Kit and method for extracting helicobacter pylori nucleic acid in excrement sample and application

Abstract

The invention discloses a kit and a method for extracting helicobacter pylori nucleic acid in an excrement sample and application. The kit comprises sample treatment liquid: guanidine isothiocyanate and lithium dodecyl sulfate; the lysis solution is prepared from magnetic beads, guanidine isothiocyanate, lauramidopropyl hydroxysulfobetaine, potassium chloride, trihydroxymethyl aminomethane and isopropanol; the washing liquid I is prepared from guanidine hydrochloride, Tween 20 and tris (hydroxymethyl) aminomethane; a washing liquid II: betaine, guaiacol and ethanol; the washing liquid III is ethanol; the eluent is tris (hydroxymethyl) aminomethane. When the kit is used for extracting the helicobacter pylori nucleic acid in the excrement sample, impurities can be fully digested, adsorption of the impurities and the magnetic beads is reduced, cross-linking of the impurities and the nucleic acid is reduced, effective adsorption of the nucleic acid and the magnetic beads is increased, and the yield and quality of the helicobacter pylori nucleic acid in the excrement sample are improved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a kit, method and application for extracting Helicobacter pylori nucleic acid from fecal samples. Background Art

[0002] Helicobacter pylori (hereinafter referred to as "Hp") is a monopolar, multi-flagellated, blunt-ended, spirally curved bacterium, 2.5-4.0 μm long and 0.5-1.0 μm wide. It often appears in a typical spiral or arc shape on the surface of gastric mucosal epithelial cells. Humans are generally susceptible to Helicobacter pylori. The global Hp infection rate in the natural population exceeds 50%, while the infection rate of Helicobacter pylori in my country is between 40% and 70%. The severity of symptoms after infection varies from individual to individual, showing obvious differences in pathogenicity. Mild cases may have no obvious symptoms for a long time, while severe cases may develop into atrophic gastritis, gastric ulcers, and even malignant tumors. Therefore, the need for detection and eradication of Helicobacter pylori is becoming increasingly prominent.

[0003] At present, the main methods for detecting Helicobacter pylori are: antigen detection, bacterial culture, breath test and PCR detection. The PCR test results can not only identify whether Helicobacter pylori is positive or not, but also provide information on the drug resistance of the strain, which has important guiding significance for the formulation of subsequent Helicobacter pylori eradication treatment plans. Among them, the object of PCR detection is Hp nucleic acid, which is mostly obtained by separating it from gastric mucosal tissue. The main method is to obtain gastric tissue mucosa through gastroscopy. This acquisition is an invasive process, which not only causes discomfort to patients but also brings a certain economic burden to patients. The convenience of sampling and patient compliance are not advantageous.

[0004] Therefore, people use in vitro samples - fecal samples for relevant tests. However, the composition of fecal samples is complex, and it is a difficult process to obtain sufficient and stable nucleic acids from fecal samples. Moreover, the studies that have been published generally believe that the Hp concentration in fecal samples is lower than that in gastric mucosal tissue. Therefore, there are higher requirements for the performance and stability of fecal sample nucleic acid extraction reagents. This application intends to conduct relevant research on the extraction of nucleic acids from fecal samples and provide a new fecal sample nucleic acid extraction scheme. Summary of the invention

[0005] In order to solve at least one of the above problems, the present invention provides a kit, method and application for extracting Helicobacter pylori nucleic acid from fecal samples. The kit can extract Helicobacter pylori nucleic acid from fecal samples with high yield and good quality.

[0006] In order to achieve the above object, the present invention adopts the following technical means: A first aspect of the present invention provides a kit for extracting Helicobacter pylori nucleic acid from a stool sample, comprising a lysis solution and a washing solution II; The lysis solution comprises magnetic beads, guanidine isothiocyanate, lauramide propyl hydroxysulfobetaine, potassium chloride, tris(hydroxymethyl)aminomethane, and isopropanol; The washing solution II comprises betaine, guaiacol and ethanol; In some embodiments of the present invention, the kit further includes a sample processing solution, a washing solution I, a washing solution III and an eluent. The sample processing solution includes guanidine isothiocyanate and lithium dodecyl sulfate; The washing solution I comprises guanidine hydrochloride, Tween20, and tris(hydroxymethyl)aminomethane; The washing liquid III comprises ethanol; The eluent includes tris(hydroxymethyl)aminomethane.

