A RNA virus nucleic acid preservative for fecal samples and a preparation method thereof

Abstract

The application discloses a kind of RNA virus nucleic acid preservative in fecal sample, including denaturant, chelating agent, pH buffering agent, osmotic pressure regulator and reducing agent;The denaturant includes guanidine salt and surfactant;The application further additionally provides a kind of RNA virus nucleic acid preservative in fecal sample preparation method, preparation above-mentioned one kind of RNA virus nucleic acid preservative in fecal sample, including the following steps: each component is mixed with ratio, pH is adjusted with pH buffering agent, with deionized water constant volume;Filter, obtain the preservative solution described in this application.The preservative solution of the application can stably store human cell DNA and viral RNA in fecal sample at room temperature for at least 7 days, which is beneficial for long-distance transportation at room temperature;Meanwhile, the preservative solution of the application also releases nucleic acid, inactivates virus, so as to prevent biological leakage and infection phenomenon;The de-inhibitor component in the preservative solution can directly combine and precipitate with various inhibitors in feces, reducing the interference to nucleic acid detection.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a fecal sample RNA virus nucleic acid preservation solution and a preparation method thereof. BACKGROUND

[0002] More and more literatures report that the nucleic acid RNA of the novel coronavirus is detected in the feces, which is the second detection approach of the novel coronavirus sample source in addition to the oral swab sample. Meanwhile, the literatures report that there is a possibility of fecal-oral transmission of the novel coronavirus. The researchers of Stanford University in the United States point out that 7 months after being infected with the novel coronavirus, the feces still contains viral genetic material. In addition, norovirus is also an infectious RNA virus in the feces. Media reports point out that the detection rate of norovirus in children under 5 years old with diarrhea in China is about 15%. Norovirus is a virus that causes non-bacterial acute gastroenteritis, has strong infectivity, is easy to mutate, and has a relatively short incubation period.

[0003] However, sometimes the RNA virus cannot be detected from the oral cavity and nasopharynx, for example, norovirus, the best detection sample of which is feces. Therefore, sometimes it is necessary to preserve and extract the viral nucleic acid in the feces to increase the detection accuracy, sensitivity and scientificity. However, after the feces are taken out of the body, the environment changes dramatically, the anaerobic environment of the intestinal tract changes to the aerobic environment of the air, a large number of strict anaerobic microorganism cells are lysed, a large amount of nucleases and proteases are released after cell death, the viral RNA in the fecal sample is easily destroyed and degraded, and the components in the fecal sample are complex, such as bile salts, bile pigments, gastrointestinal digestive juice, fibers, proteins and the like, which greatly increase the difficulty of preservation and extraction of the viral nucleic acid in the feces. The prior art cannot preserve the viral nucleic acid RNA in the feces for a long time, and low-temperature transportation is required during transportation, otherwise the viral nucleic acid RNA in the feces is easily degraded. Therefore, it is urgent to develop a nucleic acid preservation solution capable of preserving the viral RNA in the fecal sample for a long time at room temperature. SUMMARY

[0004] Therefore, the application aims to provide a fecal sample RNA virus nucleic acid preservation solution and a preparation method thereof. After being mixed with the fecal sample at room temperature, the solution can rapidly lyse the human cells and viruses in the feces, inactivate the nucleases and proteases, avoid infection caused by bacterial and viral excretion, form a precipitate with various inhibitors in the fecal sample to protect the nucleic acid, improve the nucleic acid extraction efficiency, preserve the viral RNA in the fecal sample for a long time at room temperature, maintain the stable state of the viral RNA, and avoid the degradation risk during the transportation for inspection.

[0005] To achieve the above-mentioned purposes, the technical scheme of the application is as follows:

[0006] The RNA virus nucleic acid preservative liquid in a stool sample comprises a denaturant, a chelating agent, a pH buffer, an osmotic pressure regulator and a reducing agent; the denaturant comprises guanidine salt and a surfactant.

[0007] Here, the denaturant directly lyses cells and viruses and releases nucleic acids, while inactivating viruses to avoid biological leakage from causing infection; and the preservative liquid can stably preserve human cell DNA and viral RNA in the stool sample for at least 7 days, prevents nucleic acid degradation, and facilitates long-distance transportation of the sample at room temperature.

