CN117625746A - Sample processing method - Google Patents
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- CN117625746A CN117625746A CN202311677304.7A CN202311677304A CN117625746A CN 117625746 A CN117625746 A CN 117625746A CN 202311677304 A CN202311677304 A CN 202311677304A CN 117625746 A CN117625746 A CN 117625746A
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- 238000003672 processing method Methods 0.000 title claims abstract description 11
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 38
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 38
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 38
- 238000000034 method Methods 0.000 claims abstract description 27
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 22
- 239000007788 liquid Substances 0.000 claims abstract description 19
- 238000009210 therapy by ultrasound Methods 0.000 claims abstract description 8
- 238000002156 mixing Methods 0.000 claims abstract description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 24
- 239000000243 solution Substances 0.000 claims description 18
- 239000003223 protective agent Substances 0.000 claims description 17
- 239000007853 buffer solution Substances 0.000 claims description 9
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 8
- 229930195725 Mannitol Natural products 0.000 claims description 8
- 229930006000 Sucrose Natural products 0.000 claims description 8
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 8
- 235000011187 glycerol Nutrition 0.000 claims description 8
- 239000000594 mannitol Substances 0.000 claims description 8
- 235000010355 mannitol Nutrition 0.000 claims description 8
- 239000005720 sucrose Substances 0.000 claims description 8
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 7
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 7
- 239000013504 Triton X-100 Substances 0.000 claims description 7
- 229920004890 Triton X-100 Polymers 0.000 claims description 7
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 7
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 7
- 239000004094 surface-active agent Substances 0.000 claims description 7
- 239000001763 2-hydroxyethyl(trimethyl)azanium Substances 0.000 claims description 6
- 235000019743 Choline chloride Nutrition 0.000 claims description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- 229940122426 Nuclease inhibitor Drugs 0.000 claims description 6
- 229960003178 choline chloride Drugs 0.000 claims description 6
- SGMZJAMFUVOLNK-UHFFFAOYSA-M choline chloride Chemical compound [Cl-].C[N+](C)(C)CCO SGMZJAMFUVOLNK-UHFFFAOYSA-M 0.000 claims description 6
- LQZZUXJYWNFBMV-UHFFFAOYSA-N dodecan-1-ol Chemical compound CCCCCCCCCCCCO LQZZUXJYWNFBMV-UHFFFAOYSA-N 0.000 claims description 6
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims description 6
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 6
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 6
- 239000012530 fluid Substances 0.000 claims description 4
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 3
- 229910052708 sodium Inorganic materials 0.000 claims description 3
- 239000011734 sodium Substances 0.000 claims description 3
- CHHHXKFHOYLYRE-UHFFFAOYSA-M 2,4-Hexadienoic acid, potassium salt (1:1), (2E,4E)- Chemical compound [K+].CC=CC=CC([O-])=O CHHHXKFHOYLYRE-UHFFFAOYSA-M 0.000 claims description 2
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 2
- 239000007984 Tris EDTA buffer Substances 0.000 claims description 2
- RUZMUTWCUZLWQU-UHFFFAOYSA-N [ethoxy(hydroxy)phosphoryl] ethyl hydrogen phosphate Chemical compound CCOP(O)(=O)OP(O)(=O)OCC RUZMUTWCUZLWQU-UHFFFAOYSA-N 0.000 claims description 2
- 239000000022 bacteriostatic agent Substances 0.000 claims description 2
- YQOKLYTXVFAUCW-UHFFFAOYSA-N guanidine;isothiocyanic acid Chemical compound N=C=S.NC(N)=N YQOKLYTXVFAUCW-UHFFFAOYSA-N 0.000 claims description 2
- -1 nuclease inhibitor Substances 0.000 claims description 2
- 239000008055 phosphate buffer solution Substances 0.000 claims description 2
- 239000004302 potassium sorbate Substances 0.000 claims description 2
- 229940069338 potassium sorbate Drugs 0.000 claims description 2
- 235000010241 potassium sorbate Nutrition 0.000 claims description 2
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims 1
- 239000003242 anti bacterial agent Substances 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 23
- 230000003321 amplification Effects 0.000 abstract description 11
- 238000004321 preservation Methods 0.000 abstract description 10
- 238000005336 cracking Methods 0.000 abstract description 3
- 244000052769 pathogen Species 0.000 abstract description 3
- 230000015556 catabolic process Effects 0.000 abstract description 2
- 238000006731 degradation reaction Methods 0.000 abstract description 2
- 230000001717 pathogenic effect Effects 0.000 abstract description 2
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- 230000002035 prolonged effect Effects 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 64
- 239000008223 sterile water Substances 0.000 description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 16
- 238000000605 extraction Methods 0.000 description 14
- 230000000694 effects Effects 0.000 description 8
- 241000712431 Influenza A virus Species 0.000 description 7
- 239000003761 preservation solution Substances 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 5
- 239000011324 bead Substances 0.000 description 4