[0007] In some specific embodiments of the present invention, the lysate contains 0.2-4 mg / mL magnetic beads, 1-5 M guanidine isothiocyanate, 0.1-4% lauramide propyl hydroxysulfobetaine by volume, 5-15% potassium chloride by volume, 0.1-1 M tris(hydroxymethyl)aminomethane, and 30-60% isopropanol by volume; The washing solution II contains 0.5-5 M betaine, 0.2-20% guaiacol by volume, and 75-80% ethanol by volume.

[0008] In some specific embodiments of the present invention, the sample treatment solution contains 1-3M guanidine isothiocyanate and 0.1-20% lithium dodecyl sulfate by mass volume; The washing solution I contains 1-3 M guanidine hydrochloride, 0.1-10% Tween20 by mass volume, and 1-1000 mM tris(hydroxymethyl)aminomethane; Washing liquid III: ethanol mass volume ratio 70-80%; Eluent: Tris(2-hydroxymethylaminomethane) 1-50 mM.

[0009] In some specific embodiments of the present invention, preferably, the magnetic beads are carboxylated magnetic beads with a particle size of 100-500 nm.

[0010] The second aspect of the present invention provides a method for extracting Helicobacter pylori nucleic acid from a stool sample, using the kit described in the first aspect for extraction.

[0011] In some embodiments of the present invention, the extraction of Helicobacter pylori nucleic acid from a stool sample comprises the following steps: S1. Take a stool sample, add sample processing solution, and mix thoroughly on a vortex shaker; incubate at 70-80°C; remove the reagent, cool to room temperature, centrifuge, and take the supernatant as the sample to be processed; S2. Add the sample to be processed to the sample tube containing the lysis solution and mix well; incubate at 70-80°C, invert several times to mix well; after centrifugation, place the sample tube on a magnetic rack and keep it until the liquid is clear; S3, discard the supernatant with a pipette, add washing solution I, and vortex to mix; after centrifugation, place the sample tube on a magnetic rack and keep it until the liquid is clear; S4. Use a pipette to discard the supernatant, add washing solution II, and vortex to mix; after centrifugation, place the sample tube on a magnetic rack and keep it until the liquid is clear; S5. Use a pipette to discard the supernatant, add washing solution III, and vortex to mix; after centrifugation, place the centrifuge tube on a magnetic rack and keep it until the liquid is clear; S6. Use a pipette to discard the supernatant and keep the sample tube on the magnetic rack until the magnetic beads are dry and shiny; S7, add the eluent, vortex to mix, incubate at room temperature, and invert several times to mix; after centrifugation, place the sample tube on a magnetic rack and keep it until the liquid is clear; S8. Use a pipette to transfer the supernatant to a clean sample tube free of nuclease to obtain the extracted Helicobacter pylori nucleic acid solution.

[0012] In some embodiments of the present invention, in step S1, the usage ratio of the stool sample to the sample processing solution is (2-5) g:10 mL.

[0013] In some embodiments of the present invention, in step S2, the volume ratio of the lysate to the sample to be processed is 5:3.

[0014] In some specific embodiments of the present invention, step S1, take 0.2-0.5g of fecal sample, add 1mL of sample treatment solution, and mix thoroughly on a vortex oscillator; incubate at 70-80℃ for 10-12min; take out the reagent, cool to room temperature, centrifuge at 12000rmp for 5min, and take the supernatant as the sample to be processed; step S2, add 300μL of the sample to be processed to the sample tube containing 500μL of lysis solution, and mix evenly; incubate at 70-80℃, invert and mix several times during the process; after centrifugation, place the sample tube on a magnetic rack and keep it until the liquid is clear; add 600-700μL of washing solution I in step S3, add 600-700μL of washing solution II in step S4, add 600-700μL of washing solution III in step S5, and add 100μL of elution solution in step S7.

