A method for extracting Helicobacter pylori DNA from feces

Abstract

The present invention is applicable to the field of biotechnology and provides a method for extracting Helicobacter pylori DNA from feces, which comprises the following steps: collecting a fresh fecal sample of a patient infected with Helicobacter pylori and placing it in a centrifuge tube; adding a cell lysate to the centrifuge tube and performing homogenization. The cell lysate includes: Tris-HCl, ethylenediaminetetraacetic acid, guanidine isothiocyanate, Triton X-100, isopropanol, sodium dodecyl sulfate, sodium chloride, proteinase K, modified polyethylene glycol, and modified betaine. By adding modified polyethylene glycol to the cell lysate, the capture of free DNA is enhanced, promoting further concentration and enrichment of DNA and improving the subsequent binding efficiency of DNA to magnetic beads. By adding modified betaine, the effects of removing impurities such as proteins and esters are improved, reducing the impurities adsorbed by magnetic beads in the subsequent steps. Modified polyethylene glycol and modified betaine have a synergistic effect and can improve the purity of the extracted DNA.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for extracting Helicobacter pylori DNA from feces. Background Art

[0002] Helicobacter pylori is a spiral-shaped, slightly anaerobic bacterium that can parasitize in the human stomach and be excreted in small amounts with feces. More than half of the world's population is infected with HP. Currently, the diagnosis of Helicobacter pylori mainly uses the C13 breath test, but it has a misdiagnosis rate of about 10%, so it cannot be used as the gold standard for detecting Helicobacter pylori. In addition, C13 has a certain degree of radioactivity and may cause certain harm to the human body.

[0003] Detection of Helicobacter pylori in feces can be used as a detection direction. Fecal sample collection is non-invasive, with high detection throughput, and can adapt to large-scale population screening. However, due to the presence of a large amount of impurities in feces, the efficiency of nucleic acid extraction is affected. The fecal nucleic acid extraction kits currently on the market have low concentration and purity of Helicobacter pylori DNA extracted, and the presence of more impurities will further inhibit PCR amplification. Summary of the invention

[0004] The present invention provides a method for extracting Helicobacter pylori DNA from feces, aiming to solve the above problems.

[0005] The present invention is achieved by a method for extracting Helicobacter pylori DNA from feces, comprising the following steps:

[0006] 1) Collect fresh stool samples from patients infected with Helicobacter pylori and place them in centrifuge tubes;

[0007] 2) adding cell lysis solution to the centrifuge tube for homogenization, wherein the cell lysis solution comprises: 10-200 mM Tris-HCl, 10-50 mM ethylenediaminetetraacetic acid, 3-7 mol / L guanidine isothiocyanate, 2-8% volume fraction of Triton X-100, 30-40% volume fraction of isopropanol, 15-25 g / L sodium dodecyl sulfate, 1-5 g / L sodium chloride, 0.5-1.5 g / L proteinase K, 40-60 g / L modified polyethylene glycol, and 30-50 g / L modified betaine;

[0008] 3) Add magnetic beads to the centrifuge tube, shake the centrifuge tube at 55-65℃ for 8-12 minutes, place the centrifuge tube on the magnetic separation rack for 1-3 minutes, wait until the magnetic beads in the centrifuge tube are completely adsorbed, and discard the solution in the centrifuge tube;

[0009] 4) Remove the centrifuge tube from the magnetic separation rack, add the washing solution, vortex for 5 - 15 min to fully mix the magnetic beads, then place the centrifuge tube on the magnetic separation rack for 1 - 3 min. Wait until the magnetic beads in the tube are completely adsorbed, and discard the solution in the centrifuge tube.

[0010] 5) Add the elution solution to the centrifuge tube, preheat it at 50 - 60 °C in advance, and vortex for 6 - 10 min.

[0011] 6) Transfer the centrifuge tube to the magnetic rack for adsorption for 1 - 3 min, transfer the supernatant to a new centrifuge tube, and store it at -40 to -20 °C.

[0012] Preferably, the cell lysate comprises: 50 - 150 mM Tris-HCl, 20 - 40 mM ethylenediaminetetraacetic acid, 4 - 6 mol / L guanidine isothiocyanate, 3 - 7% (v / v) Triton X-100, 33 - 37% (v / v) isopropanol, 17 - 23 g / L sodium dodecyl sulfate, 2 - 4 g / L sodium chloride, 0.8 - 1.2 g / L proteinase K, 45 - 55 g / L modified polyethylene glycol, 35 - 45 g / L modified betaine.

