A concentrate for sample nucleic acid extraction and a method for extracting nucleic acid using the same

Abstract

The application discloses a concentrated solution for sample nucleic acid extraction and a method for extracting nucleic acid by using the same, and the concentrated solution is composed of the following raw materials in percentage by weight: sodium chloride 3-5%, trehalose-25 2-4%, Tris buffer 3-5%, n-butanol 7-10%, and the rest is nuclease-free water, and the pH value is 4-6. Meanwhile, the application also discloses a method for extracting nucleic acid by using the same, and the concentration of microorganisms in an environmental sample or a water sample is improved through a one-time concentration method, so that the efficiency and concentration of nucleic acid extraction are improved, and the detection rate and sensitivity are improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial extraction, and particularly relates to a concentrated solution for sample nucleic acid extraction and a method for extracting nucleic acid by using the same. BACKGROUND

[0002] In the detection of pathogenic microorganisms in drinking water or environment, the traditional method is to collect environmental samples or water samples, and then to culture bacteria, which generally needs 7 days, and is time-consuming and laborious. Moreover, after the bacterial culture is completed, concentration is needed, and generally, the microorganism concentration method is to use an ultracentrifuge to realize concentration under the condition of 20000g for more than 2 hours, but this concentration method is not only expensive in equipment, but also long in centrifugation time, and is not conducive to the promotion of primary laboratories. SUMMARY

[0003] The present application aims to provide a concentrated solution for sample nucleic acid extraction, and to provide a method for extracting nucleic acid, which is the second invention purpose of the present application. The purpose is to improve the concentration of microorganisms in environmental samples or water samples by a certain concentration method, so as to improve the efficiency and concentration of nucleic acid extraction, and to improve the detection rate and sensitivity.

[0004] To achieve the above-mentioned purpose, the present application provides the following technical scheme:

[0005] A concentrated solution for sample nucleic acid extraction, which is composed of the following raw materials in percentage by weight: sodium chloride 3% to 5%, trehalose-25 2% to 4%, Tris buffer 3% to 5%, n-butanol 7% to 10%, and the rest is non-nuclease water, and the pH value is 4 to 6.

[0006] A method for extracting sample nucleic acid by using the concentrated solution, comprising the following steps:

[0007] 1) mixing the sample to be detected with the concentrated solution in a certain proportion, and standing for 5 to 10 minutes at room temperature;

[0008] 2) solid-liquid separation by a conventional centrifuge;

[0009] 3) removing the supernatant, and resuspending the bacterial precipitate with 280 to 300ul of purified water or physiological saline.

[0010] Preferably, when the sample to be detected is water, it can be directly mixed; when the sample to be detected is other environmental swabs, the environmental swabs are eluted with 2 to 5ml of physiological saline or purified water, the eluate is centrifuged at 500xg for 10 minutes to remove impurities, and the supernatant is the sample to be detected.

[0011] Preferably, the volume ratio of the sample to be detected to the concentrated solution is (3 to 5):1, and the mixture is mixed by vortexing.

[0012] Preferably, the centrifugal force is not less than 9000xg, the centrifugal time is 10-20min, and there is white or gray precipitate at the bottom of the centrifugal tube.

[0013] Preferably, the supernatant can be removed by using a pipette or pouring.

[0014] Specifically, in application, after resuspending the bacteria in step 3), the nucleic acid can be extracted by using a commercial nucleic acid extraction kit (magnetic bead method) according to the instructions.

[0015] The present application can quickly and effectively concentrate liquid biological samples by using one-time concentration technology, and can be widely applied to the fields of infectious disease monitoring, food safety, environmental monitoring, biochemical defense, water pollution monitoring and other rapid microbial detection.

[0016] Compared with the prior art, the present application has the following beneficial effects:

[0017] 1. The present application can be used for measuring the concentration of microbial bacteria in drinking water or cotton swab eluate, significantly improving the detection rate of bacteria in water sources or swab eluate; by standing at room temperature and centrifuging with a conventional centrifuge, a high-concentration bacterial sample can be quickly obtained, and the concentration multiple is more than 100 times;

[0018] 2. The concentrated solution of the present application is more suitable for concentrating microbial bacterial samples for clinical application in primary laboratories, effectively improving the detection accuracy of environmental swabs and improving the diagnostic sensitivity.

[0019] 3. The concentrated solution of the present application is also suitable for pre-treatment and concentration of foodborne microbial samples, improving the sensitivity of the detection method and significantly shortening the detection time. DETAILED DESCRIPTION

[0020] Figure 1 is the amplification curve of experimental examples 1-4 of the present application. DETAILED DESCRIPTION

[0021] To illustrate the effect of the application, the present application will be further described in the specific implementation mode.

[0022] Example 1

[0023] A concentrated solution for sample nucleic acid extraction, which is composed of the following raw materials in weight percentage: sodium chloride 4%, trehalose-25 3%, Tris buffer 4%, n-butanol 8%, and the balance is nuclease-free water, and the pH value is 5.

[0024] The method for extracting sample nucleic acid by using the concentrated solution comprises the following steps:

[0025] 1) Take the sample to be tested and the concentrated solution by volume ratio of 4:1 by vortex mixing, stand at room temperature for 5 min; the sample to be tested is tap water;

[0026] 2) Solid-liquid separation by conventional centrifuge; the centrifugal force is not less than 9000xg, the centrifugation time is 15 min, there is white precipitate at the bottom of the centrifuge tube;

[0027] 3) Pour to remove the supernatant, the liquid can not be completely removed, add 280 μl of physiological saline to resuspend the bacterial precipitate.

[0028] Example 2

[0029] A concentrated solution for sample nucleic acid extraction, consisting of the following raw materials by weight percentage: sodium chloride 3%, trehalose-25 2%; Tris buffer 3%, n-butanol 7%, the balance is nuclease-free water, pH value is 4.