[0008] Further, the guanidine salt is one or a combination of two or more of guanidine hydrochloride, guanidine phosphate, polyhexamethylene biguanide and guanidine isothiocyanate; the content of the guanidine salt is 2-8M.

[0009] Further, the surfactant is one or a combination of two or more of sodium dodecyl sarcosinate, SDS, Triton X-100 and cetyltrimethylammonium bromide; the content of the surfactant is 0.2-0.6M.

[0010] Further, the chelating agent is one or a combination of two or more of disodium ethylenediaminetetraacetate, sodium phosphate and diethylenetriamine pentaacetic acid; the content of the chelating agent is 0.1-1M.

[0011] Further, the pH of the preservative liquid is 7.0-12.0; the pH buffer is citric acid buffer or phosphoric acid buffer; the content of the pH buffer is 0.2%-0.8%.

[0012] Further, the osmotic pressure regulator is one or a combination of two or more of sodium chloride, potassium chloride, calcium chloride and magnesium chloride; the content of the osmotic pressure regulator is 3%-8%.

[0013] Further, the reducing agent is one or a combination of two or more of DTT, TCEP and beta-mercaptoethanol; the content of the reducing agent is 3-7mM.

[0014] Here, the content of each component is calculated based on the total amount of the RNA virus nucleic acid preservative liquid in a stool sample; the chelating agent, the osmotic pressure regulator and the reducing agent are all inhibitor components, which directly combine with various inhibitors in the stool to precipitate, thus simplifying the downstream nucleic acid extraction procedure and reducing the interference with nucleic acid detection.

[0015] The application also provides a use of the RNA virus nucleic acid preservative liquid in a stool sample, which uses the above RNA virus nucleic acid preservative liquid in a stool sample to maintain the stability of RNA virus nucleic acid and human nucleic acid in the stool sample; and / or inactivates viruses and microorganisms in the stool sample and releases nucleic acids.

[0016] Further, the fecal sample is derived from a human or a non-human animal; the mass / volume ratio of the fecal sample to the preservative solution is 1:10.

[0017] Here, the human-derived nucleic acid includes intestinal microorganism DNA and host human-derived intestinal exfoliated cell DNA.

[0018] The application further provides a preparation method of the RNA virus nucleic acid preservative solution in the fecal sample.

[0019] 1) The components are mixed in a proportion, the pH is adjusted by a pH buffer, and the volume is fixed by deionized water;

[0020] 2) Filtration, to obtain the preservative solution.

[0021] The application has the following beneficial effects: 1) The preservative solution of the application can stably preserve human cell DNA and virus RNA in the fecal sample for at least 7 days at room temperature, which is conducive to sample transportation to a third-party detection institution for virus detection; 2) The preservative solution of the application is a lysis type preservative solution, which can directly lyse cells and viruses and release nucleic acids, and inactivate viruses, so as to avoid infection phenomenon caused by biological leakage;

[0022] 3) The preservative solution of the application uses a de-inhibitor component to directly combine and precipitate a plurality of inhibitors in the feces, which simplifies the downstream nucleic acid extraction procedure and reduces interference on nucleic acid detection. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 It is a fluorescence quantitative curve of human-derived DNA in the fecal preservative solution in Example 2 of the application;

[0024] Figure 2 It is an IBV virus RNA fluorescence quantitative curve in the fecal preservative solution in Example 3 of the application;

[0025] Figure 3 It is a virus nucleic acid fluorescence quantitative curve in the fecal preservative solution at different time periods in Example 3 of the application;

[0026] Figure 4 It is a fluorescence quantitative curve of the nucleic acid stock solution extracted by the preservative solution of Company A and a 10-fold dilution sample in Example 4 of the application;

[0027] Figure 5 It is a fluorescence quantitative curve of the nucleic acid stock solution extracted by the No. 2 preservative solution and a 10-fold dilution sample in Example 4 of the application. DETAILED DESCRIPTION

[0028] In order to enable a more detailed understanding of the features and technical content of the present application, the implementation of the present application is described in detail below in conjunction with the drawings, the drawings are only used for reference and do not limit the present application.