- 238000004090 dissolution Methods 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 238000010222 PCR analysis Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 210000005178 buccal mucosa Anatomy 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 210000002615 epidermis Anatomy 0.000 description 2
- 230000000415 inactivating effect Effects 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 210000000214 mouth Anatomy 0.000 description 2
- 210000004877 mucosa Anatomy 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- 239000012981 Hank's balanced salt solution Substances 0.000 description 1
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- 238000012408 PCR amplification Methods 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
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- 150000002357 guanidines Chemical class 0.000 description 1
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Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the field of molecular biology detection, and particularly relates to a sample processing method, in particular to a rapid sample processing method. The invention provides a sample processing method, which comprises the following steps: and mixing the sample to be detected with the direct-amplification sample treatment liquid, and carrying out ultrasonic treatment to obtain a sample nucleic acid solution. By using the sample processing method, the method of combining direct-amplification sample processing liquid and ultrasonic processing is adopted, so that the cracking efficiency of pathogen nucleic acid is improved; the method has the advantages that the method has a protective effect on the released nucleic acid, so that the degradation of the sample is effectively prevented at normal temperature, a stable environment is provided for the sample, the preservation time of the nucleic acid is prolonged, and the safety of collection, transportation and detection is ensured; and the subsequent amplification can be directly carried out by hands-free access, the cost is low, and the operation is simple.
Description
Technical Field
The invention belongs to the field of molecular biology detection, and particularly relates to a sample processing method, in particular to a method for rapidly processing a sample.
Background
The nucleic acid detection has the characteristics of high sensitivity, high specificity, early diagnosis, early intervention and the like, has gradually become a detection 'gold standard' of a plurality of pathogens or disease markers, the nucleic acid detection has certain requirements on the purity and the integrity of a nucleic acid sample, the quality of the nucleic acid sample is a key for influencing the subsequent result, and when the quality of the nucleic acid can not meet the requirements, the accuracy and the detection lower limit of the subsequent detection result are influenced. The process of collection and transport of the sample, the method of extraction, and the efficiency can all have an impact on nucleic acids. And the general nucleic acid extraction has higher requirements on instruments and personnel, and the whole extraction process has longer time. Therefore, there is an urgent need to develop a cell preservation solution capable of rapidly inactivating malignant infectious viruses, simultaneously, the subsequent samples do not need nucleic acid extraction, steps in the whole process are reduced, and the subsequent nucleic acid detection is not influenced, so that the detection can be directly performed.
The sample preservation method commonly used at present mainly adopts Hank' S solution or phosphate buffer preservation solution and guanidine salt inactivation preservation solution for preservation, and has the following defects: 1. the subsequent nucleic acid extraction step is needed, the time consumption is long, and pollution is easy to generate; 2. the preservation effect quality of various preservation solutions is poor, and RNA is easy to degrade; 3. the sampled sample needs to be stored at a low temperature, and the storage time is short. The sample extraction process can be omitted by inactivating a part of sample preservation solution without extraction, the preserved sample is directly used for subsequent amplification, but the cracking capacity of the preservation solution is limited by hand-free sampling, and the sample without extraction has more inhibition matrixes because of no purification, so that the sensitivity of the method is lower than that of the sample with extraction of nucleic acid, and false negative is easy to occur. Therefore, it is of great importance to develop a sample pretreatment scheme that is hands-free and has no influence on the sensitivity.
Disclosure of Invention
In view of this, in a first aspect, the present invention provides a sample processing method, comprising the steps of:
mixing a sample to be detected with a direct-amplification sample treatment liquid, and carrying out ultrasonic treatment to obtain a sample nucleic acid solution;
the frequency of the ultrasonic treatment is 30 kHz-80 kHz, and the time is 30 s-150 s. Preferably, the frequency of the ultrasonic treatment is 40kHz and the time is 120s.
The direct-amplification sample treatment fluid has the functions of sample inactivation, nucleic acid release and preservation; the term "direct amplification" as used herein refers to a technique of releasing nucleic acid without extraction from a sample and directly amplifying the nucleic acid, and refers to a technique of directly detecting nucleic acid amplification from a sample without extracting or purifying the nucleic acid from the sample or adding a nucleic acid preserving fluid.