[0015] The third aspect of the present invention provides use of the kit described in the first aspect in extracting Helicobacter pylori nucleic acid from a stool sample.

[0016] Beneficial Effects of the Invention Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a lysis solution for extracting Hp nucleic acid from stool samples, further lyses the samples, and assists in combining nucleic acids with magnetic beads. In an environment of a certain concentration of chaotropic salts (such as guanidine isothiocyanate), inorganic salts and isopropanol, nucleic acids can be adsorbed onto magnetic beads, but the adsorption of nucleic acids by magnetic beads is not completely specific, and impurities such as proteins, lipids, sugars, and humus may also be combined with them, or these impurities may be cross-linked with nucleic acids and then brought into the next link. In this step, the impurities can be further digested by optimizing the formula of the lysis solution, reducing the adsorption of impurities with magnetic beads, reducing the cross-linking of impurities with nucleic acids, and increasing the effective adsorption of nucleic acids with magnetic beads. The present invention uses lauroamidopropyl hydroxysulfobetaine, which is a zwitterionic surfactant, which synergistically exerts a lysis and digestion effect with guanidine isothiocyanate, reduces the interaction between impurities and magnetic beads and nucleic acids, and increases the effective adsorption of nucleic acids with magnetic beads; at the same time, the washing solution 2 is optimized, and a combination of betaine and yutracol is used, which plays a key role in this step and effectively removes impurities such as sugars and humus. DETAILED DESCRIPTION

[0017] The following examples are used to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention and therefore can be considered as preferred embodiments of the present invention. However, it will be appreciated by those skilled in the art based on this specification that many modifications may be made to the specific embodiments disclosed herein and still achieve the same or similar results without departing from the spirit or scope of the present invention.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs, and the materials cited herein and those cited by them are incorporated by reference. Those skilled in the art will recognize or will learn through routine experimentation that there are many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0019] The technical solution of the present application is further described in detail below in conjunction with specific implementation methods.

[0020] Example 1 1. Reagent preparation (1) Stool sample processing solution: 2M guanidine isothiocyanate, 10% lithium dodecyl sulfate.

[0021] (2) Lysis solution: 1 mg / mL magnetic beads, 2 M guanidine thiocyanate, 2% lauramidopropyl hydroxysulfobetaine, 10% potassium chloride, 0.1 M tris(hydroxymethyl)aminomethane, and 50% isopropanol.

[0022] (3) Washing solution 1: 3 M guanidine hydrochloride, 2% Tween20, 200 mM tris(hydroxymethyl)aminomethane.

[0023] (4) Washing solution 2: betaine 1 M, guaiacol 1%, ethanol 75%.

[0024] (5) Washing solution 3: 75% ethanol.

[0025] (6) Eluent: 10 mM tris(hydroxymethyl)aminomethane.

[0026] 2. Nucleic acid extraction process (1) Take 0.5 g of stool sample, add 1 mL of stool sample treatment solution, and mix thoroughly on a vortex oscillator; (2) Incubate at 80°C for 10 min; (3) Remove the reagent and cool it to room temperature, centrifuge at 12000 rpm for 5 min, and take the supernatant as the sample to be processed; (4) Mix the lysate thoroughly, take 500 μL of the lysate into a 1.5 mL centrifuge tube, then add 300 μL of the sample to be processed and mix well; (5) Incubate at 70°C for 8 min, inverting 2-3 times to mix; (6) Briefly centrifuge and place the centrifuge tube on a magnetic rack for 1 min or until the liquid is clear; (7) Use a pipette to discard the supernatant, add 600 μL of washing solution 1, and vortex to mix for 1 min; (8) Briefly centrifuge and place the centrifuge tube on a magnetic rack for 1 min or until the liquid is clear; (9) Use a pipette to discard the supernatant, add 700 μL of washing solution 2, and vortex to mix for 1 min; (10) Briefly centrifuge and place the centrifuge tube on a magnetic rack for 1 min or until the liquid is clear; (11) Remove the supernatant with a pipette, add 600 μL of washing solution 3, and vortex to mix for 20 seconds; (12) Briefly centrifuge and place the centrifuge tube on a magnetic rack for 1 min or until the liquid is clear; (13) Use a pipette to discard the supernatant and keep the centrifuge tube on the magnetic rack for 5 minutes or until the magnetic beads are dry and shiny; (14) Add 100 μL of eluent, vortex to mix, and incubate at room temperature for 5 min, mixing 2-3 times during the process; (15) Briefly centrifuge and place the tube on a magnetic rack for 1 min or until the liquid is clear. (16) Use a pipette to transfer the supernatant, i.e., nucleic acid solution, to a clean, nuclease-free centrifuge tube for use in downstream experiments.