[0029] Example 1: Formula of stool preservation solution

[0030] The RNA virus nucleic acid preservation solution in the stool sample of the present embodiment comprises 2-8M guanidine isothiocyanate, 0.2-0.6M cetyltrimethylammonium bromide, 0.1-1M disodium ethylenediaminetetraacetate, 0.2%-0.8% citric acid buffer, 3%-8% sodium chloride and 3-7mM DTT, and the pH value is 7.5-9.5.

[0031] Within the above concentration range, 1-3 preservation solutions are respectively configured, and the specific formula is shown in Table 1.

[0032] Table 1: Formula of stool preservation solution

[0033] Component Final concentration Preservative No. 1 Preservative No. 2 Preservative No. 3 Guanylic acid isothiocyanate 2～8M 2M 4M 6M Cetyltrimethylammonium bromide 0.2～0.6M 0.3M 0.4M 0.5M Disodium ethylenediaminetetraacetate 0.1～1M 0.3M 0.6M 0.9M Citric acid buffer 0.2％～0.8％ 0.3％ 0.5％ 0.7％ Sodium chloride 3～8％ 2％ 5％ 8％ DTT 3-7 mM 3 mM 4 mM 5 mM

[0034] Example 2: Stability performance research of human DNA in stool preserved by RNA virus nucleic acid preservation solution

[0035] Sampling: Fresh stool samples were provided by 3 different volunteers, and about 1 gram of fresh stool was placed in 10ml of No. 1, 2 and 3 stool preservation solutions prepared in Example 1, respectively, and mixed well by inversion, and stored at room temperature.

[0036] Nucleic acid extraction: The sample was mixed well by oscillation, and 300ul of the stool preservation solution was aspirated into a 2ml centrifuge tube in a fume hood, and centrifuged at 12000rpm for 1min, and 200ul of supernatant was taken, and a viral DNA / RNA extraction kit (Kangwei Century, item number CW3201S-K) was used for DNA extraction. Here, the nucleic acid extraction instrument is CWE2100, and the extraction program is: mix column 1, 65℃, 2min fast, 2min medium, collect magnetic beads in column 3 twice, time 5s, mix column 1, 2min fast, 2min medium, mix column 2, 1min fast, mix column 3, 1min fast, dry 2min reagent outside, mix column 6, 56℃, 2min fast, 2min medium. The extracted nucleic acid sample was stored in a-20℃ environment.

[0037] Quantitative real-time PCR: The CW2683 Human Genomic DNA Quantification Kit was used to perform qPCR quantitative analysis on nucleic acid samples extracted from days 0-4-9. The basic reaction system consisted of 10 μl of 2*GoldStar TaqMan Mixture, 3 μl of Primer Mix, 4 μl of template, and water added to a final volume of 20 μl. The reaction conditions were 95℃ for 10 min, followed by 55 cycles of (95℃ for 10 s, 60℃ for 10 s).

[0038] The results are as follows Figure 1 As shown in Table 2, the Ct values ​​for human genome quantification of fecal samples remained stable and showed no significant changes after 0, 4, and 9 days of preservation in the three preservation solutions (p>0.05). The results indicate that preservation solutions 1, 2, and 3 can all preserve human nucleic acid DNA in fecal samples, and this human nucleic acid DNA can be used as an internal reference nucleic acid in downstream detection kits.

[0039] Table 2. qPCR results of three fecal nucleic acid preservation solutions at different time periods.

[0040]

[0041]

[0042] Example 3: Stability study of viral RNA in fecal samples in nucleic acid preservation solution

[0043] The experimental group, positive control group, and negative control group were set up according to the following steps:

[0044] 1) Experimental group: Approximately 1 gram of fresh feces from 3 volunteers was placed in 10 ml of preservation solutions 1, 2, and 3 prepared in Example 1, and in RNase-free water, respectively. The mixture was shaken and incubated at room temperature. Then, 100 μl of IBV virus was added and shaken to mix.

[0045] 2) Positive control group: Take 10 ml of preservation solution No. 2 prepared in Example 1, add 100 μl of IBV virus directly, shake to mix, and store at room temperature.

[0046] 3) Negative control group: Approximately 1 gram of fresh feces from 3 volunteers was placed in 10 ml of preservation solution No. 2 prepared in Example 1, shaken to mix, and stored at room temperature.