[0013] Preferably, the cell lysate comprises: 100 mM Tris-HCl, 30 mM ethylenediaminetetraacetic acid, 5 mol / L guanidine isothiocyanate, 5% (v / v) Triton X-100, 35% (v / v) isopropanol, 20 g / L sodium dodecyl sulfate, 3 g / L sodium chloride, 1 g / L proteinase K, 50 g / L modified polyethylene glycol, 40 g / L modified betaine.

[0014] Preferably, the preparation method of the modified polyethylene glycol is as follows: By weight, dissolve 10 - 20 parts of polyethylene glycol in 100 - 120 parts of deionized water, then add 1 - 3 parts of polyaspartic acid, stir for 30 - 50 min, and finally add 2 - 4 parts of 3-amino-1,2-propanediol. Heat to 80 - 90 °C and stir for 1 - 3 h. Extract with dichloromethane, precipitate with anhydrous ether, and vacuum dry to obtain the modified polyethylene glycol. On the one hand, by grafting amino groups onto polyethylene glycol, the reaction activity of polyethylene glycol is improved, the capture of free DNA is enhanced, the DNA is further concentrated and enriched, and the binding efficiency of subsequent DNA to magnetic beads is improved. On the other hand, the modified polyethylene glycol can protect DNA from degradation by nucleases.

[0015] Preferably, the method for preparing the modified betaine is as follows: by weight, 8-14 parts of betaine, 1-2 parts of sodium lignosulfonate, and 1-3 parts of tea saponin are heated to 100-120 °C with stirring for 30-50 min to obtain the modified betaine. The betaine is modified by sodium lignosulfonate and tea saponin, which reduces the surface tension, improves the surface activity of betaine, enhances the effects of removing impurities such as proteins and esters, reduces the impurities adsorbed by the magnetic beads in the subsequent process, and at the same time, the modified betaine reduces the damage of impurities and ions in the system to nucleic acids and protects the nucleic acids.

[0016] Preferably, the magnetic beads are silicon hydroxyl magnetic beads.

[0017] Preferably, the washing solution includes 2-4 M guanidine hydrochloride, 6-14 mM Tris, and 0.3-0.6 M NaCl, and the pH value is 8.

[0018] Preferably, the elution solution includes 20-40 mM tris(hydroxymethyl)aminomethane and 10-20 mM ethylenediaminetetraacetic acid, and the pH value is 8.

[0019] Preferably, the homogenized mixture in step 2 is centrifuged, the supernatant is taken, the modified nano-sepiolite is added to the supernatant, shaken and mixed evenly, centrifuged after standing, and the supernatant is taken for the treatment in step 3.

[0020] Preferably, the method for preparing the modified nano-sepiolite is as follows: Take nano-sepiolite, place it in a muffle furnace and program the temperature to 600-700 °C, keep it at a constant temperature and calcine for 1-2 h. After cooling, add 20-30 vt% hydrochloric acid solution and soak for 3-5 h. Filter to obtain a solid product. After the solid product is rinsed with purified water 3-5 times, it is heated to 500-600 °C, and then water vapor is introduced for pore expansion. Then it is placed in a drying oven for vacuum drying treatment, and then evenly spread on the ground electrode in a low-temperature plasma treatment device. Under the conditions of a power frequency of 20-25 kHz, a working voltage of 25-30 kV, and a discharge power of 80-100 W, it is treated for 20-30 min to obtain the modified nano-sepiolite. First, the nano-sepiolite is calcined to remove the water in different states in the nano-sepiolite structure, making the internal structure loose and porous, increasing the specific surface area. Then, the nano-sepiolite is surface-corroded and modified by hydrochloric acid to form a stable porous structure on the surface of the nano-sepiolite particles. Through water vapor pore expansion, the bamboo charcoal powder becomes more fine and porous, with a larger specific surface area. After being treated by the low-temperature plasma treatment device, the surface of the nano-sepiolite particles is rougher, the specific surface area is further increased, and a three-dimensional network space is formed, which can fully adsorb the impurities in the feces and is beneficial to obtaining high-purity DNA.

