A safe, rapid, high-throughput kit for extracting fecal genomic dna and methods of use

Abstract

This invention relates to the field of fecal genomics kit technology, and particularly to a safe, rapid, and high-throughput kit for extracting fecal genomes and its usage method. The kit includes a sample preservation solution, magnetic beads, a lysis buffer, a washing buffer 1, a washing buffer 2, and an elution buffer. The sample preservation solution is composed of guanidine isothiocyanate, sodium citrate, Triton X-100, Tween 20, propidium iodide, and purified water. The magnetic beads are hydroxyl magnetic beads. The lysis buffer is composed of guanidine hydrochloride, Tris base, Tris hydrochloric acid, Triton X-100, isopropanol, and purified water. Washing buffer 1 is composed of guanidine hydrochloride solution and isopropanol. Washing buffer 2 is composed of anhydrous ethanol and purified water. This kit can easily extract the genome from fecal samples, is simple to operate, safe and non-toxic, and can extract multiple samples at once, enabling rapid diagnosis and filling the gap in the current market for automated high-throughput kits for extracting genomes from fecal samples.

Description

Technical Field

[0001] This invention relates to the field of fecal genomics kit technology, and in particular to a safe, rapid, and high-throughput fecal genomics extraction kit and its usage method. Background Technology

[0002] Research indicates that the gut microbiota is crucial for human health. Gut microbes are closely related to various diseases, and multiple health indicators can be detected from trace amounts of feces, suggesting high-risk diseases such as polyps, colorectal cancer, hypertension, and Parkinson's disease. For diseases such as intractable diarrhea, autism, depression, obesity, allergies, and tumors, the use of microbiota transplantation technology, allowing healthy microbiota to "defeat" sub-healthy microbiota, can also have unexpected effects. As research continues, scientists have also discovered that the occurrence of various cancers is closely related to subtle changes in the gut microbiota. However, removing impurities from feces is difficult. Fecal samples contain various soluble impurities, such as bilirubin, bile salts, humic acid, and plant-derived polysaccharides, which are difficult to wash and remove. Fecal samples also have fewer exfoliated cells and lower microbial content; some samples even contain Gram-positive bacteria with thick cell walls, and incomplete lysis or elution can lead to low DNA yield and low purity.

[0003] Due to the unique nature of the gut environment, only a small fraction of gut microbiota can be cultured in vitro, severely hindering our understanding of their structure and diversity. With the continuous development of molecular biology techniques, it is now possible to study the gut microbiota directly at the DNA level. However, the complex composition of feces (containing polysaccharides, bile salts, humic substances, bile acids, and other PCR inhibitors) and the diverse types of bacteria in the gut, with varying sensitivities to different extraction methods, lead to inconsistent extraction results and efficiencies. This, in turn, results in unsatisfactory or even biased subsequent analyses.

[0004] Currently, there are many methods for extracting fecal microbial DNA, commonly including mechanical methods such as magnetic bead extraction and freeze-thaw extraction; chemical methods such as SDS extraction and phenol extraction; enzymatic methods (such as lysozyme and proteinase K); and kit-based methods. Combinations of these methods are the standard laboratory approach for extracting fecal DNA. Generally, SDS, lysozyme, and ultrasound are used to lyse cells and release DNA, followed by extraction with phenol-chloroform or magnetic beads, and finally precipitation with anhydrous ethanol. Most kit-based methods are cumbersome, requiring manual extraction, and some methods use reagents with a degree of toxicity, resulting in high costs and long processing times when handling large numbers of samples.

[0005] Therefore, we propose a safe, rapid, and high-throughput fecal genome extraction kit and its usage method to address the aforementioned issues. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by proposing a safe, rapid, and high-throughput fecal genome extraction kit and its usage method.

[0007] A safe, rapid, and high-throughput kit for extracting fecal genomes includes a sample preservation solution, magnetic beads, lysis buffer, rinsing buffer 1, rinsing buffer 2, and elution buffer.

[0008] The sample preservation solution consists of guanidine isothiocyanate, sodium citrate, Triton X-100, Tween 20, propidium iodide and purified water;

[0009] The magnetic beads are hydroxyl magnetic beads;

[0010] The lysis buffer consists of guanidine hydrochloride, Tris base, Tris hydrochloric acid, Triton X-100, isopropanol and purified water;

[0011] The rinsing solution 1 is composed of guanidine hydrochloride solution and isopropanol;

[0012] The rinsing solution 2 is composed of anhydrous ethanol and purified water;

[0013] The eluent is purified water.

[0014] Preferably, the composition and content of the sample preservation solution are as follows:

[0015] Guanidine isothiocyanate 43%-48%

[0016] Sodium citrate 0.8%-1.2%

[0017] Qulaton X-100 6%-10%

[0018] Tween 20 0.3%-0.5%

[0019] Propidium iodide 1%-2%

[0020] The remainder is purified water.