[0047] Nucleic acid extraction: After vortexing and mixing, transfer 300 μL of fecal viral RNA preservation solution to 2 ml centrifuge tubes in a fume hood. Centrifuge at 12000 rpm for 1 min, and collect 200 μL of the supernatant. Use a viral DNA / RNA extraction kit (Kangwei Century, catalog number CW3201S-K) for DNA extraction. The extraction program is as follows: Mix column 1, 65℃, 2 min rapid, 2 min medium speed; collect magnetic beads from column 3 twice, 5 s each time; mix column 1, 2 min rapid, 2 min medium speed; mix column 2, 1 min rapid; mix column 3, 1 min rapid; dry the reagent exterior for 2 min; mix column 6, 56℃, 2 min rapid, 2 min medium speed. Store the extracted nucleic acid samples at -20℃.

[0048] Quantitative Real-Time PCR: The CW3149 1.26*HyperProbe OneStep RT-qPCR Kit (UNG) was used to perform qPCR quantitative analysis on nucleic acid samples extracted on days 0, 4, and 9. The reaction mixture consisted of 19.88 μl of 1.26*HyperProbeOneStep mix, 0.12 μl of Primer Mix (100 μM), and 5 μl of template. The reaction conditions were 37℃ for 2 min, 55℃ for 2 min, 96℃ for 2 min, followed by a 45-cycle reaction (96℃ for 5 s, 58℃ for 30 s).

[0049] The results showed that, in the experimental group, IBV virus extracted from fecal samples stored in water without RNase showed complete degradation after 3 hours. Figure 2 As shown, the qPCR results showed a Ct value of N / A, and similarly, the nucleic acid qPCR results of the negative control group also showed a Ct value of N / A. For samples from experimental groups 1, 2, and 3 preserved in fecal virus preservation solution, after being stored at room temperature for 9 days, as... Figure 3 As shown, compared with the Ct values ​​on day 0 and day 4, the Ct difference detected on day 9 was within 0.7, with no significant difference (p > 0.05). The above results indicate that all three fecal viral RNA preservation solutions can effectively ensure the stability of viral RNA for at least 7 days under room temperature conditions.

[0050] Example 4: Study on the interference performance of the control group preservation solution and the preservation solution of the present invention on the inhibitory effect of RNA virus nucleic acid extraction in feces.

[0051] The experimental group, control group, and positive control group were set up according to the following steps:

[0052] 1) Experimental group: Approximately 1 gram of fresh feces from 4 volunteers was taken and placed in 10 ml of fecal viral RNA preservation solution No. 2 prepared in Example 1. The mixture was shaken and incubated at room temperature. Then, 100 μl of IBV virus was added and shaken and mixed.

[0053] 2) Positive control group: Take 10 ml of fecal virus RNA preservation solution No. 2 prepared in Example 1, add 100 μl of IBV virus directly, shake to mix, and store at room temperature.

[0054] 3) Control group: Approximately 1 gram of fresh feces from 4 volunteers was placed in 10 ml of viral RNA preservation solution from Company A, vortexed to mix, and stored at room temperature. Then, 100 μl of IBV virus was added and vortexed to mix.

[0055] Nucleic acid extraction: After vortexing and mixing, transfer 300 μL of the preservation solution to a 2 mL centrifuge tube in a fume hood. Centrifuge at 12000 rpm for 1 min, collect 200 μL of the supernatant, and perform DNA extraction using a viral DNA / RNA extraction kit (Kangwei Century, catalog number CW3201S-K). The extraction program is as follows: Mix column 1, 65℃, 2 min fast, 2 min medium; collect the magnetic beads in column 3 twice, 5 s each time; mix column 1, 2 min fast, 2 min medium; mix column 2, 1 min fast; mix column 3, 1 min fast; dry the reagent exterior for 2 min; mix column 6, 56℃, 2 min fast, 2 min medium. The extracted nucleic acid sample is diluted 10-fold. The original nucleic acid and the 10-fold diluted nucleic acid are stored at -20℃.

