A kit, method and application for extracting Helicobacter pylori nucleic acid from fecal samples

Abstract

This invention discloses a kit, method, and application for extracting Helicobacter pylori nucleic acid from fecal samples. The kit includes: sample processing solution: guanidine isothiocyanate, lithium dodecyl sulfate; lysis buffer: magnetic beads, guanidine isothiocyanate, lauramide propyl hydroxysulfonate betaine, potassium chloride, tris(hydroxymethyl)aminomethane, isopropanol; washing buffer I: guanidine hydrochloride, Tween 20, tris(hydroxymethyl)aminomethane; washing buffer II: betaine, guaiacol, ethanol; washing buffer III: ethanol; elution buffer: tris(hydroxymethyl)aminomethane. Using this kit for extracting Helicobacter pylori nucleic acid from fecal samples can fully digest impurities, reduce the adsorption of impurities to magnetic beads, reduce the cross-linking of impurities and nucleic acids, increase the effective adsorption of nucleic acids to magnetic beads, and improve the yield and quality of Helicobacter pylori nucleic acid in fecal samples.

Description

Technical Field

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

[0002] Helicobacter pylori (Hp) is a unipolar, multi-flagellated, blunt-ended, spiral-shaped bacterium, 2.5-4.0 μm long and 0.5-1.0 μm wide. On the surface of gastric mucosal epithelial cells, it typically presents as a spiral or arc shape. Humans are universally susceptible to Hp, with a global Hp infection rate exceeding 50%, while in my country, the infection rate ranges from 40% to 70%. The severity of symptoms varies among individuals, demonstrating significant differences in pathogenicity. Mild cases may be asymptomatic for a long time, while severe cases can develop into atrophic gastritis, gastric ulcers, and even malignant tumors. Therefore, the need for Hp detection and eradication is increasingly prominent.

[0003] Currently, the main methods for detecting Helicobacter pylori include antigen detection, bacterial culture, breath test, and PCR detection. PCR results not only identify whether H. pylori is positive or not but also provide information on the strain's drug resistance, which is of great guiding significance for the development of subsequent H. pylori eradication treatment plans. PCR detection targets H. pylori nucleic acid, which is mostly obtained by isolating it from gastric mucosal tissue, primarily through gastroscopy. This invasive procedure causes discomfort and financial burden for patients, and its convenience and patient compliance are not advantageous.

[0004] Therefore, people use in vitro samples—fecal samples—for relevant testing. However, fecal samples have complex components, and obtaining sufficient quantity and stable quality of nucleic acid from fecal samples is a difficult process. Moreover, current published studies generally believe that the concentration of Hp in fecal samples is lower than that in gastric mucosa tissue. Therefore, there are higher requirements for the performance and stability of nucleic acid extraction reagents from fecal samples. This application aims to conduct relevant research on nucleic acid extraction from fecal samples and provide a new nucleic acid extraction scheme for fecal samples. Summary of the Invention

[0005] To address at least one of the aforementioned problems, this invention provides a kit, method, and application for extracting Helicobacter pylori nucleic acid from fecal samples. This kit yields high-yield and high-quality Helicobacter pylori nucleic acid from fecal samples.

[0006] To achieve the above objectives, the present invention employs the following technical means:

[0007] A first aspect of the present invention provides a kit for extracting Helicobacter pylori nucleic acid from fecal samples, comprising a lysis buffer and a washing buffer II;

[0008] The lysis buffer includes magnetic beads, guanidine isothiocyanate, lauramide propyl hydroxysulfonate betaine, potassium chloride, tris(hydroxymethyl)aminomethane, and isopropanol;

[0009] The washing solution II includes betaine, guaiacol, and ethanol;

[0010] In some embodiments of the present invention, the kit further includes a sample processing solution, washing solution I, washing solution III, and elution solution.