[0027] 3. Sample Three Hp stool samples were extracted: sample 1, sample 2, and sample 3.

[0028] Example 2 The reagents, nucleic acid extraction process, and samples are the same as those in Example 1. The differences are: Lysis buffer: magnetic beads 1 mg / mL, guanidine thiocyanate 2 M, SDS 2%, potassium chloride 10%, tris hydroxymethylaminomethane 0.1 M, isopropanol 50%.

[0029] Example 3 The reagents, nucleic acid extraction process, and samples are the same as those in Example 1. The differences are: Lysis buffer: magnetic beads 1 mg / mL, guanidine thiocyanate 2 M, sodium lauroyl sarcosinate 2%, potassium chloride 10%, tris(hydroxymethyl)aminomethane 0.1 M, isopropanol 50%.

[0030] Example 4 The reagents, nucleic acid extraction process, and samples are the same as those in Example 1. The differences are: Lysis buffer: magnetic beads 1 mg / mL, guanidine isothiocyanate 2 M, lauramidopropyl hydroxysulfobetaine 2%, sodium chloride 10%, tris(hydroxymethyl)aminomethane 0.1 M, isopropanol 50%.

[0031] Example 5 The reagents, nucleic acid extraction process, and samples are the same as those in Example 1. The differences are: Lysis buffer: magnetic beads 1 mg / mL, guanidine isothiocyanate 2 M, lauramidopropyl hydroxysulfobetaine 2%, magnesium chloride 10%, tris(hydroxymethyl)aminomethane 0.1 M, isopropanol 50%.

[0032] Example 6 The reagents, nucleic acid extraction process, and samples are the same as those in Example 1. The differences are: Lysis buffer: magnetic beads 1 mg / mL, guanidine thiocyanate 2 M, SDS 2%, sodium chloride 10%, tris hydroxymethylaminomethane 0.1 M, isopropanol 50%.

[0033] Example 7 The reagents, nucleic acid extraction process, and samples are the same as those in Example 1. The differences are: Lysis buffer: magnetic beads 1 mg / mL, guanidine thiocyanate 2 M, potassium chloride 10%, tris(hydroxymethyl)aminomethane 0.1 M, isopropanol 50%.

[0034] The nucleic acid products extracted by the schemes of Examples 1 to 7 were detected using a micro-ultraviolet spectrophotometer, and the purity of the extracted products was evaluated using ratios of 260 / 280 and 260 / 230. The results are shown in Table 1.

[0035] Table 1 Purity of nucleic acid extracts from Examples 1 to 7

[0036] The nucleic acid products extracted by the schemes of Examples 1 to 7 were detected using the "Helicobacter pylori 23S rRNA Gene and GyrA Gene Mutation Detection Kit (Fluorescence PCR Method)" (Registration No.: National Medical Device Registration No. 20223400137) produced by Jiangsu Mole Biotechnology Co., Ltd., and the Ct value of the FAM channel was used to reflect the results. 23S rRNA The mutation of the gene is reflected by the Ct value of the VIC channel gyr The mutation of the gene, the Ct value of ROX reflects the production of Helicobacter pylori, and the Ct value of CY5 reflects the detection of the human internal standard. The results are shown in Table 2 below.