[0021] Preferably, the composition and content of the lysis buffer are as follows:

[0022] Guanidine hydrochloride 40%-44%

[0023] Tris base 0.4%-0.8%

[0024] Tris hydrochloric acid 0.6%-1%

[0025] Qulaton X-100 3%-5%

[0026] Isopropanol 18%-22%

[0027] Polyoxyethylene ether 4%-7%

[0028] The remainder is purified water.

[0029] Preferably, the rinsing solution 1 is a mixture of guanidine hydrochloride solution and isopropanol, wherein the concentration of guanidine hydrochloride solution is 7 mol / L and the volume ratio of guanidine hydrochloride solution to isopropanol is (3-5):1.

[0030] Preferably, the mass fraction of anhydrous ethanol in the rinsing solution 2 is 50%-75%.

[0031] Preferably, the magnetic beads are silicon dioxide magnetic beads.

[0032] Preferably, a method for using a safe, rapid, and high-throughput fecal genome extraction kit includes the following steps:

[0033] Step 1, Sample Preparation: Add 0.3-0.7 cm of [unspecified ingredient] to 4 ml of fecal preservation solution. 3 Take a human fecal sample of a certain volume, vortex to mix, and let stand for 5-10 minutes.

[0034] Step 2, Nucleic Acid Extraction Stage:

[0035] (1) Reagents are listed as follows: Plate 1: lysis buffer; Plate 2: magnetic beads and rinsing solution 1; Plate 3: rinsing solution 2; Plate 4: rinsing solution 2; Plate 5: rinsing solution 2; Plate 6: elution buffer.

[0036] (2) Add 300 μl of the supernatant of the sample treated in step 1 to the first plate position;

[0037] (3) Insert the magnetic rod sleeve into the second plate position;

[0038] (4) Extract nucleic acid using an automated nucleic acid extractor. Select the program and run it. After the automated program is completed, remove the nucleic acid sample from the 6th plate.

[0039] Preferably, when adding the supernatant of the sample after treatment in step 1 to the first plate position, the precipitate should not be removed.

[0040] Preferably, the program is selected and run within 1 hour after sample addition.

[0041] The beneficial effects of this invention are:

[0042] The kit of the present invention can easily extract the genome from fecal samples, and is simple to operate, safe and non-toxic. It can extract multiple samples at once, enabling rapid diagnosis and filling the gap in the current market for automated high-throughput extraction kits of genomes from fecal samples. Detailed Implementation

[0043] The present invention will be further explained below with reference to specific embodiments.

[0044] Example 1

[0045] A safe, rapid, and high-throughput kit for extracting fecal genomes includes the following components and their concentrations:

[0046] Sample preservation solution:

[0047] Guanidine isothiocyanate 23 g

[0048] 22 ml of purified water

[0049] Sodium citrate 0.8 g

[0050] Triton X-100 4 g

[0051] Tween 20 0.2 g

[0052] Propidium iodide 0.8 g

[0053] lysis buffer:

[0054] Guanidine hydrochloride 22 g

[0055] Tris base 0.3 g

[0056] Tris hydrochloric acid 0.4 g

[0057] 14 ml of purified water

[0058] Triton X-100 2 g

[0059] 10 g of isopropanol

[0060] 3 g of polyoxyethylene ether

[0061] Rinse solution 1:

[0062] 7M Guanidine Hydrochloride 30 ml

[0063] 10 ml of isopropanol

[0064] Rinse solution 2:

[0065] 16 ml of purified water

[0066] 24 g of anhydrous ethanol

[0067] Eluent: Purified water.

[0068] Example 2

[0069] A safe, rapid, and high-throughput kit for extracting fecal genomes includes the following components and their concentrations:

[0070] Sample preservation solution:

[0071] Guanidine isothiocyanate 21.5 g

[0072] 24.45 ml of purified water

[0073] Sodium citrate 0.4g

[0074] Triton X-100 3 g

[0075] Tween 20 0.15 g

[0076] Propidium iodide 0.5 g

[0077] lysis buffer:

[0078] Guanidine hydrochloride 20 g

[0079] Tris base 0.2 g

[0080] Tris hydrochloric acid 0.3 g

[0081] 17 ml of purified water

[0082] Triton X-100 1.5 g

[0083] Isopropanol 9 g

[0084] 2 g of polyoxyethylene ether

[0085] Rinse solution 1:

[0086] 7M Guanidine Hydrochloride 50 ml

[0087] 10 ml of isopropanol

[0088] Rinse solution 2:

[0089] 10 ml of purified water

[0090] 30 g of anhydrous ethanol

[0091] Eluent: Purified water.