Further, the direct-amplification sample processing liquid includes: protecting agent, nuclease inhibitor, surfactant, buffer system and bacteriostat.
Still further, the protective agent comprises sucrose, choline chloride, mannitol, polyvinylpyrrolidone, glycerol.
Further, in the direct-amplification sample treatment solution, the concentration of sucrose is 0.15% -1.5% (w/v), the concentration of choline chloride is 1-10 mM, the concentration of polyvinylpyrrolidone is 1-10 mM, the concentration of mannitol is 0.045% -0.45% (v/v), and the concentration of glycerol is 0.05% -0.5% (v/v).
Further, the concentration of the nuclease inhibitor is 0.01% -0.1% (v/v), the concentration of the surfactant is 0.01% -0.5% (v/v), the concentration of the bacteriostat is 0.01% -0.1% (v/v), and the concentration of the buffer system is 80 mM-120 mM.
In the definition of the present invention, the term "w/v" denotes the mass volume concentration and the term "v/v" denotes the volume fraction, wherein w is in g and v is in mL.
The nuclease inhibitor can be one or more of Rnasin, diethyl pyrophosphate, guanidine isothiocyanate and the like. In a specific embodiment, the nucleic acid inhibitor is Rnasin. The surfactant can be one or more of triton X-100, tween 20, sodium dodecyl alcohol ether sulfate, sodium dodecyl sulfate, etc. In a specific embodiment, the surfactant is triton X-100, tween 20, sodium dodecyl alcohol ether sulfate. The bacteriostat can be one or more of Proclin300, potassium sorbate and the like. In a specific embodiment, the bacteriostatic agent is Proclin300. The buffer system can be one or more of Tris-HCL buffer solution, phosphate buffer solution, TE buffer solution and the like with pH of 8.0-8.5. In a specific embodiment, the buffer system is Tris-HCl buffer at a pH of 8.0 to 8.5.
Further, mixing the sample to be tested with a direct expansion preservation solution in a container with a pipetting function; and adding the sample nucleic acid solution directly to the amplification reaction premix solution through the vessel.
Further, the container having a pipetting function may be a pipette, a centrifuge tube, or the like. Still further, the pipette is a vesicle-type pipette. Still further, the vesicle pipette is shown in fig. 1.
Further, the sample contains nucleic acid. Preferably, the nucleic acid is RNA.
By using the sample processing method, the method of combining direct-amplification sample processing liquid and ultrasonic processing is adopted, so that the cracking efficiency of pathogen nucleic acid is improved; the kit has a protective effect on the released nucleic acid, can effectively prevent the degradation of the sample at normal temperature, provides a stable environment for the sample, prolongs the preservation time of the nucleic acid, and ensures the safety of collection, transportation and detection; and the subsequent amplification can be directly carried out by hands-free access, the cost is low, and the operation is simple.
Drawings
FIG. 1 is an exemplary diagram of a vesicle pipette;
FIG. 2 shows amplification curves of samples treated with different compositions and methods (A-G are magnetic bead method, direct amplification sample treatment fluids 1-6 in order);
FIG. 3 is a graph of amplification for various days using the methods of the invention and without the methods of the invention.
Detailed Description
The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.
Example 1
The first step: preparing a protective agent.
Protective agent 1: 15g of sucrose, 1.3963g of choline chloride and 1.1114g of polyvinylpyrrolidone are sequentially added into 50mL of sterile water, 4.5mL of mannitol and 5mL of glycerol are added after dissolution is completed, and the volume is fixed to 100mL by using the sterile water;
protective agent 2: 15g of sucrose, 1.3963g of choline chloride and 3.423g of trehalose are sequentially added into 50mL of sterile water, 4.5mL of mannitol and 5mL of glycerol are added after dissolution is completed, and the volume is fixed to 100mL by using the sterile water;
protective agent 3: 15g of sucrose, 1.1114g of polyvinylpyrrolidone and 4.5mL of mannitol, 5mL of glycerol and 1mL of dimethyl sulfoxide are sequentially added into 50mL of sterile water, and after dissolution is completed, the volume is fixed to 100mL by using the sterile water;
protective agent 4: 15g of sucrose was added to 50mL of sterile water in this order, and after dissolution was completed, 4.5mL of mannitol and 5mL of glycerol were added, and the volume was fixed to 100mL with sterile water.
And a second step of: preparing a direct-expansion sample treatment liquid.