[0011] The sample processing solution includes guanidine isothiocyanate and lithium dodecyl sulfate;

[0012] The washing solution I includes guanidine hydrochloride, Tween20, and tris(hydroxymethyl)aminomethane;

[0013] The washing solution III includes ethanol;

[0014] The eluent includes tris(hydroxymethyl)aminomethane.

[0015] In some specific embodiments of the present invention, the lysis solution contains 0.2-4 mg / mL magnetic beads, 1-5 M guanidine isothiocyanate, 0.1-4% lauramidopropyl hydroxysulfonate betaine (by mass / volume), 5-15% potassium chloride (by mass / volume), 0.1-1 M tris(hydroxymethyl)aminomethane, and 30-60% isopropanol (by mass / volume).

[0016] The washing solution II contains 0.5-5M betaine, 0.2-20% guaiacol (by mass / volume), and 75-80% ethanol (by mass / volume).

[0017] In some specific embodiments of the present invention, the sample processing solution contains 1-3M guanidine isothiocyanate and 0.1-20% lithium dodecyl sulfate by mass-to-volume ratio.

[0018] The washing solution I contains 1-3M guanidine hydrochloride, 0.1-10% Tween 20 by mass / volume, and 1-1000mM tris(hydroxymethyl)aminomethane.

[0019] Washing solution III: 70-80% ethanol (mass-volume ratio);

[0020] Eluent: Tris(hydroxymethyl)aminomethane 1-50 mM.

[0021] In some specific embodiments of the present invention, preferably, the magnetic beads are carboxylated magnetic beads with a particle size of 100-500 nm.

[0022] A second aspect of the present invention provides a method for extracting Helicobacter pylori nucleic acid from fecal samples, using the kit described in the first aspect.

[0023] In some embodiments of the present invention, the extraction of Helicobacter pylori nucleic acid from fecal samples includes the following steps:

[0024] S1. Take a fecal sample, add sample processing solution, and mix thoroughly on a vortex mixer; incubate at 70-80℃; remove the reagent, cool to room temperature, centrifuge, and take the supernatant as the sample to be processed;

[0025] S2. Add the sample to be processed to the sample tube containing the lysis buffer and mix well; incubate at 70-80℃, inverting and mixing several times during the process; after centrifugation, place the sample tube on a magnetic rack and keep it until the liquid is clear.

[0026] S3. Use a pipette to discard the supernatant, 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;

[0027] S4. Use a pipette to discard the supernatant, add washing buffer II, and vortex to mix; after centrifugation, place the sample tube on a magnetic rack and keep it until the liquid is clear;

[0028] 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;

[0029] S6. Use a pipette to remove the supernatant and keep the sample tube on the magnetic rack until the magnetic beads are dry and free of water.

[0030] S7. Add elution buffer, vortex to mix, incubate at room temperature, inverting the tube several times during incubation; after centrifugation, place the sample tube on a magnetic rack and keep it until the liquid is clear.

[0031] S8. Use a pipette to transfer the supernatant into a clean, nuclease-free sample tube to obtain the extracted Helicobacter pylori nucleic acid solution.

[0032] In some embodiments of the present invention, in step S1, the ratio of fecal sample to sample processing solution is (2-5) g: 10 mL.

[0033] In some embodiments of the present invention, in step S2, the volume ratio of the lysis buffer to the sample to be processed is 5:3.

[0034] In some specific embodiments of the present invention, step S1: take 0.2-0.5g of fecal sample, add 1mL of sample processing solution, and mix thoroughly on a vortex mixer; incubate at 70-80℃ for 10-12min; remove the reagent and cool to room temperature, centrifuge at 12000rpm for 5min, and take the supernatant as the sample to be processed; step S2: add 300μL of the sample to be processed to a sample tube containing 500μL of lysis buffer, and mix thoroughly; incubate at 70-80℃, inverting and mixing several times during incubation; after centrifugation, place the sample tube on a magnetic rack and keep it until the liquid is clear; in step S3, add 600-700μL of washing buffer I; in step S4, add 600-700μL of washing buffer II; in step S5, add 600-700μL of washing buffer III; and in step S7, add 100μL of elution buffer.

