阅读说明：本技术 一种新型核酸免提取保存液 (Novel nucleic acid hand-free storage solution ) 是由 刘涛 刘硕 夏正邦 于 2021-02-09 设计创作，主要内容包括：本发明“一种新型核酸免提取保存液”,公开了一种无需进行后续核酸提取又能直接用于检测的保存液及其制备方法,该保存液包括如下组份：表面活性剂0.1-2％,酸碱缓冲剂0.1-2％,柠檬酸盐1-5％,弱酸0.1-5％,胍盐0-10％,蛋白酶K0-0.1％,螯合剂0.1-1％,防腐剂0.1-2％,其余为水,该保存液用于组织、细胞分析样本的保存,不仅包括冠状病毒、流感病毒、手足口病毒、麻疹风疹病毒,也包括其他类别的病毒和支原体、衣原体,鼻、咽拭子置于该保存液后其独特组份可以保护核酸RNA免受RNA酶的降解以及保护脱氧核糖核酸DNA免受DNA酶的降解,联合使用多种防腐剂使该试剂具有较强的抗细菌和抗真菌作用。(The invention discloses a novel nucleic acid hands-free preservation solution, and discloses a preservation solution which can be directly used for detection without subsequent nucleic acid extraction and a preparation method thereof, wherein the preservation solution comprises the following components: 0.1-2% of surfactant, 0.1-2% of acid-base buffer agent, 1-5% of citrate, 0.1-5% of weak acid, 0-10% of guanidine salt, 0-0.1% of proteinase K0, 0.1-1% of chelating agent, 0.1-2% of preservative and the balance of water, the preservative solution is used for preserving tissue and cell analysis samples, and not only comprises coronavirus, influenza virus, hand-foot-and-mouth virus and measles rubella virus, but also comprises other types of viruses, mycoplasma and chlamydia, and unique components of a nose swab and a throat swab can protect RNA from degradation of RNase and DNA from degradation of DNase after being placed in the preservative solution, and the preservative solution is combined with a plurality of preservatives to ensure that the reagent has strong antibacterial and antifungal effects.)

1. A novel nucleic acid hands-free preservation solution is characterized by comprising the following components: 0.1-2% of surfactant, 0.1-2% of acid-base buffer, 1-5% of citrate, 0.1-5% of weak acid, 0-10% of guanidine salt, 0-0.1% of protease K0, 0.1-1% of chelating agent, 0.1-2% of preservative and the balance of water.

2. The novel nucleic acid hands-free preservation solution according to claim 1, wherein the surfactant is a mixture of one or more of triton 100, triton 114, tween 40, tween 60, tween 80 and NP-40; the acid-base buffer is one or a mixture of more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate and Tris.

3. The novel nucleic acid hands-free preservation solution according to claim 1, wherein the citrate refers to one or both of sodium citrate and potassium citrate; the weak acid refers to one or two of formic acid, propionic acid and butyric acid.

4. The novel nucleic acid hands-free preservation solution according to claim 1, wherein the guanidine salt refers to one or both of guanidine isothiocyanate and guanidine hydrochloride, and the proteinase K refers to proteinase K of grade higher than diagnostic reagent grade.

5. The novel nucleic acid hands-free preservation solution according to claim 1, wherein the chelating agent is one or a mixture of more of edta.2na, edta.2k, egta.2na, egta.2k; the preservative is one or a mixture of gentamicin, merthiolate, ProClin300 and amphotericin.

6. The novel nucleic acid hands-free preservation solution according to claim 1, wherein the water is one or both of distilled water and deionized water.

7. The novel nucleic acid hands-free preservation solution according to claim 1, characterized in that the preparation method comprises the following steps: sequentially adding the surfactant, the acid-base buffer, the citrate, the weak acid, the guanidine salt, the proteinase K, the chelating agent, the preservative and the water, mixing and uniformly mixing; the preservation solution can be directly used for detection without subsequent nucleic acid extraction steps.

8. The novel nucleic acid hands-free preservation solution according to claim 1, wherein the preservation solution is used for preservation of animal, plant, bacterial, fungal, viral and cellular samples.

9. The novel nucleic acid hands-free preservation solution according to claim 1, wherein the preservation solution is used for preservation of RNA viruses and DNA viruses, and is used for nucleic acid detection, immunohistochemical detection, protein detection, colloidal gold test strips and other molecular biological detection.

10. The novel nucleic acid hands-free preservation solution according to claim 1, wherein the preservation solution is used for clinical detection reagents and scientific research reagents.

The technical field is as follows:

the invention belongs to the technical field of molecular biology, and particularly relates to novel preservation solution which can be directly used for detection without subsequent nucleic acid extraction and a preparation method thereof.

