miR-21-5p, the moderately expressed hsa-let-7d-5p and the lowly expressed hsa-let-7e-5p.

[0087] The sequences and primer sequences of the targets hsa-let-7d-5p and hsa-let-7e-5p are shown in Table 10: Table 10: Sequences and primer sequences of targets hsa-let-7d-5p and hsa-let-7e-5p

[0088] In the sequence listing, the "U" in each miRNA sequence is displayed as "t".

[0089] The test results are shown in Table 11 and Figure 6 As shown.

[0090] Table 11: Target detection results after enrichment or non-enrichment of plasma samples of different volumes

[0091] From Table 11 and Figure 6 It can be seen that when the sample volume exceeds 0.6 mL, the recovery of free miRNA from the non-enriched control group is problematic, resulting in abnormal final detection results, with Ct values ​​increasing or detection failure.

[0092] After enrichment using enrichment combination 6, stable high recoveries were observed across plasma sample volumes ranging from 0.2 mL to 10 mL, with good consistency and linearity as expected. With a significant increase in plasma volume, the detection results for low-abundance hsa-let-7d-5p and extremely low-abundance hsa-let-7e-5p in plasma were also improved.

[0093] Therefore, the above-mentioned enrichment combination 6 / kit can efficiently recover miRNA from large volumes (up to 10 mL) of plasma / serum samples, thereby enabling the quantitative detection of low-expression free miRNA, which is beneficial for screening studies of liquid biopsy miRNA biomarkers.

[0094] Example 6: Room temperature storage effect of miRNA precipitate treated with precipitant Those skilled in the art will know that miRNA samples are easily degraded and have a limited shelf life. However, the inventors unexpectedly discovered that after enrichment using enrichment combination 6 of Example 3, miRNAs could be stored at room temperature for a longer period. The inventors' analysis revealed that this was due to the precipitant performing its function.

[0095] To demonstrate this unexpected technical effect, the inventors grouped miRNA precipitate samples treated with 50% TCA precipitant and stored them at 20℃, 30℃, and 40℃ respectively. On days 3 and 7, the samples were resuspended using the corresponding resuspension solutions, and miRNA was further extracted (e.g., ...). Figure 7 As shown in the figure, the extracted miRNA was detected by RT-PCR, with the target being hsa-miR-21-5p. The extraction and detection methods were the same as in Example 1. In the non-enrichment control group, miRNA was extracted directly from plasma samples. The detection results are shown in Table 12 and... Figure 8 As shown.

[0096] Table 12: Preservation effect of miRNA samples treated with precipitant at room temperature

[0097] From Table 12 and Figure 8 It was found that the Ct values ​​of the non-enrichment control group increased to varying degrees after storage under all conditions, indicating that miRNAs were degraded. However, after treatment with the precipitant TCA, free miRNAs in plasma samples could be stably stored at 20°C for at least 7 days; even when the storage temperature was increased to 30°C or 40°C, the stability of miRNAs remained good, and the degradation was minimal.

[0098] To further verify the above technical effects, the inventors used Streck RNA Complete BCT blood collection tubes to collect blood, centrifuged the samples, and collected plasma, which was then aliquoted into 200 μL volumes. The plasma samples were treated with 50% TCA precipitant, and the treated miRNA precipitates were stored at 20℃, 30℃, and 40℃, respectively. On days 3 and 7, the samples were resuspended using the corresponding resuspension solutions, and miRNA was further extracted. The extracted miRNA was then analyzed by RT-PCR, with the target being hsa-miR-21-5p. The extraction and detection methods were the same as in Example 1. In the non-enrichment control group, miRNA was directly extracted from plasma samples stored in Streck RNA Complete BCT blood collection tubes. The detection results are shown in Table 13. Figure 9 As shown.

[0099] Table 13: Room temperature storage effect of miRNA samples treated with precipitant in blood collection tubes

[0100] From Table 13 and Figure 9 It can be seen that the Streck RNA Complete BCT blood collection tube helps improve the stability of miRNA to some extent, allowing it to be stably stored at 20℃ for 3 days. However, when the storage time is longer or the temperature is higher, significant degradation still occurs. Treatment with the precipitant TCA resulted in better miRNA stability under all conditions.

