1. A sample processing method for rapidly extracting biological nucleic acid and/or protein is characterized by comprising the following steps:

a) adding the refrigerant gas and the biological sample to be ground into the grinding device before, after or simultaneously, and adding an appropriate volume of inhibitor solution or H containing the extract₂O or other liquid;

b) under the refrigeration action of the refrigeration gas, grinding the biological sample after the refrigeration gas is in a liquid state and/or is dried;

2. according to claim 1a), a liquid is added to aid the milling, especially a solution containing the inhibitor to be extracted.

3. According to claim 1b), the biological sample can be crushed or ground when the refrigerating gas is in liquid state, or ground after the refrigerating gas is dried, and the biological sample can be repeatedly ground by adding the refrigerating gas once or more than once until the required powder state is achieved.

4. The conventional RNA or protein or any co-mentioned kit of DNA, RNA and protein can be changed into a room temperature rapid extraction kit by freeze-drying and grinding, and specifically, the kit comprises but is not limited to, reduced incubation time and/or reduced centrifugation time and/or added inhibitor to be extracted and/or reduced irrelevant or non-functional reagent and/or simplified experiment steps and the like.

5. The improved kit of claim 4, wherein: grinding biological sample by freeze drying; adding the solution to be extracted to allow the extract (DNA and/or RNA) to enter; immobilizing the extract to be extracted on a substrate, including but not limited to a microcolumn, a magnetic sphere, etc.; washing once or more than once to remove impurities; the extract can be obtained by one or more times of elution.

6. The method of claim 4, wherein: when the extract is protein, adding protein extract, and/or shortening incubation time and centrifugation time, and directly centrifuging to obtain protein.

7. According to claim 4, the DNA, RNA and protein optionally contain protein, after extraction of DNA and/or RNA, proteins are precipitated with organic substances, in particular acetone; washing the protein; solubilised proteins or dried proteins.

8. A kit for carrying out the method according to any one of claims 1 to 9, and a vector describing the method according to any one of claims 1 to 8.

9. A computer readable carrier carrying a computer program comprising instructions for carrying out the method according to any one of claims 1 to 9.

10. An automated nucleic acid extraction device, the device comprising: a liquid filling/pipetting assembly, a centrifugation assembly, a grinding assembly and a controller electrically connected with the assemblies; the controller comprises a computer readable carrier according to claims 1-9.

[ background of the invention ]

With the development of genome sequencing technology, the sequence, structure and function research of biological genome is rapidly developed, and obtaining biological genome DNA with high purity, high content and high integrity is the first prerequisite for the application of genome sequencing technology. With the development of biology, researchers have gained increased interest in the study of RNA produced by transcription of genes and proteins that are the products of translation of RNA.

More and more DNA, RNA and protein and extraction methods of the three have been developed. Specifically, the conventional methods for extracting biological genomic DNA mainly include CTAB and SDS methods, and the common methods for extracting biological RNA mainly include Trizol method, guanidine thiocyanate/phenol method, SDS/phenol method, guanidine hydrochloride method, and the like. Some formulations of denatured and non-denatured proteins were also developed.

The quality and quantity of the extractions for DNA, RNA and proteins are not only related to the formulation from which they are extracted but also to the manner and/or method of processing the biological sample. Thus, some methods for processing biological samples are emerging continuously, such as: mechanical methods, including high speed tissue mashers, glass homogenizers, and the like; physical methods including repeated freeze thawing, cold and hot alternation, ultrasonic method, and pressure crushing method; chemical and biological methods including organic solvent extraction, aqueous solution extraction, enzymatic methods, and the like.

The methods and methods widely used in laboratories are mainly mechanical methods, enzymatic methods and liquid nitrogen milling methods. Each of these methods has its advantages and disadvantages. The mechanical method has the advantages that: the efficiency is high, and the automation is facilitated; the disadvantages are as follows: the cost is high. An enzyme method comprises the following steps: the efficiency is high, and the trouble is saved; the disadvantages are as follows: high cost and easy inactivation of enzyme. The traditional liquid nitrogen grinding method has the advantages that: the cost is low; the disadvantages are as follows: the method has the advantages of low efficiency, difficult grinding, low extracted product amount and the like, and particularly, the traditional liquid nitrogen grinding method requires that a biological sample is ground under the protection of liquid nitrogen, the liquid nitrogen flows due to the liquid nitrogen, and the biological sample moves along with the flowing of the liquid nitrogen and is difficult to grind in the grinding process.

