阅读说明：本技术 一种生物样本核酸释放保存剂 (Biological sample nucleic acid release preservative ) 是由 王珺飞 李文天 于 2021-08-04 设计创作，主要内容包括：本发明“一种生物样本核酸释放保存剂”属于分子生物学技术领域,公开了一种可裂解常见的检测样本且能直接用于下游的PCR检测或高通量测序且裂解后所释放的样本核酸可以稳定的长期保存的方法,该保存剂包含了的Tween20(0.01%-10%,V/V)、糖原(2-500ng/μl)、甲酰胺(2-500ng/μl)、人工合成的25nt的poly A(2-500ng/μl)、氢氧化钾(0.01mM-50mM),甲酚红(0.01%-10%,质量/体积),溶剂为无核酸酶水,使用该方法可肉眼对PCR体系进行质量控制,耗时短,效率高,操作简便,生产成本低,保存时间长,极具市场推广价值。(The invention discloses a method for cracking common detection samples and directly using for downstream PCR detection or high-throughput sequencing, wherein the sample nucleic acid released after cracking can be stably stored for a long time, the preservative comprises Tween20 (0.01-10%, V/V), glycogen (2-500 ng/mu l), formamide (2-500 ng/mu l), artificially synthesized 25nt poly A (2-500 ng/mu l), potassium hydroxide (0.01 mM-50 mM), cresol red (0.01-10%, mass/volume) and a solvent of nuclease-free water, the method can visually control the quality of a PCR system, has the advantages of short time consumption, high efficiency, simple operation, low production cost and long storage time, has great market popularization value.)

1. A biological sample nucleic acid release preservative is characterized in that the preservative comprises Tween20 (0.01-10%, V/V), glycogen (2-500 ng/mu l), formamide (2-500 ng/mu l), artificially synthesized 25nt poly A (2-500 ng/mu l), potassium hydroxide (0.01-50 mM), cresol red (0.01-10%, mass/volume) and a solvent of nuclease-free water.

2. The biological sample nucleic acid release preservative agent for releasing nucleic acid according to claim 1, wherein a sample to be tested is added into the biological sample nucleic acid release preservative agent according to different operation methods and mixed uniformly.

3. The biological sample nucleic acid release preservative agent for releasing nucleic acid according to claim 2, wherein the sample to be tested comprises an oral swab, a throat swab, a vaginal swab, an anal swab, a bacterial colony, blood, urine and fungus.

4. The biological sample nucleic acid release preservative for releasing nucleic acid according to claim 2, wherein the operation method comprises immersing and mixing an oral swab, a pharyngeal swab, a vaginal swab and an anal swab in the biological nucleic acid release preservative; centrifuging the urine sample at 3000rpm/min, discarding the supernatant, mixing the urine residue with 0.5-3ml of the biological sample nucleic acid release preservative, and mixing the blood and the biological sample nucleic acid release preservative according to the volume ratio of 1: 24-149; after the colony is picked up, the colony is mixed with 0.5 to 3ml of the biological sample nucleic acid release preservative and then is shaken vigorously for 40 seconds.

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a novel preservative which can be directly used for detection without subsequent nucleic acid extraction and can stably store sample nucleic acid (including DNA and RNA) released after cracking for a long time and a preparation method thereof.

Background

Nucleic acid refers to a general term of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), is a biological macromolecular polymer polymerized by a plurality of nucleotide monomers, is one of the most basic substances of life, is an essential constituent substance of all known life forms, is the most important substance of all biological molecules, is widely present in all animal and plant cells and microorganisms, consists of nucleotide, and the nucleotide monomers consist of pentose, phosphate and nitrogenous base; if the five carbon sugar is ribose, the polymer formed is RNA; if the five carbon sugar is deoxyribose, the polymer formed is DNA.

In recent years, nucleic acid detection has been increasingly used in various fields, and often involves processing various samples (including tissues, nasal swabs, throat swabs, blood, serum, saliva, urine, etc.) for detection and gene amplification. In general, various brands of nucleic acid extraction kits are used to perform the above-mentioned processing of various samples and extraction of nucleic acids.

The nucleic acid detection has the natural advantages of high sensitivity and high specificity, the extraction, purification and storage of nucleic acid in a sample directly influence the downstream detection quality, and the nucleic acid extraction kit is used for extracting one sample of nucleic acid, so that not only is the time consumed, but also the operation steps are complex, and the consumed cost is not low.