[0037] Table 2 Ct values ​​of nucleic acid extracts detected in Examples 1 to 7

[0038] The results showed that Example 1 had the best effect, in which the use of lauramidopropyl hydroxysulfobetaine could improve the purity of nucleic acid products and increase the yield of Helicobacter pylori nucleic acid.

[0039] Example 8 Three Hp stool samples were extracted: sample 1, sample 2, and sample 3.

[0040] The reagents and nucleic acid extraction process are the same as those in Example 1.

[0041] Example 9 The reagents, nucleic acid extraction process, and samples are the same as those in Example 8. The differences are: Washing solution 2: 0.5 M sodium acetate, 1% NP40, 75% ethanol.

[0042] Example 10 The reagents, nucleic acid extraction process, and samples are the same as those in Example 8. The differences are: Washing solution 2: 0.5 M sodium acetate, 1% TritonX-100, 75% ethanol.

[0043] Embodiment 11 The reagents, nucleic acid extraction process, and samples are the same as those in Example 8. The differences are: Washing solution 2: 0.5 M sodium acetate, 1% guaiacol, 75% ethanol.

[0044] Example 12 The reagents, nucleic acid extraction process, and samples are the same as those in Example 8. The differences are: Washing solution 2: betaine 1M, ethanol 75%.

[0045] The nucleic acid products extracted by the schemes of Examples 8 to 12 were detected using a micro-ultraviolet spectrophotometer, and the purity of the extracted products was evaluated using ratios of 260 / 280 and 260 / 230. The results are shown in Table 3.

[0046] Table 3 Purity of nucleic acid extracts from Examples 8 to 12

[0047] The nucleic acid products extracted by the schemes of Examples 8 to 12 were detected using the "Helicobacter pylori 23S rRNA Gene and gyrA Gene Mutation Detection Kit (Fluorescence PCR Method)" (Registration No.: National Medical Device Registration No. 20223400137) produced by Jiangsu Mole Biotechnology Co., Ltd., and the Ct value result of the FAM channel was used to reflect the 23S rRNA The mutation of the gene is reflected by the Ct value of the VIC channel gyr The mutation of the gene, the Ct value of ROX reflects the production of Helicobacter pylori, and the Ct value of CY5 reflects the detection of the human internal standard. The results are shown in Table 4 below.

[0048] Table 4 Ct values ​​of nucleic acid extracts detected in Examples 8-12

[0049] The results showed that Example 8 had the best effect, in which the use of guaiacol could improve the purity of the nucleic acid product and also increase the yield of Helicobacter pylori nucleic acid.

[0050] Embodiment 13 Eight samples numbered 1 to 8 were taken, and the commercial reagent: QIAamp Fast DNA Stool Mini Kit (manufacturer: QIAGEN) and the method of Example 1 were used to extract the eight Hp samples, and the "Helicobacter pylori 23S rRNA Gene and GyrA Gene Mutation Detection Kit (Fluorescence PCR Method)" (Registration Certificate No.: National Medical Device Registration No. 20223400137) produced by Jiangsu Mole Biotechnology Co., Ltd. was used to detect the products extracted from the eight samples, and the Ct value results of the FAM channel were used to react 23S rRNA The mutation of the gene is reflected by the Ct value of the VIC channel gyrThe mutation of the gene, the Ct value of ROX reflects the production of Helicobacter pylori, and the Ct value of CY5 reflects the detection of the human internal standard. The results are shown in Table 5 below.

[0051] Table 58 Ct values ​​of nucleic acid extracts from samples

[0052] The results show that: in the extraction experiment of 8 samples, the Helicobacter pylori positivity rate corresponding to the commercial reagent was 50%, and the positive rate corresponding to the present application was 100%. The present application can significantly improve the positive detection rate of Helicobacter pylori; in the samples where Helicobacter pylori was detected by both parties, the Ct value corresponding to Helicobacter pylori of the present application was 0.93-2.47 higher than that of the commercial reagent, that is, the nucleic acid yield of Helicobacter pylori obtained by the present application is 2-4 times that of the commercial reagent.

[0053] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as references separately. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined in the present application.