[0030] The method for extracting sample nucleic acid by using the concentrated solution, comprising the following steps:

[0031] 1) Take the sample to be tested and the concentrated solution by volume ratio of 3:1 by vortex mixing, stand at room temperature for 10 min; the sample to be tested is an environmental swab, which is eluted with 5 ml of physiological saline or purified water, the eluate is centrifuged at 500xg for 10 min to remove impurities, and the supernatant is the sample to be tested.

[0032] 2) Solid-liquid separation by conventional centrifuge; the centrifugal force is not less than 9000xg, the centrifugation time is 10 min, there is gray precipitate at the bottom of the centrifuge tube;

[0033] 3) Pour to remove the supernatant, the liquid can not be completely removed, add 300 μl of physiological saline to resuspend the bacterial precipitate.

[0034] Example 3

[0035] A concentrated solution for sample nucleic acid extraction, consisting of the following raw materials by weight percentage: sodium chloride 5%, trehalose-25 4%; Tris buffer 5%, n-butanol 10%, the balance is nuclease-free water, pH value is 6.

[0036] The method for extracting sample nucleic acid by using the concentrated solution, comprising the following steps:

[0037] 1) Take the sample to be tested and the concentrated solution by volume ratio of 5:1 by vortex mixing, stand at room temperature for 10 min; the sample to be tested is tap water.

[0038] 2) Solid-liquid separation by conventional centrifuge; the centrifugal force is not less than 9000xg, the centrifugation time is 10 min, there is white precipitate at the bottom of the centrifuge tube;

[0039] 3) Pour to remove supernatant, the liquid can not be completely removed, add 300 μl of physiological saline to resuspend the bacterial precipitate.

[0040] Test Example:

[0041] Test Method:

[0042] Experimental Group 1:

[0043] 4 ml of environmental swab eluate and 1 ml of concentrated solution were mixed;

[0044] In this experimental group, the environmental swab eluate was eluted with 4 ml of physiological saline, and the eluate was centrifuged at 500 x g for 10 min to remove impurities.

[0045] Experimental Group 2:

[0046] In this experimental group, 8 ml of tap water (containing 100 PFU of bacterial particles) was mixed with 2 ml of concentrated solution; the rest of the preparation method was the same as in Example 1.

[0047] Experimental Group 3:

[0048] 4 ml of environmental swab eluate and 1 ml of PBS were mixed;

[0049] In this experimental group, the environmental swab eluate was the same as in Experimental Group 1.

[0050] Experimental Group 4:

[0051] In this experimental group, 8 ml of tap water (containing 100 PFU of bacterial particles) was mixed with 2 ml of PBS; the rest of the preparation method was the same as in Example 1.

[0052] 200 ul of the concentrated samples obtained from the above 4 groups were taken respectively, and the commercial magnetic bead method was used for nucleic acid extraction, the corresponding amplification reaction system was configured according to the fluorescent PCR method of “SN / T 2206.11-2014: Staphylococcus aureus multiplex real-time fluorescent PCR method”, and the Gentier 48R type real-time fluorescent quantitative PCR detection system (Xi'an Tianlong Science and Technology Co., Ltd.) was used for machine operation. The experimental results are shown in Table 1 and Table 2: Figure 1

[0053] Table 1 Experimental Results

[0054] grouping Ct values grouping Ct values Experiment 1 26.2 Experiment 2 29.4 Experiment 3 31.3 Experiment 4 36.5

[0055] The test results show that the Ct value of the sample treated by the sample concentrated solution of the present application is 5-7 smaller than that of the untreated sample. According to the mathematical principle calculation formula of fluorescent quantitative PCR: Ct = klgX0 + b (X0 is the amount of initial nucleic acid, ​e is amplification efficiency; b is a constant), if the amplification efficiency e is 100%, then 1 Ct value difference represents a 2-fold difference in sample content; that is, the concentrated liquid of the present application concentrates the sample by about 32 times to 128 times.

Claims

1. A concentrated solution for extracting nucleic acids from samples, characterized in that, It consists of the following raw materials by weight percentage: sodium chloride 3%–5%, trehalose 2%–4%; Tris buffer 3%–5%, n-butanol 7%–10%, and the balance being nuclease-free water with a pH of 4–6.

2. The method for extracting nucleic acid from a sample using the concentrate according to claim 1, characterized in that, Includes the following steps: 1) Take the sample to be tested and mix it with the concentrated solution in a certain proportion, and let it stand at room temperature for 5 to 10 minutes; 2) Solid-liquid separation in conventional centrifuges; 3) Remove the supernatant and resuspend the bacterial precipitate in 280-300 μl of purified water or physiological saline.

3. The method for extracting nucleic acid from a sample using a concentrated solution as described in claim 2, characterized in that, When the test sample is water, it can be mixed directly; when the test sample is other environmental swabs, the environmental swabs are eluted with 2-5 ml of physiological saline or purified water. The eluent is centrifuged at 500×g for 10 min to remove impurities, and the supernatant is the test sample.

4. The method for extracting nucleic acid from a sample using a concentrated solution as described in claim 2, characterized in that, The volume ratio of the sample to be tested to the concentrate is (3-5):1, and the mixture is homogenized by vortexing.

5. The method for extracting nucleic acid from a sample using a concentrated solution as described in claim 2, characterized in that, The centrifugal force should be no less than 9000×g, the centrifugation time should be 10 to 20 minutes, and there should be white or gray precipitate at the bottom of the centrifuge tube.

6. The method for extracting nucleic acid from a sample using a concentrated solution as described in claim 2, characterized in that, Simply remove the supernatant using a pipette or by pouring it out.

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