[0035] A third aspect of the present invention provides the application of the kit described in the first aspect in the extraction of Helicobacter pylori nucleic acid from fecal samples.

[0036] Beneficial effects of the present invention

[0037] Compared to existing technologies, this invention offers the following advantages: It provides a lysis buffer for extracting Helicobacter pylori (Hp) nucleic acid from fecal samples, further lysing the sample and facilitating the binding of nucleic acids to magnetic beads. Under certain concentrations of dissociative salts (such as guanidine isothiocyanate), inorganic salts, and isopropanol, nucleic acids can adsorb onto magnetic beads. However, the adsorption of nucleic acids by magnetic beads is not entirely specific; impurities such as proteins, lipids, sugars, and humic substances may also bind to them, or these impurities may cross-link with nucleic acids and be carried to the next step. In this step, the formulation of the lysis buffer can be optimized to further digest impurities, reduce the adsorption of impurities to magnetic beads, decrease the cross-linking of impurities with nucleic acids, and increase the effective adsorption of nucleic acids to magnetic beads. This invention uses lauramidopropyl hydroxysulfonyl betaine, an amphoteric surfactant, which synergistically works with guanidine isothiocyanate to promote lysis and digestion, reducing the interaction between impurities and magnetic beads and nucleic acids, and increasing the effective adsorption of nucleic acids to magnetic beads. Simultaneously, the washing solution 2 is optimized, employing a combination of betaine and eugenol, which plays a crucial role in this step, effectively removing impurities such as sugars and humic substances. Detailed Implementation

[0038] The following examples are used to illustrate preferred embodiments of the invention. Those skilled in the art will understand that the techniques disclosed in the examples represent techniques discovered by the inventors that can be used to implement the invention, and therefore can be considered preferred embodiments for implementing the invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of the invention.

[0039] 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 this invention pertains, and all materials disclosed herein and cited therein are incorporated herein by reference. Many equivalent techniques of specific embodiments of the invention described herein will be recognized or can be understood by ordinary experimentation by those skilled in the art. These equivalents will be included in the claims.

[0040] The technical solution of this application will be further described in detail below with reference to specific embodiments.

[0041] Example 1

[0042] 1. Reagent preparation

[0043] (1) Fecal sample processing solution: guanidine isothiocyanate 2M, lithium dodecyl sulfate 10%.

[0044] (2) Lysis buffer: 1 mg / mL magnetic beads, 2 M guanidine isothiocyanate, 2% lauramide propyl hydroxysulfonate betaine, 10% potassium chloride, 0.1 M tris(hydroxymethyl)aminomethane, 50% isopropanol.

[0045] (3) Washing solution 1: Guanidine hydrochloride 3M, Tween 20 2%, Tris(hydroxymethyl)aminomethane 200mM.

[0046] (4) Washing solution 2: betaine 1M, guaiacol 1%, ethanol 75%.

[0047] (5) Washing solution 3: 75% ethanol.

[0048] (6) Eluent: Tris(hydroxymethyl)aminomethane 10mM.

[0049] 2. Nucleic acid extraction process

[0050] (1) Take 0.5g of fecal sample, add 1mL of fecal sample processing solution, and mix thoroughly on a vortex mixer;

[0051] (2) Incubate at 80℃ for 10 min;

[0052] (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.