[ summary of the invention ]

The invention aims to overcome the defects in the prior art and provide a sample processing method, an extraction kit and an extraction device which can rapidly extract biological RNA, protein and DNA, RNA and protein.

In order to achieve the above purpose, a preferable scheme is to select a refrigerating liquefied gas, absorb a large amount of heat in the process of gasification, rapidly cool the surrounding environment in a short time, and provide a low-temperature freezing environment in a short time. Liquid nitrogen is preferably selected as the refrigerant gas in view of safety, cost price and ready availability. The invention provides a new liquid nitrogen grinding method, namely a liquid nitrogen freeze-drying grinding method. The method utilizes the advantages of the traditional liquid nitrogen grinding method: refrigeration to prevent chemical reactions, more specifically degradation reactions of the extract, while maintaining the integrity of the extract; meanwhile, the defects of the traditional grinding method are also avoided: the biological sample flows with the flow of liquid nitrogen. Of course, the biological sample can also be mashed or ground under the protection of liquid nitrogen. After freeze-drying, the biological sample can be directly ground, so that the grinding process becomes simple, and the grinding time can be shortened by easy operation, and at the same time, the ground biological sample particles reach a very fine state.

Theoretically, first, below 0 ℃, the ground biological sample is in a solid state, and the rate at which chemical reactions between solids, including degradation reactions, occur is very small. Secondly, according to the van t hough rule: when the reaction temperature is increased by 10K, the reaction rate is 2-4 times of the original rate; on the contrary, the reaction rate is changed to 1/4-1/2 times when the temperature is reduced by 10K. In practical operation, the whole grinding process can be completed within about 3-5 minutes, and the time of degradation reaction is short. Combining the above 3 factors, the degradation reaction of the biological sample to be extracted is negligible.

Under freeze-drying conditions, the biological sample is ground to a very fine powder. This can greatly increase the contact area of the extract to be extracted with the extracting solution, so that the extract to be extracted easily enters the extracting solution, and thus can shorten the extraction time and increase the amount of the extract to be extracted. More precisely, the extract to be extracted is RNA, DNA and/or protein.

To confirm the feasibility of the above implementation, the temperature dependence was determined after thorough cooling of the mortar with liquid nitrogen at room temperature of about 22 ℃. As can be seen from fig. 1, the temperature near the mortar wall is very low in the range of the first 5 minutes, so that the rate of degradation reaction of the extract to be extracted is negligible. Even if a small amount of heat is generated when the biological sample is ground, liquid nitrogen may be further added to continuously lower the temperature according to actual conditions. Thus, the present invention is feasible.

As a preferred method, the liquid nitrogen is added once or more, and a particularly preferred number is 1 to 5, and a more preferred number is 2 to 3.

In the practice of the invention, a set of grinding apparatus is selected, preferably a common but not limited mortar apparatus.

The liquid nitrogen and the biological sample can be added into the mortar simultaneously, the liquid nitrogen can be added firstly and then the biological sample can be added, or the liquid nitrogen can be added firstly and then the biological sample can be added, so that the mortar and the biological sample are cooled, the grinding is started after the liquid nitrogen is dried, and the biological sample is ground into powder. The biological sample can be continuously ground by adding liquid nitrogen more than once, so that the particles of the powder become finer.

For biological samples with high water content, such as plant tissue, it can be freeze dried directly with liquid nitrogen and ground into fine powder. Or grinding together with the plant tissue by adding a small amount of a solution containing an extract inhibitor. Generally, for biological samples with high water content, liquid nitrogen freeze-drying can be directly used for grinding.

For biological samples with low water content, such as animal tissues and cells, the grinding can be carried out by direct freeze-drying or by adding a suitable volume of liquid, especially a solution containing an inhibitor for preventing the degradation of the extract, together with a biological sample with low water content. The effect of adding the solution containing the extract inhibitor is as follows: 1. preventing the degradation of the extract; 2. the formation of ice crystals is beneficial to the crushing of a biological sample with low water content; 3. the ground biological sample is advantageously dispersed in the powder formed by the addition of the solution. More particularly, in relation to DNA extraction, it is desirable to add a chelating agent or mixture of chelating agents, which are well known to those skilled in the art, including but not limited to N-acetyl-L-cysteine, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), ethylenediamine-N, N ' -disuccinic acid (EDDS), 1, 2-bis (o-aminophenoxy) ethane-N, N, N ', N ' -tetraacetic acid (BAPTA), and phosphonate chelating agents (e.g., including but not limited to nitrilotris (methylene) phosphonic acid (NTMP), ethylenediaminetetra (methylenephosphonic acid) (EDTMP), diethylenetriaminepenta (methylene) phosphonic acid (DTPMP), 1-hydroxyethylidene-1, 1-diphosphonic acid (HEDP), and the like). In some embodiments, the chelating agent comprises EDTA or DTAP. In some embodiments, aqueous EDTA is commonly used.

