CN-121975795-A - Application of haloacetic acid in enrichment of small-molecule nucleic acid in biological sample

Abstract

The application discloses application of halogenated acetic acid in enriching small molecular nucleic acid in a biological sample, and belongs to the technical field of molecular biology. The application utilizes the halogenated acetic acid to enrich the small molecular nucleic acid in the liquid biological sample, not only can eliminate the influence of interfering substances, but also can ensure that the enriched small molecular nucleic acid sample is stably stored at room temperature, and can directly carry out amplification detection without extraction, thereby having very important application value.

Inventors

• ZHANG PAN
• ZOU RUIYANG
• FENG QIUYING
• LIU YUNLONG
• YUAN ZHIWEN
• ZHANG JILIAN
• WANG LEI
• ZHONG JUNYU
• GUO HUI
• CHENG HE

Assignees

• 觅瑞（杭州）生物科技有限公司

Dates

Publication Date

20260505

Application Date

20260206

Claims (10)

1. 1. Use of haloacetic acid in the preparation of a precipitant for precipitating small molecule nucleic acids in a liquid biological sample.
2. 2. A precipitating agent for precipitating small molecule nucleic acids in a liquid biological sample, comprising haloacetic acid, with or without other components.
3. 3. A kit for enriching small molecule nucleic acids in a liquid biological sample comprising the precipitant of claim 2.
4. 4. The kit of claim 3, further comprising a suspension for suspending the precipitate comprising the small molecule nucleic acid separated after the liquid biological sample is treated with the precipitating agent.
5. 5. The kit of claim 4, wherein the resuspension is an alkaline solution.
6. 6. A method for enriching small molecular nucleic acid in a liquid biological sample, which is characterized by comprising the step of adding halogenated acetic acid into the liquid biological sample, and separating to obtain a precipitate containing the small molecular nucleic acid after adding the halogenated acetic acid.
7. 7. The method of claim 6, further comprising the step of resuspending the precipitate with a suspension to obtain a suspension comprising the small molecule nucleic acid.
8. 8. The method of claim 7, wherein the sediment is stored at 20-40 ℃ and the sediment is resuspended within 7 days using the resuspension.
9. 9. A method for detecting small molecular nucleic acid in a liquid biological sample based on RT-PCR, which is characterized by comprising the following steps: Obtaining a resuspension comprising the small molecule nucleic acid using the method of claim 7 or 8; Selecting one of the following treatments to obtain a test substance comprising the small molecule nucleic acid: (i) Treating the resuspension matter by using a small molecule nucleic acid extraction kit to obtain the object to be detected containing the small molecule nucleic acid, (Ii) According to the volume of the added heavy suspension, carrying out cracking on the heavy suspension after dilution or undiluted to obtain the to-be-detected object containing the small molecule nucleic acid; and carrying out RT-PCR detection by taking the object to be detected as a template.
10. 10. The method of claim 9, wherein the lysing is selected from one of the following treatments: (i) Incubating for 5-15 min at 94-98 ℃; (ii) After protease is added, the mixture is incubated for 10-30 min at 50-65 ℃ and then incubated for 5-15 min at 94-98 ℃.

Description

Application of haloacetic acid in enrichment of small-molecule nucleic acid in biological sample Technical Field The application relates to the technical field of molecular biology, in particular to application of halogenated acetic acid in enrichment of small molecular nucleic acid in biological samples. Background MicroRNA (miRNA) is an endogenous non-coding small molecular RNA with the length of about 18-25 nucleotides, has important regulation and control functions in cells, and participates in various biological processes such as cell proliferation, differentiation, apoptosis, metabolism and the like. In recent years, free miRNA exists in blood plasma, and the expression profile of the miRNA changes in the occurrence and development processes of various diseases such as tumor, cardiovascular diseases and the like, so that the blood plasma serum free miRNA is expected to become a novel biomarker for disease diagnosis, prognosis evaluation and treatment monitoring. The existing common free miRNA extraction method mainly comprises two steps of 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 targets with lengths exceeding 50nt can generally be recovered well. However, mirnas are not substantially more than 25nt in length, and thus their recovery is generally not high. To maximize miRNA recovery, the extraction kit typically limits loading volumes, e.g., QIAGEN MIRNEASY Serum/PLASMA ADVANCED KIT allows up to 0.6mL plasma, and, e.g., promega Maxwell RSC miRNA from PLASMA AND Serum kit allows up to 0.5mL plasma. Under the condition of limited total volume of the reagent, the precipitation capability of miRNA (micro ribonucleic acid) can be enhanced by realizing higher lysate ratio and binding solution ratio, such as ethanol, isopropanol and the like with ultrahigh concentration. However, in such an ultra-high concentration ethanol and isopropanol reagent environment, molecular impurities in the sample are easily adsorbed to a solid phase together with miRNA, thereby resulting in reduced purity and eventually interfering with sample detection stability. In addition, when the sample volume is less than 1mL, the free miRNA which is low expressed in the blood plasma serum is difficult to detect, and when a large volume sample is directly used, excessive impurities are easy to be brought, and the detection reaction is inhibited to cause detection failure. Therefore, there is a need to develop a technique for free miRNA enrichment of plasma/serum samples to concentrate large volumes of samples to fit miRNA extraction kits. Meanwhile, most of interferents are not enriched, and inhibition interference of impurities in an initial sample on miRNA detection is greatly weakened. Disclosure of Invention In order to solve at least one of the above technical problems, the inventors found that when a liquid biological sample such as plasma/serum is treated with haloacetic acid such as trichloroacetic acid, small molecule nucleic acids such as free miRNA can be enriched and the influence of interferents in the initial sample on the subsequent detection of small molecule nucleic acids can be reduced, and at the same time, small molecule nucleic acids enriched with haloacetic acid such as trichloroacetic acid can be stably stored at room temperature (20 to 40 ℃) for 7 days and can be directly used for RT-PCR without extraction, thereby completing the present invention. In a first aspect, the application provides the use of haloacetic acid in the preparation of a precipitant for precipitating small molecule nucleic acids in a liquid biological sample. As described above, the present inventors have unexpectedly found and verified through a large number of experiments that haloacetic acids such as trichloroacetic acid can enrich small-molecule nucleic acids in biological samples. Haloacetic acids are a class of organic compounds produced by substitution of one or more hydrogen atoms on the alkyl portion of acetic acid (particularly the alpha-carbon atom, i.e., the carbon atom directly attached to the carboxyl group) with a halogen atom (fluorine, chlorine, bromine, iodine). In some embodiments of the application, the halogenated acetic acid includes, but is not limited to, chloroacetic acid, dichloroacetic acid, trichloroacetic acid, trifluoroacetic acid, tribromoacetic acid. In the application, the liquid biological sample is selected from a body fluid sample, a tissue processing fluid sample, a cell suspension sample, an exosome suspension sample, a water body and other environmental liquid samples. Wherein the body fluid sample includes, but is not limited to, serum, plasma, urine, cerebrospinal fluid. The tissue treatment fluid sample is a fluid sample obtained by homogenizing, cracking and the like of a fresh/frozen/paraffin-embedded tissue sample. Such cells include, but