Several reagents for direct processing of test samples without purification of nucleic acids are known, and application No. 202010130611.3 discloses a reagent for lysis of samples, which can be used in combination with a PCR reagent to directly detect viral nucleic acids. Patent application No. 201510973871.6 also discloses a method for directly detecting viral nucleic acids in common sample types by denaturing viral coat proteins with a protein denaturing agent to release viral nucleic acids, followed by PCR using specific primers and probes. The publication No. 108823201B discloses an RNA nucleic acid releasing agent and its application, which can release RNA in a sample, and then can replace guanidine salt or urea for RNA extraction by a water boiling method, thereby simplifying the purification process of RNA extract before PCR. The reagent for preserving sample nucleic acid, such as RNAlater of Sermer Fei company (ThermoFisher), which is commercially available, can stably preserve sample RNA at 4 ℃ for one month and at room temperature for 7 days, but the RNA sample of RNAlater cannot be directly used for downstream PCR detection, and must be subjected to complicated purification steps before PCR amplification. However, the above technical solutions still have the following problems.

The technical scheme is mainly used for detecting viruses, bacteria and the like, and has a narrow application range.

The above-mentioned technical solutions do not provide a method for long-term storage or transport of nucleic acids released by the sample upon lysis.

The technical scheme can not directly carry out PCR amplification, and also needs traditional nucleic acid purification, thus having complex operation.

In summary, the prior art has obvious defects and inconveniences in practical use, and can realize direct amplification but cannot store nucleic acid for a long time, and can store nucleic acid for a long time but cannot directly amplify nucleic acid, so that improvement is needed.

Disclosure of Invention

In order to solve the technical defects, the invention aims to provide a preservative which can be directly used for detection and preservation without subsequent nucleic acid extraction and a preparation method thereof, not only can crack common detection samples (oral swabs, throat swabs, vaginal swabs, anal swabs, blood, urine, fungi and the like), but also can be directly used for downstream PCR detection or high-throughput sequencing, and meanwhile, the released sample nucleic acids (including DNA and RNA) can be stably preserved for a long time after cracking, wherein the DNA can be preserved for half a year at room temperature and preserved for one year at 2-8 ℃ and preserved for a long time below-20 ℃; RNA can be stored for one month at room temperature, half a year at 2-8 ℃, and long-term storage below-20 ℃; in quality control, when a sample to be detected is not added, the biological nucleic acid release preservative is pink or purple, and the color of the added sample is orange yellow or orange green after complete treatment, so that whether the sample is completely cracked and the nucleic acid is released can be known by naked eyes, and when the treated sample is subsequently added into a PCR reaction system, the color of the system is pink, and meanwhile, the quality control can be carried out on the sample addition of PCR; the method has the advantages of short time consumption, high efficiency, simple operation, low production cost, long storage time and great market popularization value.

In order to achieve the aim, the invention provides a preservative for releasing nucleic acid of a biological sample, which can be directly used for detecting and preserving without subsequent nucleic acid extraction, and the preservative comprises Tween20 (0.01-10%, V/V), glycogen (2-500 ng/mu l), formamide (2-500 ng/mu l), artificially synthesized 25nt poly A (2-500 ng/mu l), potassium hydroxide (0.01 mM-50 mM), cresol red (0.01-10%, mass/volume) and a solvent of nuclease-free water.

The invention also provides a use method of the nucleic acid extraction-free reagent, which comprises the following steps.

And (3) placing the sample to be detected in the biological sample nucleic acid release preservative, uniformly mixing to obtain a mixed solution, and directly carrying out nucleic acid detection on the mixed solution according to different operation methods.

The biological sample nucleic acid release preservative agent is used for releasing nucleic acid, and the sample to be detected comprises an oral swab, a throat swab, a vaginal swab, urine, an anal swab, a bacterial colony, blood and a fungus sample.

The operation method of the biological sample nucleic acid release preservative for nucleic acid release comprises the following steps: immersing the biological sample nucleic acid release preservative in an oral swab, a throat swab, a vaginal swab and an anal swab, and uniformly mixing; centrifuging the urine sample at 3000rpm/min, discarding the supernatant, mixing the urine residue with 0.5-3ml of the biological sample nucleic acid release preservative, and mixing the blood and the biological sample nucleic acid release preservative according to the volume ratio of 1: 24-149; after the colony is picked up, the colony is mixed with 0.5 to 3ml of the biological nucleic acid release preservative and then is shaken vigorously for 40 seconds.