More particularly, it concerns the extraction of RNA by adding reducing agents and/or RNase A inhibitors or mixtures of RNA inhibitors, such as: including but not limited to some of the reducing agents disclosed in U.S. patent specification 6825340 or U.S. patent specification 677720. In some embodiments, the commonly used RNase a inhibitor is Diethylpyrocarbonate (DEPC).

More particularly, it relates to protein extraction, wherein some protease inhibitors or mixtures of protease inhibitors are added, such as: including but not limited to phenylmethylsulfonyl fluoride (PMSF), ethylenediaminetetraacetic acid (EDTA), pepsin inhibitor (peptin), leupeptin (leupeptin), trypsin inhibitor (aprotinin), nelfinavir, saquinavir, indinavir, amprenavir, ritonavir, lopinavir and complex formulations, sodium fluoride, sodium orthovanadate, sodium glycerate, sodium pyrophosphate, and the like. In some embodiments, an aqueous solution of PMSF is used.

As a preferred scheme, the inhibitor to be extracted is added with the concentration more than or equal to 0; an especially preferred inhibitor concentration is typically between 0.1 and 200 times the normal recommended working concentration; more preferably, the inhibitor concentration is between 1-fold and 2-fold. Specifically, the DNase I inhibitor is 20mM EDTA; DEPC aqueous solution with RNase A inhibitor concentration of 0.2%; the concentration of the protease inhibitor was 50ug/ml PMSF.

As a preferred scheme, the volume of the solution containing the inhibitor to be extracted is more than or equal to 0 ul; one particularly preferred volume of inhibitor solution is between 10ul and 1000 ul; one more preferred volume of inhibitor solution is 20 ul-200 ul; more specifically, the volume of inhibitor solution was 80ul per microcolumn.

By the above operation, the biological sample can be completely ground in about 5 minutes.

The carrier for describing the method for processing the biological sample is typically a paper specification, and may be any carrier for describing an electronic version of the method (including but not limited to a removable magnetic disk, an optical disk, an electronic ink screen, a network resource, an address thereof, and the like), as long as the method can be known by reading the carrier.

In another aspect, the present invention provides a computer-readable medium carrying a computer program for implementing the method for processing a sample of biological tissue DNA. The computer is understood in a broad sense and includes but is not limited to a single chip microcomputer, a PLC, a single chip microcomputer, an industrial personal computer, a PC and the like. The computer readable carrier includes, but is not limited to, any form of Flash, EEPROM, magnetic disk (floppy or hard disk), optical disk, and the like. The computer program may be written in any language, such as assembly, JAVA, VB, VC, C + +, Python, as long as the associated system is controlled to implement the method.

In the research of the sample processing method for extracting the DNA of the biological tissue, the inventor surprisingly discovers that after the biological sample is ground by a liquid nitrogen freeze-drying method, the particles of the tissue sample reach a very small degree, so that the surface area of the particles contacting with the extracting solution is greatly increased, and the extract to be extracted: RNA, DNA and protein enter the extracting solution quickly, so that the extracting time is greatly shortened, and the amount of the extract to be extracted is increased. The traditional kit for extracting RNA or protein or DNA, RNA and protein together can be changed into a kit for extracting RNA or protein at room temperature.

The invention provides a processing method, an extraction kit and an extraction device for rapidly extracting a sample by random co-extraction of RNA or protein or DNA, RNA and protein of a biological sample, and at least has the following beneficial effects: 1. the operation is simple; 2. the extraction time is greatly shortened, and the working efficiency is improved; 3. improving the quality and quantity of the extract; 4. the traditional kit is improved.

[ description of the drawings ]

FIG. 1 is a graph of time versus temperature after precooling of a grinding system;

FIG. 2 is a photograph of scindapsus aureus leaves and mouse liver after being ground by liquid nitrogen freeze-drying;

FIG. 3 is a comparative photograph of freeze-dried ground and treated mouse liver before and after centrifugation;

FIG. 4 gel electrophoresis chart of RNA extraction from ground mouse liver by liquid nitrogen freeze-drying method;

FIG. 5 gel electrophoresis chart of RNA extraction result of scindapsus aureus leaf processed by liquid nitrogen freeze-drying method;

[ detailed description ] embodiments

The invention is further described below in conjunction with the drawings and the specific embodiments, which are provided only to assist in understanding the invention.