[0092] Example 3

[0093] A safe, rapid, and high-throughput kit for extracting fecal genomes includes the following components and their concentrations:

[0094] Sample preservation solution:

[0095] Guanidine isothiocyanate 24 g

[0096] 19.15 ml of purified water

[0097] Sodium citrate 0.6 g

[0098] Triton X-100 5 g

[0099] Tween 20 0.25 g

[0100] 1 g of propidium iodide

[0101] lysis buffer:

[0102] Guanidine hydrochloride 22 g

[0103] Tris base 0.4 g

[0104] Tris hydrochloric acid 5 g

[0105] 5.6 ml of purified water

[0106] Triton X-100 2.5 g

[0107] Isopropanol 11 g

[0108] Polyoxyethylene ether 3.5 g

[0109] Rinse solution 1:

[0110] 7M Guanidine Hydrochloride 40 ml

[0111] 10 ml of isopropanol

[0112] Rinse solution 2:

[0113] 20 ml of purified water

[0114] 20 g of anhydrous ethanol

[0115] Eluent: Purified water.

[0116] In Examples 1-3, the magnetic beads were selected from silicon dioxide magnetic beads.

[0117] The extraction instrument was a fully automated nucleic acid extractor developed by Xingchun (Changzhou) Biotechnology Co., Ltd., model Purfirer96; the total extraction time was 45 minutes.

[0118] Extracting the genome from a fecal sample includes the following steps:

[0119] 1. Reagent preparation: Prepare the solutions required for the experiment according to Example 1, including sample preservation solution, lysis buffer, wash buffer 1, wash buffer 2, and elution buffer;

[0120] 2. Sample preparation:

[0121] Add 0.5 cm to 4 ml of fecal preservation solution. 3 Take a human fecal sample of a certain volume, vortex to mix, and let stand for 5-10 minutes.

[0122] 3. Nucleic acid extraction stage:

[0123] Reagents are listed as follows: Plate 1: lysis buffer; Plate 2: magnetic beads and rinse buffer 1; Plate 3: rinse buffer 2; Plate 4: rinse buffer 2; Plate 5: rinse buffer 2; Plate 6: elution buffer.

[0124] Add 300 μl of the supernatant of the sample treated in (1) to the first plate position. Note: Do not remove the precipitate when transferring the supernatant, otherwise the DNA purity may be reduced.

[0125] Insert the magnetic rod sleeve into the second plate position. Note: Please run the program within 1 hour after adding the sample.

[0126] Select the program and run it. After the automated program finishes, remove the nucleic acid sample from plate 6 for the next experiment.

[0127] 4. Subsequent DNA testing of the sample:

[0128] Purity testing: The purity of nucleic acid samples was tested using Nanodrop.

[0129] Turn on the ultraviolet spectrophotometer and the corresponding computer software;

[0130] Use a pipette to draw 2 ml of deionized water onto the UV spectrophotometer for calibration and zeroing. After calibration, use clean tissue paper to blot out the deionized water.

[0131] Use a pipette to draw 2 μl of nucleic acid sample and spot it onto the UV spectrophotometer. Click "Measure" to display the corresponding data and curves, thus completing the NanoDrop measurement of this nucleic acid sample.

[0132] Table 1. Results of genomic samples extracted by UV spectrophotometry

[0133]

[0134] Concentration detection: The extracted DNA was quantitatively determined using the dsDNA HS Assay Kit for Qubit (StarPure Biotechnology, catalog number NK901-01).

[0135] Preparation of working solution: The working solution ratio is 1:200, that is, 1 μl dsDNA Reagent (component A) plus 199 μl dsDNA HS buffer (component B) and mixed well (this is one working solution; N working solutions need to be prepared for measuring N samples).

[0136] After the power-on self-test, click on DNA on the screen to enter the next interface, select DNA HS, and follow the on-screen prompts to first create a standard curve.

[0137] To begin measuring sample concentration: Take 1 μl of sample and add it to the prepared working solution, making a total of 200 μl. Mix well and let stand in the dark for 2 minutes before testing. (Note: Select the actual sample volume.) The test will be completed in a few seconds, and two circles will be displayed on the screen. The larger circle represents the sample concentration, and the smaller circle represents the actual concentration after dilution.

[0138] Table 2. Results of genomic samples extracted by Qubit detection

[0139]

[0140] Quantitative fluorescence detection:

[0141] Five μl of each sample was used as a template for quantitative real-time PCR detection of the human internal reference gene and the bacterial 16S gene. The extracted DNA samples were analyzed using Kangwei Century qPCR reagent (catalog number CW0957), with primers for the human internal reference gene B-actin and the bacterial 16S internal reference gene.

[0142] Table 3. B-actin primer sequence information

[0143]

[0144] Table 4. Primer sequence information for the bacterial 16S internal reference gene

[0145]

[0146] DNA nucleic acid extracted from fecal preservation solution without fecal sample was used as a negative control. The reaction solution system was prepared according to the product instructions and dispensed into 8-tube quantitative PCR tubes.