The nucleic acid (including DNA and RNA) released by the biological sample nucleic acid release preservative can be stably stored for a long time, wherein the DNA can be stored for a half year at room temperature and stored for a year at 2-8 ℃, and can be stored for a long time below-20 ℃; RNA can be stored for one month at room temperature, half a year at 2-8 ℃, and long-term storage below-20 ℃.

According to the biological sample nucleic acid release preservative, through the special action mechanism of each component, the biological sample nucleic acid release preservative can have strong cell lysis capacity within the temperature range of 2-30 ℃, and after the action, the lysis is not only fast, but also no additional heating is needed, and no subsequent nucleic acid extraction is needed.

According to the biological sample nucleic acid release preservative, quality control can be performed when samples are added for PCR, when the samples are not added, the biological sample nucleic acid release preservative is pink or purple, and when the samples are added, the biological sample nucleic acid release preservative is changed into orange yellow or orange green after being completely treated, so that whether the samples are completely cracked and release nucleic acid can be known by naked eyes; meanwhile, when the processed sample is added into a PCR reaction system subsequently, the color of the system is changed into pink, and meanwhile, the quality control can be carried out on the sample adding of the PCR.

Drawings

FIG. 1 shows the results of a nucleic acid detection experiment using human buccal swab beta-actin.

FIG. 2 shows the results of detection of nucleic acids released from human buccal swabs (sample A) at room temperature (25 ℃) and human buccal swabs (sample A) in a refrigerator (2 ℃ -8 ℃).

FIG. 3 shows the results of detection of nucleic acids released from human buccal swab (sample B) at room temperature (25 ℃) and human buccal swab (sample B) in a refrigerator (2 ℃ to 8 ℃).

FIG. 4 shows the results of detection of nucleic acids released from human buccal swab (sample C) at room temperature (25 ℃) and human buccal swab (sample C) in a refrigerator (2 ℃ to 8 ℃).

Detailed Description

The following detailed description further illustrates the context of the "a biological sample nucleic acid release preservative" of the present invention, but should not be construed as limiting the invention. Modifications or substitutions to methods, conditions, steps and applications of the invention may be made without departing from the spirit and substance of the invention.

The invention discloses a preservative for nucleic acid release of a biological sample, which can be directly used for detection and preservation without subsequent nucleic acid extraction, and comprises Tween20 (0.01-10%, V/V), glycogen (2-500 ng/mu l), formamide (2-500 ng/mu l), artificially synthesized 25nt poly A (2-500 ng/mu l), potassium hydroxide (0.01 mM-50 mM), cresol red (0.01-10%, mass/volume) and nuclease-free water as a solvent.

The invention uses Tween20 with the concentration range of 0.01-10% as a main cell lysate and a mild surfactant, which can disintegrate cell membranes, nuclear membranes, mitochondrial membranes and chloroplast membranes to release inclusion without denaturing protein, thereby being beneficial to ensuring that the activity of enzyme in downstream PCR is not influenced; because the Tween20 has mild action, it can also keep the activity of DNase and RNase in cells.

In order to ensure that DNA and RNA are not degraded, glycogen, formamide and artificially synthesized 25nt Poly A are innovatively added into a release agent, the concentration of the polyA is in a range of 2-500 ng/mu l, and the three protective agents have the following three functions: the RNase can be competitively rob, so that the target RNA is protected; the target RNA is protected from being adsorbed by the static electricity of the tube wall, and the effective concentration of the target RNA is ensured to be invariable all the time; formamide is also an accelerator for PCR amplification, and can promote PCR reaction.

In addition, in order to further increase the stability of the solution system and the efficiency of disintegrating cell walls (fungi), potassium hydroxide with the concentration of 0.01mM-50mM is also contained in the reagent, and the cell walls of the fungi are easier to break walls in an alkaline environment, so that nucleic acid substances are released.

In order to realize quality control, an indicator with 0.01-10% cresol red is added, the color of the releasing agent is pink or grape purple when a sample is not added, and the releasing agent is orange yellow or orange green after the sample is added and completely treated, so that whether the sample is completely cracked and releases nucleic acid can be obviously known by naked eyes; meanwhile, when the processed sample is subsequently added into a PCR reaction system, the color of the system is changed into pink.