[0021] Compared with the prior art, the embodiments of the present application mainly have the following beneficial effects:

[0022] The method for extracting Helicobacter pylori DNA from feces provided by the present invention adds modified polyethylene glycol to the cell lysate to enhance the capture of free DNA, promote further concentration and enrichment of DNA, and improve the subsequent binding efficiency of DNA to magnetic beads. At the same time, the modified polyethylene glycol can protect DNA and prevent it from being degraded by nucleases. By adding modified betaine, the surface activity of betaine is increased, and the effects of removing impurities such as proteins and esters are improved, reducing the impurities adsorbed by magnetic beads in the subsequent process. The modified polyethylene glycol and modified betaine have a synergistic effect and can improve the purity of the extracted DNA. Brief Description of the Drawings

[0023] Figure 1 is a flowchart of a method for extracting Helicobacter pylori DNA from feces provided by the present invention;

[0024] Figure 2 is a flowchart of another method for extracting Helicobacter pylori DNA from feces provided by the present invention. Detailed Description of the Embodiments

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this application belongs; the terms used in the description of the present application in the specification are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "including" and "having" and any variations thereof in the specification and claims of this application and the above description of the drawings are intended to cover non-exclusive inclusion. The terms "first", "second", etc. in the specification and claims of this application or the above drawings are used to distinguish different objects and not to describe a specific order.

[0026] Reference to "embodiment" herein means that a particular feature, structure, or characteristic described in connection with the embodiment can be included in at least one embodiment of this application. The phrase appears in various places in the specification and does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment mutually exclusive with other embodiments. Those skilled in the art will explicitly and implicitly understand that the embodiments described herein can be combined with other embodiments.

[0027] Example 1

[0028] The embodiment of the present invention provides a method for extracting Helicobacter pylori DNA from feces, as Figure 1 shown, including the following steps:

[0029] 1) Collect a fresh fecal sample from a patient infected with Helicobacter pylori and place it in a centrifuge tube;

[0030] 2) Add cell lysate into a centrifuge tube and homogenize it. The cell lysate includes: 10 mM Tris-HCl, 10 mM ethylenediaminetetraacetic acid, 3 mol / L guanidine isothiocyanate, 2% (v / v) Triton X-100, 30% (v / v) isopropanol, 15 g / L sodium dodecyl sulfate, 1 g / L sodium chloride, 0.5 g / L proteinase K, 40 g / L modified polyethylene glycol, 30 g / L modified betaine;

[0031] 3) Add silica hydroxyl magnetic beads into the centrifuge tube, shake the centrifuge tube at 55 °C for 8 min, place the centrifuge tube on a magnetic separation rack for 1 min. Wait until the magnetic beads in the centrifuge tube are completely adsorbed, and discard the solution in the centrifuge tube;

[0032] 4) Take out the centrifuge tube from the magnetic separation rack, add washing solution, vortex for 5 min to fully mix the magnetic beads, then place the centrifuge tube on the magnetic separation rack for 1 min. Wait until the magnetic beads in the tube are completely adsorbed, and discard the solution in the centrifuge tube;

[0033] 5) Add elution buffer into the centrifuge tube, preheat it at 50 °C in advance, and vortex for 6 min;

[0034] 6) Transfer the centrifuge tube to the magnetic rack and adsorb for 1 min, transfer the supernatant to a new centrifuge tube, and store it at -40 °C.

[0035] Among them, the preparation method of the modified polyethylene glycol is as follows: By weight, dissolve 10 parts of polyethylene glycol in 100 parts of deionized water, then add 1 part of polyaspartic acid, stir for 30 min, and finally add 2 parts of 3-amino-1,2-propanediol. Heat to 80 °C and stir for 1 h, extract with dichloromethane, precipitate with anhydrous ether, and dry in vacuum to obtain modified polyethylene glycol.

[0036] Specifically, the preparation method of the modified betaine is as follows: By weight, mix 8 parts of betaine, 1 part of sodium lignosulfonate and 1 part of tea saponin, heat to 100 °C while stirring, and react for 30 min to obtain modified betaine.