Background art:

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. Viruses (Virus) are non-cellular organisms that are small in size, simple in structure, contain only one nucleic acid (DNA or RNA) and must be parasitic in living cells and proliferate in a replicative manner, such as the novel coronavirus 2019-nCoV, hepatitis b Virus, influenza Virus, and the like.

For the preservation of viruses, common components such as phosphate, sodium chloride and the like are mostly adopted in the traditional method, the defects are that the traditional method can only be used for preservation, and the nucleic acid still needs to be extracted again before the subsequent detection, namely, the traditional preservation-extraction-detection step is one of the traditional preservation-extraction-detection steps, and the operation is troublesome. Therefore, it is of great significance to research a reagent which combines preservation and extraction instead of a conventional preservation solution for detecting samples such as new coronavirus, and at present, partial substitutes are used for preserving virus samples. The invention patent "a virus preservation solution free from nucleic acid extraction (application No. 202010148767.4)" discloses a virus preservation solution free from nucleic acid extraction, which can preserve viruses, but has the following disadvantages: 1. tween 20 is easier to foam, and because the volume of a common sampling tube is small, foam is easy to overflow out of the tube to cause cross infection, and meanwhile, Tween 20 influences the appearance and transmittance of the solution; 2. the acid protease is expensive and difficult to popularize and use in a large area. The invention patent "a nucleic acid extraction-free reagent and a use method thereof (application number is 202011191934. X)" discloses a technology of the nucleic acid extraction-free reagent, but has the following defects: 1. chelex-100 and digitonin are expensive and not readily available; 2. only Tris-HCl single buffer agent is available, and the buffering capacity is not strong.

The invention content is as follows:

in order to solve the technical defects, the invention discloses a novel nucleic acid hands-free preservation solution, and aims to provide a preservation solution which can be directly used for detection without subsequent nucleic acid extraction and a preparation method thereof, wherein the preservation solution comprises the following components: 0.1 to 2 percent of surfactant, 0.1 to 2 percent of acid-base buffer, 1 to 5 percent of citrate, 0.1 to 5 percent of weak acid, 0 to 10 percent of guanidine salt, 0 to 0.1 percent of protease K0, 0.1 to 1 percent of chelating agent, 0.1 to 2 percent of preservative, the rest is water, the preparation method comprises the preparation method of the nucleic acid hands-free storage solution, the virus storage solution is used for storing tissue and cell analysis samples, the virus storage solution not only comprises coronavirus, influenza virus, hand-foot-and-mouth virus and measles rubella virus, but also comprises other types of viruses, mycoplasma and chlamydia, the collected representative viruses are stored in the storage solution, the unique components of the nose swab and the throat swab can protect RNA from degradation of RNA enzyme and DNA from degradation of DNA enzyme after the nose swab and the throat swab are placed in the novel nucleic acid hands-free storage solution, and the reagent has stronger antibacterial and antifungal effects by using a plurality of preservatives in combination.

The technical scheme adopted by the invention is as follows:

a novel nucleic acid hands-free preservation solution is characterized by comprising the following components: 0.1-2% of surfactant, 0.1-2% of acid-base buffer, 1-5% of citrate, 0.1-5% of weak acid, 0-10% of guanidine salt, 0-0.1% of protease K0, 0.1-1% of chelating agent, 0.1-2% of preservative and the balance of water.

A novel nucleic acid hands-free preservation solution is characterized in that the surfactant is one or a mixture of more of triton 100, triton 114, Tween 40, Tween 60, Tween 80 and NP-40.

A novel nucleic acid hands-free preservation solution is characterized in that the acid-base buffer is one or a mixture of more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate and Tris.

A novel nucleic acid hands-free preservation solution is characterized in that citrate refers to one or two of sodium citrate and potassium citrate.

A novel nucleic acid hands-free preservation solution is characterized in that the weak acid refers to one or two of formic acid, propionic acid and butyric acid.

A novel nucleic acid hands-free preservation solution is characterized in that guanidine salt refers to one or two of guanidine isothiocyanate and guanidine hydrochloride.

A novel nucleic acid hands-free preservation solution is characterized in that the proteinase K is proteinase K of a grade higher than a diagnostic reagent grade.

A novel nucleic acid hands-free preservation solution is characterized in that the chelating agent is one or a mixture of more of EDTA.2Na, EDTA.2K, EGTA.2Na and EGTA.2K.

A novel nucleic acid hands-free preservation solution is characterized in that the preservative is one or a mixture of gentamicin, merthiolate sodium, ProClin300 and amphotericin.

A novel nucleic acid hands-free preservation solution is characterized in that water refers to one or two of distilled water and deionized water.

A novel nucleic acid hands-free preservation solution is characterized in that the preparation method comprises the following operation steps: and sequentially adding the surfactant, the acid-base buffer, the citrate, the weak acid, the guanidine salt, the proteinase K, the chelating agent, the preservative and the water, mixing and uniformly mixing.