In order to verify the effect of the preservative released from the nucleic acid of the biological sample, the present invention also provides several examples, wherein the results of the tests are shown in FIG. 1, FIG. 2, FIG. 3 and FIG. 4.

Example 1: nucleic acid detection of human buccal swab beta-actin.

The detection method comprises the following steps.

Step one.

Taking one oral swab, directly extending the swab into 1ml of the sample releasing agent, turning upside down for several times, and discarding the swab after the color of the reagent is changed from grape purple to orange green to obtain a uniformly mixed solution.

And step two.

2 mul, 4 mul and 8 mul of processed samples are respectively taken to configure a qPCR system, three parallel experiments are carried out on samples with each volume, FAM channels are used for detecting mRNA of human beta-actin, the primer design (detection primer and probe of mRNA of human beta-actin: HB-F CGAGCACAGAGCCTCGC; HB-R CATCATCCATGGTGAGCTGG; HB-P FAM-ATCCGCCGCCCGTCCA-MGB) is a cross-intron primer, and only the mRNA of human beta-actin can be specifically amplified but the coding DNA of the mRNA can not be amplified.

Secondly, PCR amplification.

The detection equipment ABI 7500, the reaction conditions are as follows: reacting at 50 deg.C for 5min, at 95 deg.C for 2min, at 95 deg.C for 10s, at 60 deg.C for 30s, and performing 45 cycles, and collecting fluorescence at 60 deg.C.

And thirdly, experimental results.

Referring to FIG. 1, it can be seen that the biological sample nucleic acid release preservative of the present invention has excellent lysis release effect, and the Ct of PCR decreases by 1 with the doubling of the addition amount of the release agent, indicating that the release agent has no inhibition effect on PCR.

Example 2: human buccal swab nucleic acids released at room temperature (25 ℃) and in a refrigerator (2 ℃ to 8 ℃) were detected.

Step one.

Taking oral swabs (sample A, sample B and sample C) of three different persons, respectively and directly putting the swabs of the different samples into different 1ml of the sample releasing agent, turning upside down for several times until the color of the reagent is changed from grape purple to orange green, then discarding the swabs to obtain a uniformly mixed solution, averagely dividing each sample into two parts, placing one part in a refrigerator at room temperature (25 ℃) and placing the other part in a refrigerator at 2-8 ℃.

And step two.

It was PCR tested according to the exemplary first step two and PCR amplification method on day 1, day 2, day 3 through day 15, respectively.

And thirdly, experimental results.

Referring to FIG. 2, FIG. 3, FIG. 4, it can be seen that the preservation of nucleic acid released at room temperature (25 ℃) and in a refrigerator (2 ℃ -8 ℃) is not very different, which indicates that the nucleic acid sample can be preserved for 15 days at room temperature, and the condition meets the actual living requirements.

In summary, the present invention aims to provide a preservative which can be directly used for detection and preservation without subsequent nucleic acid extraction and a preparation method thereof, which can not only crack common detection samples (oral swab, throat swab, vaginal swab, anal swab, blood, urine, fungi, etc.), but also be directly used for downstream PCR detection or high throughput sequencing, and meanwhile, the sample nucleic acids (including DNA and RNA) released after cracking can be stably preserved for a long time, wherein the DNA can be preserved for half a year at room temperature, and can be preserved for a long time below-20 ℃ at 2-8 ℃; RNA can be stored for one month at room temperature, half a year at 2-8 ℃, and long-term storage below-20 ℃; in quality control, when a sample to be detected is not added, the biological nucleic acid release preservative is pink or purple, and the color of the added sample is orange yellow or orange green after complete treatment, so that whether the sample is completely cracked and the nucleic acid is released can be known by naked eyes, and when the treated sample is subsequently added into a PCR reaction system, the color of the system is pink, and meanwhile, the quality control can be carried out on the sample addition of PCR; the method has the advantages of short time consumption, high efficiency, simple operation, low production cost, long storage time and great market popularization value.

It is obvious to those skilled in the art that the above processing time, sample and temperature can be adjusted according to the specific needs according to the technical scheme and concept of the present invention, and the obvious changes or modifications from this are still within the protection scope of the present invention.