Example 1 measurement of mortar temperature after thorough Cooling vs. time

The mortar was thoroughly cooled with liquid nitrogen at room temperature of 22 c, and the mortar temperature was recorded once per minute at the beginning of drying of the liquid nitrogen, with time on the abscissa and temperature on the ordinate, to produce a time-temperature curve (fig. 1). The biological sample can be ground to a very fine powder state, typically in about 5 minutes. Meanwhile, in order to obtain better grinding effect, liquid nitrogen is added, so that the whole grinding process is carried out at a very low temperature. And/or some biological samples are added with a solution of the inhibitor to be extracted, the chemical reaction rate between solids is very small, the chemical reaction can be reduced by low temperature, and in addition, the whole grinding process time is as short as about 5 minutes, so that the extract including DNA, RNA and protein can not be degraded and can be kept in an intact state.

Example 2 extraction of mouse liver RNA

As described above, approximately 22.3mg of mouse liver tissue was placed in a mortar and an appropriate amount of liquid nitrogen was added, and 80ul of 0.2% DEPC H was added after drying with liquid nitrogen₂O solution is applied to the liver tissue of the mouse, and when the liquid is about to coagulate, the liquid about to coagulate and the liver of the mouse are ground into powder. Then, liquid nitrogen was further added to further grind the powder into very fine powder (see right side of FIG. 2). Transferring the powder into lysate A (Normal temperature rapid biological RNA extraction kit I (Beijing, China International technology for Biotechnology, Inc.)), grinding the powder and lysate A to form a relatively uniform suspension, centrifuging at 12000rpm for 1 min (see FIG. 3), and grinding the liver tissue of the mouse into very small pieces by liquid nitrogen freeze-drying grinding methodThe fine powder is homogeneously distributed in lysate a. The whole RNA extraction process was completed in 10 minutes and the RNA was eluted with 100ul of eluent. The results were determined on a biaosharp (table 1) and detected by gel electrophoresis (fig. 4). Therefore, the method can rapidly extract the mouse liver RNA by a liquid nitrogen freeze-drying method.

Table 2: quality of extracted mouse liver RNA

Name of living being	Mouse liver
		RNA concentration	337.9ng/ul
A260/A280	2.037

Example 3 improvement on patent CN 105063018A

Approximately 100mg of young scindapsus aureus leaves were taken, placed in a mortar and an appropriate amount of liquid nitrogen was added, in which the scindapsus aureus leaves were crushed and ground, as described previously. After drying with liquid nitrogen, the scindapsus aureus leaves were further ground into a powder, and then liquid nitrogen was further added to further grind the powder into a very fine powder (see fig. 2, left). The powder was transferred to 600ul of lysate A (2% PVP40, 2% cetyltrimethylammonium bromide (CTAB), 200mM NaAc, 2M NaCl, 20mM EDTA) and shaken, 300ul of phenol was added and shaken. Centrifuging at 12000rpm for 1 min, collecting supernatant, adding 0.5 times of anhydrous ethanol, transferring into RNA purification column, centrifuging at 12000rpm for 30 s, cleaning for 2 times, and eluting. The whole extraction process was carried out at room temperature for about 10 minutes, and 100ul of the eluent was used to elute the RNA. The results were determined on a biaosharp (table 2) and detected by gel electrophoresis (fig. 5). Therefore, the traditional kit can be improved into the kit for rapidly extracting RNA at normal temperature by a liquid nitrogen freeze-drying method.

Table 2: quality of extracted scindapsus aureus leaf RNA

Name of living being	Scindapsus aureus leaf
		RNA concentration	316.7ng/ul
A260/A280	2.058

The result shows that the RNA concentration and purity of the RNA are high, and the method can be used for subsequent molecular biology experimental operation. More remarkably, the method is simple to operate, time-saving and efficient, and the original kit can be improved. In addition, application or patent numbers: 202011070133.8 the biological DNA is extracted quickly by freeze drying the biological sample. Because the DNA of the eukaryote is positioned in the cell nucleus, the DNA can be quickly extracted, which indicates that the tissue of the biological sample is well crushed after the freeze drying treatment of liquid nitrogen. Any two or three of DNA, RNA and protein can be extracted by a corresponding extraction method, and the details are not repeated herein. Sources of reagents used in the present invention:

although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, combinations, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.