Preparing a direct-expansion sample treatment solution 1 by adopting a protective agent 1: 1mL of a protective agent 1, 5. Mu.L of triton X-100, 5. Mu.L of Tween 20, 10. Mu.L of RNasin and 10. Mu.L of Proclin300,1.2608g of Tris-HCL solid are added into 50mL of sterile water, the pH is regulated, and the volume is fixed to 100mL by using the sterile water, so that a direct-expansion sample treatment liquid 1 is prepared.
Preparing a direct-expansion sample treatment solution 2 by adopting a protective agent 1: 5mL of a protective agent 1, 50. Mu.L of triton X-100, 50. Mu.L of Tween 20, 50. Mu.L of RNasin and 50. Mu.L of Proclin300,1.576g of Tris-HCL solid were added to 50mL of sterile water, the pH was adjusted, and the volume was fixed to 100mL with sterile water to prepare a direct-amplification sample treatment solution 2.
Preparing a direct-expansion sample treatment solution 3 by adopting a protective agent 1: 10mL of a protective agent 1, 250. Mu.L of triton X-100, 250. Mu.L of Tween 20, 100. Mu.L of RNasin and 100. Mu.L of Proclin300,1.8912g of Tris-HCL solid were added to 50mL of sterile water, the pH was adjusted, and the volume was fixed to 100mL with sterile water to prepare a direct-amplification sample treatment solution 3.
Preparing a direct-expansion sample treatment solution 4-6 by adopting a protective agent 2-4: 1mL of protective agent 2-4 is added to 3 parts of 50mL of sterile water respectively, then 5 mu L of triton X-100, 5 mu L of Tween 20, 10 mu L of RNasin and 10 mu L of Proclin300,1.2608g of Tris-HCL solid are added, pH is regulated, and the volume is fixed to 100mL by using the sterile water, so that a direct-expansion sample treatment solution 4-6 is prepared.
The prepared direct-spread sample treatment solution is used for 1-6 to 1 x 10 5 Influenza a virus samples at a concentration of copies/mL were treated and diluted to 1 x 10 4 The copies/mL, followed by sonication (40 kHz,120 s);
in addition, nucleic acid extraction and purification reagent S10015 of Sanxiang biotechnology Co., ltd is adopted to carry out magnetic bead method nucleic acid extraction on the influenza A virus sample;
the detection is carried out by using a general type nucleic acid detection kit for influenza A virus of Sanxiang biotechnology Co., ltd, the reaction system is 20 mu L of the processed sample and 30 mu L of PCR reaction liquid, and the detection is carried out on an SLAN-96P full-automatic medical PCR analysis system.
The results are shown in FIG. 2, wherein the curve A in the figure is the result of sample amplification extracted by the magnetic bead method, and the curve B-G is the result of sample amplification processed by the sample processing liquids 1-6 respectively.
As can be seen from FIG. 2, the above 6 sample processing liquids can be directly amplified without additional nucleic acid extraction steps, and can meet the detection requirements. However, the Ct value of the formula of the sample treatment liquid 1-3 is relatively higher, the fluorescence increment is higher, the release effect on nucleic acid is better, the difference between the result of the sample treatment liquid and the result of the magnetic bead extraction control group is small, and the sample treatment liquid is basically consistent with the extraction effect of the magnetic bead method.
Example 2
(1) The throat swab is adopted to gently scrape the upper epidermis mucosa cells of the lingual surface or the buccal mucosa (cheek on two sides in the oral cavity) of a human, 9 parts of 2mL of sample treatment liquid 1 in the example 1 is respectively adopted to infiltrate the swab, and the numbers are 1 to 9 respectively; the tube cap was closed, mixed upside down for 90 seconds and then centrifuged instantaneously.
(2) Will be 1 x 10 6 Influenza a virus stock at a concentration of copies/mL was diluted to 1 x 10 5 The copies/mL was added to the treatment liquid No. 1-9 of the above (1) to obtain sample No. 1-9.
(3) The samples 1 to 9 in (2) were subjected to physical treatment under the conditions shown in Table 1 as the sample to be loaded directly:
TABLE 1
(4) Sample numbers 1-9 were detected using a universal nucleic acid detection kit for influenza A virus from san-xiang biotechnology Co., ltd, 20. Mu.L of sample+30. Mu.L of PCR reaction solution was used, and detection was performed on a SLAN-96P full-automatic medical PCR analysis system, and the detection results are shown in Table 2.