[0037] In this example, the washing solution includes 2 M guanidine hydrochloride, 6 mM Tris and 0.3 M NaCl, with a pH value of 8. The elution buffer includes 20 mM tris(hydroxymethyl)aminomethane and 10 mM ethylenediaminetetraacetic acid, with a pH value of 8.

[0038] Example 2

[0039] The embodiment of the present invention provides a method for extracting Helicobacter pylori DNA from feces, as Figure 1 shown, including the following steps:

[0040] 1) Collect a fresh fecal sample from a patient infected with Helicobacter pylori and place it in a centrifuge tube;

[0041] 2) Add cell lysate to the centrifuge tube and homogenize it. The cell lysate includes: 50 mM Tris-HCl, 20 mM ethylenediaminetetraacetic acid, 4 mol / L guanidine isothiocyanate, 3% (v / v) Triton X-100, 33% (v / v) isopropanol, 17 g / L sodium dodecyl sulfate, 2 g / L sodium chloride, 0.8 g / L proteinase K, 45 g / L modified polyethylene glycol, 35 g / L modified betaine;

[0042] 3) Add silica hydroxyl magnetic beads to the centrifuge tube, shake the centrifuge tube at 55 °C for 8 min, place the centrifuge tube on a magnetic separation rack for 1 min. After the magnetic beads in the centrifuge tube are completely adsorbed, discard the solution in the centrifuge tube;

[0043] 4) Take out the centrifuge tube from the magnetic separation rack, add washing solution, vortex for 5 min to fully mix the magnetic beads, then place the centrifuge tube on the magnetic separation rack for 1 min. After the magnetic beads in the tube are completely adsorbed, discard the solution in the centrifuge tube;

[0044] 5) Add elution buffer to the centrifuge tube, preheat it at 50 °C in advance, and vortex for 6 min;

[0045] 6) Transfer the centrifuge tube to the magnetic rack to adsorb for 1 min, transfer the supernatant to a new centrifuge tube, and store it at -40 °C.

[0046] Among them, the preparation method of the modified polyethylene glycol is as follows: By weight, dissolve 10 parts of polyethylene glycol in 100 parts of deionized water, then add 1 part of polyaspartic acid, stir for 30 min, and finally add 2 - 3 parts of 3-amino-1,2-propanediol, heat to 80 °C and stir for 1 h, extract with dichloromethane, precipitate with anhydrous ether, and dry in vacuum to obtain modified polyethylene glycol.

[0047] Specifically, the preparation method of the modified betaine is as follows: By weight, mix 8 parts of betaine, 1 part of sodium lignosulfonate and 1 part of tea saponin, heat to 100 °C while stirring, and react for 30 min to obtain modified betaine.

[0048] In this example, the washing solution includes 2 M guanidine hydrochloride, 6 mM Tris and 0.3 M NaCl, with a pH value of 8, and the elution buffer includes 20 mM tris(hydroxymethyl)aminomethane and 10 mM ethylenediaminetetraacetic acid, with a pH value of 8.

[0049] Example 3

[0050] The embodiment of the present invention provides a method for extracting Helicobacter pylori DNA from feces, as Figure 1As shown, it includes the following steps:

[0051] 1) Collect fresh fecal samples from patients infected with Helicobacter pylori and place them in centrifuge tubes;

[0052] 2) Add cell lysate to the centrifuge tube and homogenize. The cell lysate includes: 100 mM Tris-HCl, 30 mM ethylenediaminetetraacetic acid, 5 mol / L guanidine isothiocyanate, 5% (v / v) Triton X-100, 35% (v / v) isopropanol, 20 g / L sodium dodecyl sulfate, 3 g / L sodium chloride, 1 g / L proteinase K, 50 g / L modified polyethylene glycol, 40 g / L modified betaine;

[0053] 3) Add silica hydroxyl magnetic beads to the centrifuge tube, shake the centrifuge tube at 60 °C for 10 min, place the centrifuge tube on a magnetic separation rack for 2 min. After the magnetic beads in the centrifuge tube are completely adsorbed, discard the solution in the centrifuge tube;

[0054] 4) Take out the centrifuge tube from the magnetic separation rack, add washing solution, vortex and shake for 10 min to fully mix the magnetic beads, then place the centrifuge tube on the magnetic separation rack for 2 min. After the magnetic beads in the tube are completely adsorbed, discard the solution in the centrifuge tube;

[0055] 5) Add elution buffer to the centrifuge tube, preheat it at 55 °C in advance, and vortex and shake for 8 min;

[0056] 6) Transfer the centrifuge tube to the magnetic rack and adsorb for 2 min, transfer the supernatant to a new centrifuge tube, and store it at -30 °C.