[0056] Quantitative real-time PCR: The CW3149 1.26*HyperProbe OneStep RT-qPCR Kit (UNG) was used to perform qPCR quantitative analysis on nucleic acid samples extracted from days 0-4-9. The reaction mixture consisted of 19.88 μl of 1.26*HyperProbeOneStep mix, 0.12 μl of Primer Mix (100 μM), and 5 μl of template. The reaction conditions were 37℃ for 2 min, 55℃ for 2 min, 96℃ for 2 min, followed by a 45-cycle reaction (96℃ for 5 s, 58℃ for 30 s). The results are shown in Table 3.

[0057] Table 3 shows the qPCR results of the inhibitor removal performance of Company A's preservation solution and the No. 2 preservation solution of this invention.

[0058]

[0059]

[0060] In this embodiment, compared with the original nucleic acid solution, the nucleic acid extracted from Company A's viral RNA preservation solution, when diluted 10-fold, did not show an increase in Ct value; in fact, the Ct value decreased. The fluorescence quantitative curve is shown below. Figure 4As shown, the fecal background RNA virus nucleic acid stock solution extracted using the company's virus preservation solution contains a certain concentration of inhibitors.

[0061] Furthermore, in this embodiment, the Ct value of the fecal background RNA virus nucleic acid extracted from preservation solution No. 2, after a 10-fold dilution, was significantly higher than that of the original nucleic acid solution, with a difference of approximately 3. The fluorescence quantitative curve is shown below. Figure 5 As shown, the results are consistent with the theoretical values, indicating that during the 10-fold dilution of sample No. 2 in this embodiment, no other inhibitors inhibited the dilution process, and no inhibitors were present in the extracted nucleic acid stock solution.

[0062] The specific part numbers and models of the reagents and instruments mentioned above are not limited or described in detail, as they are common knowledge and can be understood by those skilled in the art.

[0063] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A preservation solution for RNA virus nucleic acid in fecal samples, characterized in that, It consists of the following formula: Guanidine isothiocyanate 2~8M; Cetyltrimethylammonium bromide 0.2~0.6M; Disodium ethylenediaminetetraacetate 0.1~1M; Citric acid buffer 0.2%~0.8%; Sodium chloride 3-8%; DTT 3~7mM; The pH value is 7.5~9.

5.

2. The RNA virus nucleic acid preservation solution in fecal samples according to claim 1, characterized in that, It consists of the following formula: Guanidine isothiocyanate 4M; Hexadecyltrimethylammonium bromide 0.4M; Disodium ethylenediaminetetraacetate 0.6M; Citric acid buffer 0.5%; Sodium chloride 5%; DTT 4mM.

3. The RNA virus nucleic acid preservation solution in fecal samples according to claim 1, characterized in that, It consists of the following formula: Guanidine isothiocyanate 6M; Hexadecyltrimethylammonium bromide 0.5M; Disodium ethylenediaminetetraacetate 0.9M; Citric acid buffer 0.7%; Sodium chloride 8%; DTT 5mM.

4. A preservation solution for RNA virus nucleic acid in fecal samples, characterized in that, It consists of the following formula: Guanidine isothiocyanate 2M; Hexadecyltrimethylammonium bromide 0.3M; Disodium ethylenediaminetetraacetate 0.3M; Citric acid buffer 0.3%; Sodium chloride 2%; DTT 3mM; The pH value is 7.5~9.

5.

5. The use of a preservation solution for RNA virus nucleic acid in fecal samples, characterized in that, The RNA virus nucleic acid in the fecal sample is preserved using a fecal sample RNA virus nucleic acid preservation solution as described in any one of claims 1 to 4; and / or the virus in the fecal sample is inactivated and the nucleic acid is released.

6. The use of the RNA virus nucleic acid preservation solution in fecal samples according to claim 5, characterized in that, The fecal sample was obtained from a human or a non-human animal; the mass-to-volume ratio of the fecal sample to the preservation solution was 1:

10.

7. A method for preparing a preservation solution for RNA virus nucleic acid in fecal samples, characterized in that, The preparation of a fecal sample RNA virus nucleic acid preservation solution as described in any one of claims 1 to 4 comprises the following steps: 1) Mix the components according to their proportions, adjust the pH with a pH buffer, and bring the volume to a final volume with deionized water; 2) Filtration, to obtain the preservation solution.

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