[0053] (4) Mix the lysis buffer thoroughly, take 500 μL of the lysis buffer into a 1.5 mL centrifuge tube, then add 300 μL of the sample to be processed and mix thoroughly;

[0054] (5) Incubate at 70℃ for 8 minutes, inverting and mixing 2-3 times during the process;

[0055] (6) Brief centrifugation: Place the centrifuge tube on a magnetic rack and keep it for 1 minute or until the liquid is clear;

[0056] (7) Discard the supernatant with a pipette, add 600 μL of washing solution 1, and vortex mix for 1 min;

[0057] (8) Brief centrifugation: Place the centrifuge tube on a magnetic rack and keep it for 1 minute or until the liquid is clear;

[0058] (9) Discard the supernatant with a pipette, add 700 μL of washing solution 2, and vortex mix for 1 min;

[0059] (10) Brief centrifugation: Place the centrifuge tube on a magnetic rack and keep it for 1 minute or until the liquid is clear;

[0060] (11) Discard the supernatant with a pipette, add 600 μL of washing solution 3, and vortex mix for 20 s;

[0061] (12) Brief centrifugation: Place the centrifuge tube on a magnetic rack and keep it for 1 minute or until the liquid is clear;

[0062] (13) Use a pipette to remove the supernatant and keep the centrifuge tube on the magnetic rack for 5 minutes or until the magnetic beads are dry and free of water.

[0063] (14) Add 100 μL of elution buffer, vortex to mix, and incubate at room temperature for 5 min, mixing 2-3 times during the incubation period;

[0064] (15) Brief centrifugation: Place the centrifuge tube on a magnetic rack and keep it for 1 minute or until the liquid is clear;

[0065] (16) Use a pipette to transfer the supernatant, i.e. the nucleic acid solution, to a clean, nuclease-free centrifuge tube for use in downstream experiments.

[0066] 3. Sample

[0067] Three Hp fecal samples were extracted: Sample 1, Sample 2, and Sample 3.

[0068] Example 2

[0069] The reagents, nucleic acid extraction procedures, and samples are consistent with those in Example 1. The difference is:

[0070] Lysis buffer: 1 mg / mL magnetic beads, 2 M guanidine isothiocyanate, 2% SDS, 10% potassium chloride, 0.1 M tris(hydroxymethyl)aminomethane, 50% isopropanol.

[0071] Example 3

[0072] The reagents, nucleic acid extraction procedures, and samples are consistent with those in Example 1. The difference is:

[0073] Lysis buffer: 1 mg / mL magnetic beads, 2 M guanidine isothiocyanate, 2% sodium lauroyl sarcosinate, 10% potassium chloride, 0.1 M tris(hydroxymethyl)aminomethane, 50% isopropanol.

[0074] Example 4

[0075] The reagents, nucleic acid extraction procedures, and samples are consistent with those in Example 1. The difference is:

[0076] Lysis buffer: 1 mg / mL magnetic beads, 2 M guanidine isothiocyanate, 2% lauramide propyl hydroxysulfonate betaine, 10% sodium chloride, 0.1 M tris(hydroxymethyl)aminomethane, 50% isopropanol.

[0077] Example 5

[0078] The reagents, nucleic acid extraction procedures, and samples are consistent with those in Example 1. The difference is:

[0079] Lysis buffer: 1 mg / mL magnetic beads, 2 M guanidine isothiocyanate, 2% lauramidopropyl hydroxysulfonate betaine, 10% magnesium chloride, 0.1 M tris(hydroxymethyl)aminomethane, 50% isopropanol.

[0080] Example 6

[0081] The reagents, nucleic acid extraction procedures, and samples are consistent with those in Example 1. The difference is:

[0082] Lysis buffer: 1 mg / mL magnetic beads, 2 M guanidine isothiocyanate, 2% SDS, 10% sodium chloride, 0.1 M tris(hydroxymethyl)aminomethane, 50% isopropanol.

[0083] Example 7

[0084] The reagents, nucleic acid extraction procedures, and samples are consistent with those in Example 1. The difference is:

[0085] Lysis buffer: 1 mg / mL magnetic beads, 2 M guanidine isothiocyanate, 10% potassium chloride, 0.1 M tris(hydroxymethyl)aminomethane, 50% isopropanol.

[0086] The nucleic acid products extracted using the methods in Examples 1 to 7 were tested using a micro-ultraviolet spectrophotometer. The purity of the extracted products was evaluated using the 260 / 280 and 260 / 230 ratios. The results are shown in Table 1.