[0057] Among them, the preparation method of the modified polyethylene glycol is as follows: By weight, dissolve 15 parts of polyethylene glycol in 110 parts of deionized water, then add 2 parts of polyaspartic acid, stir for 40 min, and finally add 3 parts of 3-amino-1,2-propanediol, heat to 85 °C and stir for 2 h, extract with dichloromethane, precipitate with anhydrous ether, and vacuum dry to obtain modified polyethylene glycol.

[0058] Specifically, the preparation method of the modified betaine is as follows: By weight, mix 11 parts of betaine, 1.5 parts of sodium lignosulfonate and 2 parts of tea saponin, heat to 110 °C while stirring, and react for 40 min to obtain modified betaine.

[0059] In this example, the washing solution includes 3 M guanidine hydrochloride, 10 mM Tris and 0.45 M NaCl, with a pH value of 8, and the elution buffer includes 30 mM tris(hydroxymethyl)aminomethane and 15 mM ethylenediaminetetraacetic acid, with a pH value of 8.

[0060] Example 4

[0061] An embodiment of the present invention provides a method for extracting Helicobacter pylori DNA from feces, as Figure 1 shown, which includes the following steps:

[0062] 1) Collect fresh fecal samples from patients infected with Helicobacter pylori and place them in a centrifuge tube;

[0063] 2) Add cell lysate to the centrifuge tube and homogenize. The cell lysate includes: 150 mM Tris-HCl, 40 mM ethylenediaminetetraacetic acid, 6 mol / L guanidine isothiocyanate, 7% (v / v) Triton X-100, 37% (v / v) isopropanol, 23 g / L sodium dodecyl sulfate, 4 g / L sodium chloride, 1.2 g / L proteinase K, 55 g / L modified polyethylene glycol, 45 g / L modified betaine;

[0064] 3) Add silica hydroxyl magnetic beads to the centrifuge tube, shake the centrifuge tube at 65 °C for 12 min, place the centrifuge tube on a magnetic separation rack for 3 min. After the magnetic beads in the centrifuge tube are completely adsorbed, discard the solution in the centrifuge tube;

[0065] 4) Take out the centrifuge tube from the magnetic separation rack, add washing solution, vortex and shake for 15 min to fully mix the magnetic beads, then place the centrifuge tube on the magnetic separation rack for 3 min. After the magnetic beads in the tube are completely adsorbed, discard the solution in the centrifuge tube;

[0066] 5) Add elution buffer to the centrifuge tube, preheat it at 60 °C in advance, and vortex and shake for 10 min;

[0067] 6) Transfer the centrifuge tube to the magnetic rack and adsorb for 3 min, transfer the supernatant to a new centrifuge tube, and store it at -20 °C.

[0068] Among them, the preparation method of the modified polyethylene glycol is as follows: By weight, dissolve 20 parts of polyethylene glycol in 120 parts of deionized water, then add 3 parts of polyaspartic acid, stir for 50 min, and finally add 4 parts of 3-amino-1,2-propanediol, heat to 90 °C and stir for 3 h, extract with dichloromethane, precipitate with anhydrous ether, and vacuum dry to obtain modified polyethylene glycol.

[0069] Specifically, the preparation method of the modified betaine is as follows: By weight, mix 14 parts of betaine, 2 parts of sodium lignosulfonate and 3 parts of saponin, heat to 120 °C while stirring, and react for 50 min to obtain modified betaine.

[0070] In this embodiment, the washing solution includes 4 M guanidine hydrochloride, 14 mM Tris and 0.6 M NaCl, with a pH value of 8, and the elution buffer includes 40 mM tris(hydroxymethyl)aminomethane and 20 mM ethylenediaminetetraacetic acid, with a pH value of 8.