Claims

1. A kit for extracting Helicobacter pylori nucleic acid from stool samples, characterized in that: The kit includes lysis buffer and washing buffer II; The lysis solution comprises magnetic beads, guanidine isothiocyanate, lauramide propyl hydroxysulfobetaine, potassium chloride, tris(hydroxymethyl)aminomethane, and isopropanol; The washing solution II comprises betaine, guaiacol and ethanol.

2. A kit for extracting Helicobacter pylori nucleic acid from stool samples according to claim 1, characterized in that: The kit also includes sample treatment solution, washing solution I, washing solution III and elution solution. The sample processing solution includes guanidine isothiocyanate and lithium dodecyl sulfate; The washing solution I comprises guanidine hydrochloride, Tween20, and tris(hydroxymethyl)aminomethane; The washing liquid III comprises ethanol; The eluent includes tris(hydroxymethyl)aminomethane.

3. The kit for extracting Helicobacter pylori nucleic acid from stool samples according to claim 1, characterized in that: The lysate contains 0.2-4 mg / mL magnetic beads, 1-5 M guanidine isothiocyanate, 0.1-4% lauramide propyl hydroxysulfobetaine by volume, 5-15% potassium chloride by volume, 0.1-1 M tris(hydroxymethyl)aminomethane, and 30-60% isopropanol by volume; The washing solution II contains 0.5-5 M betaine, 0.2-20% guaiacol by volume, and 75-80% ethanol by volume.

4. A kit for extracting Helicobacter pylori nucleic acid from stool samples according to claim 2, characterized in that: The sample treatment solution contains 1-3M guanidine isothiocyanate and 0.1-20% lithium dodecyl sulfate by mass volume; The washing solution I contains 1-3 M guanidine hydrochloride, 0.1-10% Tween20 by mass volume, and 1-1000 mM tris(hydroxymethyl)aminomethane; Washing liquid III: ethanol mass volume ratio 70-80%; Eluent: Tris(2-hydroxymethylaminomethane) 1-50 mM.

5. A kit for extracting Helicobacter pylori nucleic acid from stool samples according to any one of claims 1 to 4, characterized in that: The magnetic beads are carboxylated magnetic beads with a particle size of 100-500nm.

6. A method for extracting Helicobacter pylori nucleic acid from a stool sample, characterized in that: The extraction is performed using the kit described in claim 5.

7. The method according to claim 6, characterized in that The steps include: S1. Take a stool sample, add sample processing solution, and mix thoroughly on a vortex shaker; incubate at 70-80°C; remove the reagent, cool to room temperature, centrifuge, and take the supernatant as the sample to be processed; S2. Add the sample to be processed to the sample tube containing the lysis solution and mix well; incubate at 70-80°C, invert several times to mix well; after centrifugation, place the sample tube on a magnetic rack and keep it until the liquid is clear; S3, discard the supernatant with a pipette, add washing solution I, and vortex to mix; after centrifugation, place the sample tube on a magnetic rack and keep it until the liquid is clear; S4. Use a pipette to discard the supernatant, add washing solution II, and vortex to mix; after centrifugation, place the sample tube on a magnetic rack and keep it until the liquid is clear; S5. Use a pipette to discard the supernatant, add washing solution III, and vortex to mix; after centrifugation, place the centrifuge tube on a magnetic rack and keep it until the liquid is clear; S6. Use a pipette to discard the supernatant and keep the sample tube on the magnetic rack until the magnetic beads are dry and shiny; S7, add the eluent, vortex to mix, incubate at room temperature, and invert several times to mix; after centrifugation, place the sample tube on a magnetic rack and keep it until the liquid is clear; S8. Use a pipette to transfer the supernatant to a clean sample tube free of nuclease to obtain the extracted Helicobacter pylori nucleic acid solution.

8. The method according to claim 7, characterized in that In step S1, the usage ratio of the stool sample to the sample processing solution is (2-5) g:10 mL.

9. The method according to claim 7, characterized in that: In the step S2, the volume ratio of the lysate to the sample to be processed is 5:

3.

10. Use of the kit according to any one of claims 1 to 5 for extracting Helicobacter pylori nucleic acid from stool samples.

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