TABLE 2
| No. 1 | No. 2 | No. 3 | No. 4 | No. 5 | No. 6 | No. 7 | No. 8 | No. 9 | |
| Ct value | 27.90 | 30.01 | 31.27 | 30.29 | 28.44 | 30.49 | 33.19 | NoCt | 38.27 |
As can be seen from the table, the ultrasonic frequency is 30 kHz-80 kHz, and the ultrasonic time is 30 s-150 s, so that the sample can be treated, and a good detection effect can be obtained. The Ct value of the ultrasonic probe can be more than 6 in advance compared with the comparative example which is not subjected to ultrasonic treatment, and the detection effect of the ultrasonic probe is more excellent compared with the comparative example of the rest ultrasonic conditions.
Example 3 preservation Effect test
(1) The throat swab is adopted to gently scrape the upper epidermis mucosa cells of the lingual surface or the buccal mucosa (cheek on two sides in the oral cavity) of a human, 4 parts of 2mL of sample treatment liquid 1 in the example 1 is adopted to infiltrate the swab, and the numbers are 10 to 13 respectively; the tube cap was closed, mixed upside down for 90 seconds and then centrifuged instantaneously.
(2) Will be 1 x 10 6 Influenza a virus stock at a concentration of copies/mL was diluted to 1 x 10 5 The copies/mL was added to the treatment liquid No. 10-13 of the above (1) to obtain a sample No. 10-13.
(3) Sample nos. 10-13 were placed and treated under the conditions shown in table 3:
TABLE 3 Table 3
(4) The sample after treatment was directly used as a sample loading, and was detected by using the universal nucleic acid detection kit for influenza A virus of Santa Clara Biotechnology, inc., the reaction system was 20. Mu.L of the PCR reaction solution of +30. Mu.L of the sample after treatment in (4), and the detection was performed on a SLAN-96P full-automatic medical PCR analysis system, and the PCR amplification result was shown in FIG. 3. From the figure, it can be seen that the ultrasound-treated samples were better than the non-ultrasound samples both after 7 days of non-preservation and after preservation. And the method still has good detection effect after being stored for 7 days at 37 ℃.
Claims (10)
1. A sample processing method comprising the steps of:
and mixing the sample to be detected with the direct-amplification sample treatment liquid, and carrying out ultrasonic treatment to obtain a sample nucleic acid solution.
2. The method according to claim 1, wherein the ultrasonic treatment has a frequency of 30kHz to 80kHz for 30S to 150S.
3. The method according to claim 2, wherein the ultrasonic treatment has a frequency of 40kHz and a time of 120S.
4. The sample processing method according to claim 1, wherein the direct-amplification sample processing liquid comprises: protecting agent, nuclease inhibitor, surfactant, buffer system and bacteriostat.
5. The method of claim 4, wherein the protective agent comprises sucrose, choline chloride, mannitol, polyvinylpyrrolidone, glycerol.
6. The method according to claim 5, wherein the concentration of sucrose in the sample processing liquid is 0.15% to 1.5% (w/v), the concentration of choline chloride is 1 to 10mM, the concentration of polyvinylpyrrolidone is 1 to 10mM, the concentration of mannitol is 0.045% to 0.45% (v/v), and the concentration of glycerol is 0.05% to 0.5% (v/v).
7. The method according to any one of claims 4 to 6, wherein the concentration of the nuclease inhibitor is 0.01 to 0.1% (v/v), the concentration of the surfactant is 0.01 to 0.5% (v/v), the concentration of the bacteriostatic agent is 0.01 to 0.1% (v/v), and the concentration of the buffer system is 80mM to 120mM.
8. The method according to claim 7, wherein the nuclease inhibitor is at least one selected from the group consisting of RNasin, diethyl pyrophosphate, and guanidine isothiocyanate; the surfactant is at least one selected from triton X-100, tween 20, sodium dodecyl alcohol ether sulfate and sodium dodecyl sulfate; the buffer system is at least one selected from Tris-HCL buffer solution, phosphate buffer solution and TE buffer solution; the antibacterial agent is at least one selected from Proclin300 and potassium sorbate.
9. The method according to claim 1, wherein the sample to be measured is mixed with a direct expansion preserving fluid in a container having a pipetting function.
10. The method of claim 1, wherein the sample nucleic acid solution contains RNA.
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| CN202311677304.7A CN117625746A (en) | 2023-12-07 | 2023-12-07 | Sample processing method |
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| CN202311677304.7A CN117625746A (en) | 2023-12-07 | 2023-12-07 | Sample processing method |
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