[0071] Example 5

[0072] An embodiment of the present invention provides a method for extracting Helicobacter pylori DNA from feces, as Figure 1 shown, which includes the following steps:

[0073] 1) Collect a fresh fecal sample from a patient infected with Helicobacter pylori and place it in a centrifuge tube;

[0074] 2) Add cell lysate to the centrifuge tube and homogenize it. The cell lysate includes: 200 mM Tris-HCl, 50 mM ethylenediaminetetraacetic acid, 7 mol / L guanidine isothiocyanate, 8% (v / v) Triton X-100, 40% (v / v) isopropanol, 25 g / L sodium dodecyl sulfate, 5 g / L sodium chloride, 1.5 g / L proteinase K, 60 g / L modified polyethylene glycol, 50 g / L modified betaine;

[0075] 3) Add silica hydroxyl magnetic beads to the centrifuge tube, shake the centrifuge tube at 65 °C for 12 min, place the centrifuge tube on a magnetic separation rack for 3 min. Wait until the magnetic beads in the centrifuge tube are completely adsorbed, and discard the solution in the centrifuge tube;

[0076] 4) Take out the centrifuge tube from the magnetic separation rack, add washing solution, vortex and shake for 15 min to fully mix the magnetic beads. Then place the centrifuge tube on the magnetic separation rack for 3 min. Wait until the magnetic beads in the tube are completely adsorbed, and discard the solution in the centrifuge tube;

[0077] 5) Add elution solution to the centrifuge tube, preheat it at 60 °C in advance, and vortex and shake for 10 min;

[0078] 6) Transfer the centrifuge tube to the magnetic rack and adsorb for 3 min. Transfer the supernatant to a new centrifuge tube and store it at -20 °C.

[0079] Among them, the preparation method of the modified polyethylene glycol is as follows: By weight, dissolve 20 parts of polyethylene glycol in 120 parts of deionized water, then add 3 parts of polyaspartic acid, stir for 50 min, and finally add 4 parts of 3-amino-1,2-propanediol. Heat to 90 °C and stir for 3 h. Extract with dichloromethane, precipitate with anhydrous ether, and vacuum dry to obtain modified polyethylene glycol.

[0080] Specifically, the preparation method of the modified betaine is as follows: By weight, mix 14 parts of betaine, 2 parts of sodium lignosulfonate and 3 parts of saponin. Stir and heat to 120 °C and react for 50 min to obtain modified betaine.

[0081] In this embodiment, the washing solution comprises 4M guanidine hydrochloride, 14 mM Tris, and 0.6M NaCl, with a pH value of 8, and the elution solution comprises 40 mM tris(hydroxymethyl)aminomethane and 20 mM ethylenediaminetetraacetic acid, with a pH value of 8.

[0082] Example 6

[0083] An embodiment of the present invention provides a method for extracting Helicobacter pylori DNA from feces, as Figure 2 shown, comprising the following steps:

[0084] 1) Collect a fresh fecal sample from a patient infected with Helicobacter pylori and place it in a centrifuge tube;

[0085] 2) Add a cell lysate to the centrifuge tube and homogenize it. The cell lysate comprises: 5 mol / L guanidine isothiocyanate, 5% (v / v) Triton X-100, 35% (v / v) isopropanol, 20 g / L sodium dodecyl sulfate, 3 g / L sodium chloride, 1 g / L proteinase K, 50 g / L modified polyethylene glycol, 40 g / L modified betaine;

[0086] 3) Centrifuge the homogenized mixture, take the supernatant, add modified nano-sepiolite to the supernatant, shake and mix well, centrifuge after standing, and take the supernatant and place it in another centrifuge tube;

[0087] 4) Add silica hydroxyl magnetic beads to the other centrifuge tube, shake the centrifuge tube at 60 °C for 10 min, place the centrifuge tube on a magnetic separation rack for 2 min, wait until the magnetic beads in the centrifuge tube are completely adsorbed, and discard the solution in the centrifuge tube;

[0088] 5) Take the centrifuge tube out of the magnetic separation rack, add the washing solution, vortex and shake for 10 min to fully mix the magnetic beads, then place the centrifuge tube on the magnetic separation rack for 2 min, wait until the magnetic beads in the tube are completely adsorbed, and discard the solution in the centrifuge tube;

[0089] 6) Add the elution solution to the centrifuge tube, preheat it at 55 °C in advance, and vortex and shake for 8 min;

[0090] 7) Transfer the centrifuge tube to the magnetic rack and adsorb for 2 min, transfer the supernatant to a new centrifuge tube, and store it at -30 °C.