[0087] Table 1 Purity of nucleic acid extracts from Examples 1 to 7

[0088]

[0089] The nucleic acid products extracted using the methods in Examples 1 to 7 were detected using the "Helicobacter pylori 23S rRNA Gene and gyrA Gene Mutation Detection Kit (Fluorescent PCR Method)" (Registration Certificate No.: 20223400137) manufactured by Jiangsu Mole Biotechnology Co., Ltd., with the Ct value of the FAM channel as the reaction. 23S rRNA Gene mutation status is reflected by the Ct value of the VIC channel. gyr Gene mutation status was assessed using the ROX Ct value to reflect Helicobacter pylori production, and the CY5 Ct value to reflect the detection of human internal standards. The results are shown in Table 2 below.

[0090] Table 2. Ct values ​​for nucleic acid extracts from Examples 1 to 7.

[0091]

[0092] The results showed that Example 1 had the best effect, in which the use of lauramide propyl hydroxysulfonate betaine improved the purity of nucleic acid products and increased the yield of Helicobacter pylori nucleic acid.

[0093] Example 8

[0094] Three Hp fecal samples were extracted: Sample 1, Sample 2, and Sample 3.

[0095] The reagents and nucleic acid extraction procedures are the same as in Example 1.

[0096] Example 9

[0097] The reagents, nucleic acid extraction procedures, and samples are consistent with those in Example 8. The difference is:

[0098] Washing solution 2: Sodium acetate 0.5M, NP40 1%, ethanol 75%.

[0099] Example 10

[0100] The reagents, nucleic acid extraction procedures, and samples are consistent with those in Example 8. The difference is:

[0101] Washing solution 2: Sodium acetate 0.5M, Triton X-100 1%, ethanol 75%.

[0102] Example 11

[0103] The reagents, nucleic acid extraction procedures, and samples are consistent with those in Example 8. The difference is:

[0104] Washing solution 2: Sodium acetate 0.5M, guaiacol 1%, ethanol 75%.

[0105] Example 12

[0106] The reagents, nucleic acid extraction procedures, and samples are consistent with those in Example 8. The difference is:

[0107] Washing solution 2: 1M betaine, 75% ethanol.

[0108] The nucleic acid products extracted using the methods in Examples 8 to 12 were tested using a micro-ultraviolet spectrophotometer. The purity of the extracted products was evaluated using the 260 / 280 and 260 / 230 ratios. The results are shown in Table 3.

[0109] Table 3 Purity of nucleic acid extracts from Examples 8 to 12

[0110]

[0111] The nucleic acid products extracted using the methods in Examples 8 to 12 were detected using the "Helicobacter pylori 23S rRNA Gene and gyrA Gene Mutation Detection Kit (Fluorescent PCR Method)" (Registration Certificate No.: 20223400137) manufactured by Jiangsu Mole Biotechnology Co., Ltd., with the Ct value of the FAM channel as the reaction. 23S rRNA Gene mutation status is reflected by the Ct value of the VIC channel. gyr Gene mutation status was assessed using the ROX Ct value to reflect Helicobacter pylori production, and the CY5 Ct value to reflect the detection of human internal standards. The results are shown in Table 4 below.

[0112] Table 4. Ct values ​​for nucleic acid extracts in Examples 8-12

[0113]

[0114] The results showed that Example 8 had the best effect, in which the use of guaiacol improved the purity of nucleic acid products and increased the yield of Helicobacter pylori nucleic acid.