[0101] Therefore, treating plasma samples with 50% w / v TCA precipitant can precipitate miRNAs, ensuring that miRNAs can be stored and transported at room temperature without the need for special sample collection / preservation tubes.

[0102] Example 7 Inhibition of RNase activity by precipitant Those skilled in the art also know that free miRNAs in plasma samples are easily degraded, mainly because RNase (RNAse) is present in plasma / serum samples. In Example 6, the inventors demonstrated that 50% w / v precipitant TCA allowed treated free miRNAs to be stored at room temperature (20~40℃) for a relatively long time. The inventors analyzed that this might be because the precipitant could reduce RNase activity. To verify this hypothesis, the inventors treated plasma samples with the aforementioned precipitant TCA and different RNase inhibitors, and used an RNase detection kit (fluorescent probe method) (Hanhai New Enzyme, HBP003002) to test the RNase activity after treatment, with untreated plasma samples as a control (no inhibition control).

[0103] Specifically, add 20 μL of RNAse inhibitor to 180 μL of plasma sample (i.e., sample volume: RNAse inhibitor volume = 9:1) and vortex to mix. When the RNAse inhibitor is the precipitant TCA, the sample will become a suspension after addition, and should be vortexed to mix.

[0104] Because the initial RNAse activity in the plasma samples was too high, exceeding the detection limit of the RNAse detection kit, each sample was diluted 50,000 times before testing. The diluted RNAse activity concentration of each sample was calculated based on the standards in the kit, and then multiplied by 50,000 to obtain the original RNAse activity concentration. The results are shown in Table 14. Figure 10 As shown.

[0105] Table 14: Effects of different treatments on the inhibition of RNase activity in plasma samples

[0106] From Table 14 and Figure 10The concentration of RNase activity in the plasma sample was as high as 156.5 ng / mL. Treatment with different RNase inhibitors resulted in varying degrees of inhibition. EDTA and guanidine isothiocyanate showed relatively low inhibition of RNase activity, while mouse RNAsIn showed relatively high inhibition, but the residual rate remained very high. However, using 50% w / v TCA as a precipitant showed the best inhibitory effect on RNase activity, with a residual RNase activity rate of only 2.88%.

[0107] The results of this embodiment also show that TCA can be used as a nuclease inhibitor, or as an active ingredient or one of the active ingredients in the preparation of a nuclease inhibitor.

[0108] Example 8: Direct amplification of enriched free miRNA samples without extraction Based on the results of Example 4 above, the inventors believe that the addition of a precipitant can separate free miRNA from interfering substances, and even low-abundance miRNA can be effectively enriched (Example 5). The inventors attempted to bypass nucleic acid extraction and purification of the enriched miRNA samples, instead performing RT-PCR directly after dilution and lysis, as shown in Example 5. Figure 11 As shown.

[0109] In this embodiment, the enrichment operation was also performed using enrichment combination 6 from Example 3. The specific steps are the same as in Example 1. The volume of the resuspension added at the end was the same as the volume of the plasma sample used for miRNA extraction, which was 200 μL.

[0110] After resuspending, the samples were diluted. To test different dilution factors, nuclease-free water was used to perform one 1-fold, 2-fold, 5-fold, 10-fold, 25-fold, or 50-fold dilution of the resuspended samples.

[0111] After dilution, the samples were lysed, and three lysis conditions were tested: no lysis, lysis condition 1, and lysis condition 2. Lysis condition 1 involved incubating the sample at 95°C for 10 min. Lysis condition 2 involved adding 5 μL of proteinase K (NEB, P8107S) to the resuspended sample, vortexing to mix, and then incubating at 55°C for 15 min followed by incubation at 95°C for 10 min.

[0112] After lysis, 5 μL of each sample was taken for RT-PCR detection. The detection method was the same as in Example 1, and the detection target was also hsa-miR-21-5p. The detection results are shown in Table 15. Figure 12 As shown.