[0091] Among them, the preparation method of the modified polyethylene glycol is as follows: By weight, dissolve 15 parts of polyethylene glycol in 110 parts of deionized water, then add 2 parts of polyaspartic acid, stir for 40 min, finally add 3 parts of 3-amino-1,2-propanediol, raise the temperature to 85 °C and stir for 2 h, extract with dichloromethane, precipitate with anhydrous ether, and vacuum dry to obtain the modified polyethylene glycol.

[0092] Specifically, the preparation method of the modified betaine is as follows: by weight, 11 parts of betaine, 1.5 parts of sodium lignosulfonate and 2 parts of saponin are heated to 110 °C with stirring and reacted for 40 min to obtain the modified betaine.

[0093] In this example, the washing solution includes 3M guanidine hydrochloride, 10 mM Tris and 0.45M NaCl, with a pH value of 8, and the elution solution includes 30 mM tris(hydroxymethyl)aminomethane and 15 mM ethylenediaminetetraacetic acid, with a pH value of 8.

[0094] Comparative Example 1: Compared with Example 3, the modified polyethylene glycol is replaced with ordinary polyethylene glycol.

[0095] Comparative Example 2: Compared with Example 3, the modified betaine is replaced with ordinary betaine.

[0096] Comparative Example 3: Compared with Example 3, the modified polyethylene glycol is replaced with ordinary polyethylene glycol and the modified betaine is replaced with ordinary betaine.

[0097] Comparative Example 4: Commercially available chemical kits for extracting fecal DNA are used for extraction.

[0098] Test

[0099] Fresh fecal samples of patients infected with Helicobacter pylori are collected and divided into 10 parts. The DNA is extracted by the methods of Examples 1-6 and Comparative Examples 1-4 respectively. The extracted DNA is tested for concentration and purity by an ultra-micro spectrophotometer, and the Helicobacter pylori gene amplification is detected. The results are shown in Table 1 below:

[0100] Table 1 Results of DNA concentration and purity test and Helicobacter pylori gene amplification detection

[0101]

[0102] It can be seen from the above results that the method for extracting Helicobacter pylori DNA from feces used in the present invention has relatively high concentration and purity of the extracted DNA, and a relatively small amplification Ct value. In particular, by adding modified polyethylene glycol and modified betaine, the two have a synergistic effect and can improve the DNA extraction efficiency.

[0103] It should be noted that for the foregoing embodiments, for the sake of simple description, they are all expressed as a series of action combinations. However, those skilled in the art should know that the present invention is not limited by the described action sequence, because according to the present invention, certain steps may be performed in other sequences or simultaneously. Secondly, those skilled in the art should also know that the embodiments described in the specification are all preferred embodiments, and the actions and modules involved are not necessarily essential to the present invention.

[0104] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting the protection scope of the invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on these embodiments, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those of ordinary skill in the art can still, without conflict and without creative efforts, combine, add or delete the features in the embodiments of the present invention according to the circumstances, or make other adjustments, so as to obtain different technical solutions that essentially do not deviate from the concept of the present invention, and these technical solutions also fall within the scope of protection of the present invention.