[0115] Example 13

[0116] Eight samples, numbered 1 to 8, were collected. *Helicobacter pylori* (Hp) samples were extracted using the commercially available reagent, QIAamp Fast DNA Stool Mini Kit (manufacturer: QIAGEN), and the method described in Example 1. The extracted products from the eight samples were detected using the *Helicobacter pylori 23S rRNA Gene and gyrA Gene Mutation Detection Kit (Fluorescent PCR Method)* (Registration Certificate No.: 20223400137) manufactured by Jiangsu Mole Biotechnology Co., Ltd. The Ct values ​​of the FAM channel were used to determine the results. 23S rRNA Gene mutation status is reflected by the Ct value of the VIC channel. gyrGene mutation status was assessed using the ROX Ct value to reflect Helicobacter pylori production, and the CY5 Ct value to reflect the detection of human internal standards. The results are shown in Table 5 below.

[0117] Table 58 shows the Ct values ​​of nucleic acid extracts from samples.

[0118]

[0119] The results showed that in the extraction experiments of 8 samples, the positive rate of Helicobacter pylori corresponding to the commercial reagent was 50%, while the positive rate corresponding to the present application was 100%, indicating that the present application can significantly improve the positive detection rate of Helicobacter pylori. In samples where Helicobacter pylori was detected in both reagents, the Ct value of Helicobacter pylori corresponding to the present application was 0.93-2.47 earlier than that of the commercial reagent, which means that the nucleic acid yield of Helicobacter pylori obtained by the present application was 2-4 times that of the commercial reagent.

[0120] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by this application.

Claims

1. A kit for extracting Helicobacter pylori nucleic acid from fecal samples, characterized in that, The kit includes lysis buffer and wash buffer II; The lysis buffer consists of the following reagents: magnetic beads 0.2-4 mg / mL, guanidine isothiocyanate 1-5 M, lauramide propyl hydroxysulfonate betaine 0.1-4% (w / v), potassium chloride 5-15% (w / v), tris(hydroxymethyl)aminomethane 0.1-1 M, and isopropanol 30-60% (w / v). The washing solution II is composed of the following reagents: betaine 0.5-5M, guaiacol 0.2-20% (w / v), and ethanol 75-80% (w / v). The kit also includes sample processing solution, washing solution I, washing solution III, and elution solution; The sample processing solution consists of the following reagents: guanidine isothiocyanate 1-3M, lithium dodecyl sulfate 0.1-20% by mass / volume ratio; The washing solution I is composed of the following reagents: guanidine hydrochloride 1-3M, Tween 20 0.1-10% by mass / volume, and tris(hydroxymethyl)aminomethane 1-1000mM; Washing solution III: 70-80% ethanol (mass-volume ratio); Eluent: Tris(hydroxymethyl)aminomethane 1-50 mM.

2. The Helicobacter pylori nucleic acid extraction kit from fecal samples according to claim 1, characterized in that: The kit also includes magnetic beads, which are carboxylated magnetic beads with a particle size of 100-500 nm.

3. A method for extracting Helicobacter pylori nucleic acid from fecal samples, characterized in that: Extraction was performed using the kit described in claim 2.

4. The method according to claim 3, characterized in that, Includes the following steps: S1. Take a fecal sample, add sample processing solution, and mix thoroughly on a vortex mixer; incubate at 70-80℃; 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 buffer and mix well; incubate at 70-80℃, inverting and mixing several times during the process; after centrifugation, place the sample tube on a magnetic rack and keep it until the liquid is clear. S3. Use a pipette to discard the supernatant, 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 buffer 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 remove the supernatant and keep the sample tube on the magnetic rack until the magnetic beads are dry and free of water. S7. Add elution buffer, vortex to mix, incubate at room temperature, inverting the tube several times during incubation; 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 into a clean, nuclease-free sample tube to obtain the extracted Helicobacter pylori nucleic acid solution.

5. The method according to claim 4, characterized in that, In step S1, the ratio of fecal sample to sample processing solution is (2-5) g: 10 mL.

6. The method according to claim 4, characterized in that, In step S2, the volume ratio of the lysis buffer to the sample to be processed is 5:

3.

7. The use of the kit according to any one of claims 1-2 in the extraction of Helicobacter pylori nucleic acid from fecal samples.

Images