A novel nucleic acid hands-free preservation solution is characterized in that the preservation solution can be directly used for detection without a subsequent nucleic acid extraction step.

A novel nucleic acid hands-free preservation solution is characterized in that the preservation solution is used for preserving animal, plant, bacteria, fungi, virus and cell samples.

A novel nucleic acid hands-free preservation solution is characterized in that the preservation solution is used for preserving RNA viruses and DNA viruses.

A novel nucleic acid hands-free preservation solution is characterized in that the preservation solution is used for nucleic acid detection, immunohistochemical detection, protein detection, colloidal gold test strips and other molecular biological detection.

A novel nucleic acid hands-free preservation solution is characterized in that the preservation solution is used for clinical detection reagents and scientific research reagents.

The preparation method of the novel nucleic acid hands-free storage solution in the novel nucleic acid hands-free storage solution comprises the following operation steps: and sequentially adding the surfactant, the acid-base buffer, the citrate, the weak acid, the guanidine salt, the proteinase K, the chelating agent, the preservative and the water, mixing uniformly, filtering by a 50-300-mesh sieve, and hermetically storing to obtain the novel nucleic acid hands-free storage solution.

It should be noted that, in the preparation process of the novel nucleic acid hands-free preservation solution, the reagents should be added in sequence in batches.

For those skilled in the art, according to the technical scheme and concept of the present invention, the amount and ratio of the above components can be adjusted according to specific requirements, so as to prepare the nucleic acid hands-free preservation solution with different characteristics.

Compared with the prior art, the novel nucleic acid hands-free storage solution has the following advantages:

1. the product does not contain toxic substances, is not easy to cause harm to human bodies, and has little influence on human bodies and environment.

2. The preservation solution can be directly used for nucleic acid detection without subsequent nucleic acid extraction.

3. Compared with the preservation solution, the preservation solution is simple, convenient and rapid.

Description of the drawings:

FIG. 1 is a schematic view of the present invention showing how "a novel nucleic acid hands-free preservation solution" preserves lentiviruses, FIG. 1-1 shows a state in which a phosphate buffer solution (tube A) and a novel nucleic acid hands-free preservation solution (tube B) are not added with lentiviruses according to an embodiment, and FIG. 1-2 shows a state in which a phosphate buffer solution (tube A) and a novel nucleic acid hands-free preservation solution (tube B) are added with a sampling swab containing lentiviruses according to an embodiment;

FIG. 2 is a schematic view of a fluorescence microscope for preserving lentivirus with the novel nucleic acid hands-free preservation solution of the present invention, FIG. 2-1 is a fluorescence microscope observation result of preserving lentivirus with phosphate buffer solution for 3 minutes in an embodiment, and FIG. 2-2 is a fluorescence microscope observation result of preserving lentivirus with the novel nucleic acid hands-free preservation solution for 3 minutes in an embodiment.

The specific implementation mode is as follows:

the following detailed description further illustrates the contents of the "novel nucleic acid hands-free preservation solution" 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.

Example one

The invention relates to a novel nucleic acid hands-free preservation solution for preserving lentivirus, which comprises the following components: 0.7% of surfactant, 1% of acid-base buffer, 3% of citrate, 2% of weak acid, 0% of guanidine salt, 0% of protease K, 0.15% of chelating agent, 0.13% of preservative and the balance of water. The following examples use phosphate buffer at the same temperature for the same treatment time and the same virus as a control.

The preparation method of the novel nucleic acid hands-free preservation solution comprises the following steps: taking 7ml of the surfactant, 10g of acid-base buffer, 30g of citrate, 20ml of weak acid, 0g of guanidine salt, 0g of protease K, 1.5g of chelating agent and 1.3g of preservative, supplementing water to 1000ml, mixing the components in sequence, fully mixing the components uniformly, filtering the mixture by a 100-mesh sieve, and storing the mixture in a sealed manner to obtain the novel nucleic acid hands-free storage solution.

3ml of phosphate buffer solution is taken, and the same lentivirus is preserved for 3 minutes; taking 3ml of the novel nucleic acid hands-free storage solution prepared by the steps, and storing the same lentivirus for 3 minutes; the results are shown in the attached figures 1 and 2.

The experimental result shows that when the same lentivirus is stored, the phosphate buffer solution in the embodiment I is compared with the novel nucleic acid hands-free storage solution under the same condition, the novel nucleic acid hands-free storage solution has no virus, and the phosphate buffer solution virus still exists; therefore, the novel nucleic acid hands-free storage solution can completely crack the lentivirus, and the phosphate buffer solution cannot crack the lentivirus, namely the novel nucleic acid hands-free storage solution can be directly used for detection without subsequent nucleic acid extraction.

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.