[0113] Table 15: Effects of dilution factor and lysis conditions on the detection results of miRNA amplification without extraction

[0114] From Table 15 and Figure 12 It is evident that after enriching free miRNAs in plasma samples using enrichment combination 6, miRNA detection can be achieved using a direct amplification method without extraction. A feasible direct amplification procedure requires a sample dilution factor of at least 5 times, combined with appropriate lysis treatment. The optimal direct amplification procedure is direct amplification procedure 12, i.e., a 10-fold sample dilution followed by lysis condition 2.

[0115] To verify the effect of enrichment combination 6 on extraction-free direct amplification, the inventors compared the direct amplification process 12 with no enrichment treatment (i.e., directly diluting the plasma sample 10 times with nuclease-free water and then processing it using lysis condition 2). The results are shown in Table 16.

[0116] Table 16: Effect of enrichment treatment on extraction-free direct amplification

[0117] As shown in Table 16, after processing with enrichment combination 6, the corresponding miRNA targets can be detected by direct amplification without extraction. However, when the enrichment step is removed, the corresponding miRNA targets cannot be detected by direct amplification without extraction, resulting in detection failure.

[0118] Therefore, by using the above-mentioned enrichment combination 6 to enrich free miRNA in plasma / serum samples, the extraction step can be eliminated, saving reagents and operational steps. Based on this, enrichment combination 6 (50% w / v TCA and 50mM NaOH) can be used to prepare a kit for direct amplification without extraction. In use, 50% w / v TCA is first added to liquid samples such as blood samples, centrifuged to obtain a precipitate, and then resuspended in 50mM NaOH. Without undergoing nucleic acid extraction and purification, the resuspended solution is directly loaded for PCR amplification after lysis. Furthermore, the kit also includes lysis reagents such as proteinase K, as well as necessary reagents for PCR amplification.

[0119] Furthermore, it should be understood that after reading the foregoing teachings of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. Application of haloacetic acid in the preparation of precipitants for precipitating small molecule nucleic acids in liquid biological samples.

2. A precipitant for precipitating small molecule nucleic acids in liquid biological samples, characterized in that, Includes haloacetic acid, with or without other components.

3. A kit for enriching small-molecule nucleic acids in liquid biological samples, characterized in that, Includes the precipitant as described in claim 2.

4. The reagent kit according to claim 3, characterized in that, It also includes a resuspension solution for resuspending a precipitate containing the small molecule nucleic acid obtained after the liquid biological sample has been treated with the precipitant.

5. The reagent kit according to claim 4, characterized in that, The resuspension solution is an alkaline solution.

6. A method for enriching small-molecule nucleic acids in liquid biological samples, characterized in that, The method includes the step of adding haloacetic acid to the liquid biological sample, and after adding haloacetic acid, separating a precipitate containing the small molecule nucleic acid.

7. The method according to claim 6, characterized in that, The method further includes the step of resuspending the precipitate using a resuspension solution to obtain a resuspension containing the small molecule nucleic acid.

8. The method according to claim 7, characterized in that, The precipitate is stored at 20-40°C and resuspended using the resuspension solution within 7 days.

9. A method for detecting small nucleic acid molecules in liquid biological samples based on RT-PCR, characterized in that, Includes the following steps: A heavy suspension containing the small molecule nucleic acid is obtained using the method of claim 7 or 8; Choose one of the following processes to obtain an analyte containing the small molecule nucleic acid: (i) The resuspended sample is treated with a small molecule nucleic acid extraction kit to obtain the analyte containing the small molecule nucleic acid. (ii) Depending on the volume of resuspension added, the resuspension is diluted or not diluted and then lysed to obtain the analyte containing the small molecule nucleic acid; Using the analyte as a template, RT-PCR detection was performed.

10. The method according to claim 9, characterized in that, The pyrolysis is selected from one of the following processing methods: (i) Incubate at 94~98℃ for 5~15 min; (ii) After adding the protease, incubate at 50-65℃ for 10-30 min, and then incubate at 94-98℃ for 5-15 min.

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