[0147] Ultra SYBR Mixture: 10 μl

[0148] Bactin-F / 341F (10μM): 0.4 μl

[0149] Bactin-R / 517R (10μM): 0.4 μl

[0150] RNase-Free ddH2O: 4.2 μl

[0151] Add 5 μl of the nucleic acid sample extracted in step 3, mix well, and centrifuge briefly;

[0152] Turn on the PCR instrument and set the program parameters as shown in Table 3 below;

[0153] Table 5 PCR Procedure

[0154]

[0155] Set the channel and sample information, and set the reaction system to 20 μl; select the fluorescence channel: SYBR channel.

[0156] Once the settings are complete, save the file and run the reaction program; after running, analyze the results.

[0157] Table 6. Quantitative Real-Time PCR Detection of Genomic Samples from Feces

[0158]

[0159] Experimental results: See Tables 1, 2, and 6. Tables 1 and 2 show that the genome extraction concentration and purity of the kit of this invention are better than those of a kit from a domestic company. Table 6 shows that the concentration of the human internal reference gene B-actin and the bacterial 16S internal reference gene extracted by this invention is higher (lower Ct value) than those extracted by the kit from a domestic company.

[0160] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A safe, rapid, and high-throughput kit for extracting fecal genomes, characterized in that, Includes sample preservation solution, magnetic beads, lysis buffer, wash buffer 1, wash buffer 2, and elution buffer; The sample preservation solution consists of guanidine isothiocyanate, sodium citrate, Triton X-100, Tween 20, propidium iodide and purified water; The magnetic beads are hydroxyl magnetic beads; The lysis buffer consists of guanidine hydrochloride, Tris base, Tris hydrochloric acid, Triton X-100, isopropanol and purified water; The rinsing solution 1 is composed of guanidine hydrochloride solution and isopropanol; The rinsing solution 2 is composed of anhydrous ethanol and purified water; The eluent is purified water.

2. The safe, rapid, and high-throughput fecal genome extraction kit according to claim 1, characterized in that, The composition and content of the sample preservation solution are as follows: Guanidine isothiocyanate 43%-48% Sodium citrate 0.8%-1.2% Qulaton X-100 6%-10% Tween 20 0.3%-0.5% Propidium iodide 1%-2% The remainder is purified water.

3. The safe, rapid, and high-throughput fecal genome extraction kit according to claim 1, characterized in that, The composition and content of the lysis buffer are as follows: Guanidine hydrochloride 40%-44% Tris base 0.4%-0.8% Tris hydrochloric acid 0.6%-1% Qulaton X-100 3%-5% Isopropanol 18%-22% Polyoxyethylene ether 4%-7% The remainder is purified water.

4. The safe, rapid, and high-throughput fecal genome extraction kit according to claim 1, characterized in that, The rinsing solution 1 is a mixture of guanidine hydrochloride solution and isopropanol, wherein the concentration of guanidine hydrochloride solution is 7 mol / L and the volume ratio of guanidine hydrochloride solution to isopropanol is (3-5):

1.

5. The safe, rapid, and high-throughput fecal genome extraction kit according to claim 1, characterized in that, The mass fraction of anhydrous ethanol in the rinsing solution 2 is 50%-75%.

6. The safe, rapid, and high-throughput fecal genome extraction kit according to claim 1, characterized in that, The magnetic beads are silicon dioxide magnetic beads.

7. A method of using the safe, rapid, high-throughput fecal genome extraction kit according to any one of claims 1-6, characterized in that, Includes the following steps: Step 1, sample preparation: add 0.3-0.7 cm 3 volume of human fecal sample into 4 ml of fecal preservative, vortex to mix, and let stand for 5-10 minutes; Step 2, Nucleic Acid Extraction Stage: (1) Reagents are listed as follows: Plate 1: lysis buffer; Plate 2: magnetic beads and rinsing solution 1; Plate 3: rinsing solution 2; Plate 4: rinsing solution 2; Plate 5: rinsing solution 2; Plate 6: elution buffer. (2) Add 300 μl of the supernatant of the sample treated in step 1 to the first plate position; (3) Insert the magnetic rod sleeve into the second plate position; (4) Extract nucleic acid using an automated nucleic acid extractor. Select the program and run it. After the automated program is completed, remove the nucleic acid sample from the 6th plate.

8. The method of using the safe, rapid, high-throughput fecal genome extraction kit according to claim 7, characterized in that, Do not remove the precipitate when adding the supernatant of the sample treated in step 1 to plate 1.

9. The method of using the safe, rapid, high-throughput fecal genome extraction kit according to claim 7, characterized in that, Select the program and run it. Run the program on the machine within 1 hour after adding the sample.

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