Claims

1. A method for extracting Helicobacter pylori DNA from feces, characterized in that: The steps include: Collect fresh stool samples from patients infected with Helicobacter pylori and place them in centrifuge tubes; Adding cell lysis solution into a centrifuge tube for homogenization, the cell lysis solution comprises: 10-200 mM Tris-HCl, 10-50 mM ethylenediaminetetraacetic acid, 3-7 mol / L guanidine isothiocyanate, 2-8% volume fraction of Triton X-100, 30-40% volume fraction of isopropanol, 15-25 g / L sodium dodecyl sulfate, 1-5 g / L sodium chloride, 0.5-1.5 g / L proteinase K, 40-60 g / L modified polyethylene glycol, and 30-50 g / L modified betaine; Add magnetic beads to the centrifuge tube, shake the centrifuge tube at 55-65℃ for 8-12 minutes, place the centrifuge tube on the magnetic separation rack for 1-3 minutes, wait until the magnetic beads in the centrifuge tube are completely adsorbed, and discard the solution in the centrifuge tube; Take the centrifuge tube out of the magnetic separation rack, add washing solution, vortex and shake for 5-15 minutes to fully mix the magnetic beads, then place the centrifuge tube on the magnetic separation rack for 1-3 minutes, wait until the magnetic beads in the tube are completely adsorbed, and discard the solution in the centrifuge tube; Add eluent to the centrifuge tube, preheat to 50-60℃, and vortex for 6-10 minutes; Transfer the centrifuge tube to a magnetic rack and adsorb for 1-3 minutes. Transfer the supernatant to a new centrifuge tube and store at -40 to -20°C. The preparation method of the modified polyethylene glycol is as follows: by weight, 10-20 parts of polyethylene glycol are dissolved in 100-120 parts of deionized water, then 1-3 parts of polyaspartic acid are added, stirred for 30-50 minutes, and finally 2-4 parts of 3-amino-1,2-propylene glycol are added, the temperature is raised to 80-90° C. and stirred for 1-3 hours, extracted with dichloromethane, precipitated with anhydrous ether, and vacuum dried to obtain the modified polyethylene glycol; The preparation method of the modified betaine is as follows: by weight, 8-14 parts of betaine, 1-2 parts of sodium lignin sulfonate and 1-3 parts of tea saponin are heated to 100-120° C. while stirring, and reacted for 30-50 minutes to obtain the modified betaine.

2. The method for extracting Helicobacter pylori DNA from feces according to claim 1, characterized in that The cell lysis solution comprises: 50-150 mM Tris-HCl, 20-40 mM ethylenediaminetetraacetic acid, 4-6 mol / L guanidine isothiocyanate, 3-7% volume fraction of Triton X-100, 33-37% volume fraction of isopropanol, 17-23 g / L sodium dodecyl sulfate, 2-4 g / L sodium chloride, 0.8-1.2 g / L proteinase K, 45-55 g / L modified polyethylene glycol, and 35-45 g / L modified betaine.

3. The method for extracting Helicobacter pylori DNA from feces according to claim 2, characterized in that The cell lysis solution comprises: 100 mM Tris-HCl, 30 mM ethylenediaminetetraacetic acid, 5 mol / L guanidine isothiocyanate, 5% volume fraction of Triton X-100, 35% volume fraction of isopropanol, 20 g / L sodium dodecyl sulfate, 3 g / L sodium chloride, 1 g / L proteinase K, 50 g / L modified polyethylene glycol, and 40 g / L modified betaine.

4. The method for extracting Helicobacter pylori DNA from feces according to claim 1, characterized in that The magnetic beads are silanol magnetic beads.

5. The method for extracting Helicobacter pylori DNA from feces according to claim 1, characterized in that: The washing solution includes 2-4 M guanidine hydrochloride, 6-14 mM Tris and 0.3-0.6 M NaCl, and the pH value is 8.

6. The method for extracting Helicobacter pylori DNA from feces according to claim 1, characterized in that: The eluent includes 20-40 mM tris(hydroxymethyl)aminomethane and 10-20 mM ethylenediaminetetraacetic acid, and has a pH value of 8.

7. The method for extracting Helicobacter pylori DNA from feces according to claim 1, characterized in that: The mixed solution homogenized in step 2 is centrifuged, and the supernatant is taken. The modified nano-sepiolite is added to the supernatant, and the mixture is shaken and mixed. The mixture is allowed to stand and then centrifuged. The supernatant is taken and processed in step 3.

8. The method for extracting Helicobacter pylori DNA from feces according to claim 7, characterized in that: The preparation method of the modified nano sepiolite is as follows: taking nano sepiolite, placing it in a muffle furnace and heating it to 600-700°C, calcining it at a constant temperature for 1-2h, adding 20-30vt% hydrochloric acid solution after cooling, soaking it for 3-5h, filtering to obtain a solid product, washing the solid product with purified water for 3-5 times, heating it to 500-600°C, introducing water vapor to expand the pores, placing it in a drying oven for reduced pressure drying treatment, and then evenly spreading it on a ground electrode in a low-temperature plasma treatment device, treating it for 20-30min under the conditions of a power frequency of 20-25kHz, a working voltage of 25-30kV, and a discharge power of 80-100W to obtain the modified nano sepiolite.

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