Kit for extracting nucleic acid of pathogenic microorganisms in liquid sample by paramagnetic particle method

Abstract

The invention provides a magnetic bead method liquid sample pathogenic microorganism nucleic acid extraction kit, which is characterized by comprising protease K, a nucleic acid protection solution, a lysis strengthening solution, a lysis binding solution, magnetic beads MXVB, a washing solution 1, a washing solution 2 and an eluent, wherein the cracking combination liquid is prepared from tris (hydroxymethyl) aminomethane, guanidine isothiocyanate, ammonium thiocyanate, tween-80, lauryl sodium sulfate, sodium chloride and isopropanol; the nucleic acid protection liquid comprises tris (hydroxymethyl) aminomethane hydrochloride and formamide; the cracking reinforcing liquid is lauryl sodium sulfate. According to the kit, DNA of bacteria, fungi and viruses can be rapidly extracted and purified, the extraction efficiency of pathogenic microorganism nucleic acid is improved, good stability is achieved, the nucleic acid quality is guaranteed, and development of a pathogenic microorganism detection technology is facilitated.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to a kit for extracting nucleic acid of pathogenic microorganisms from liquid samples using a magnetic bead method. Background Art

[0002] Liquid sample pathogen detection refers to the process of identifying and quantitatively analyzing pathogens in liquid samples (such as blood, urine, cerebrospinal fluid, etc.) through specific technical means. This technology is crucial for clinical diagnosis, infection control, public health and safety. At present, pathogen detection technologies include traditional microbial culture detection, molecular biology detection, immunological detection, etc. Although these technologies are relatively mature, they still have some shortcomings, such as the long cycle and positive cycle of traditional microbial culture detection; molecular biology detection methods are prone to false positives and false negatives; and immunological detection may not be able to accurately detect when the antigen concentration in the sample is low, and the sensitivity is limited. Especially in the case of mild infection, the amount of antigen released by the pathogen into the liquid sample is very small. Therefore, the effective acquisition of pathogenic microbial nucleic acid is the basis of molecular detection and one of the important links in the subsequent confirmation of the type of pathogenic microorganism.

[0003] There are many factors that affect the effective acquisition of pathogenic microorganism nucleic acids. One of the most important factors is that during the nucleic acid extraction process, due to the large variety of infectious clinical samples and the complex composition of the samples, more proteins, sugars, lipids and other substances will precipitate along with the nucleic acids, making it difficult to ensure the total amount and purity of nucleic acid extraction. In particular, the stability of pathogenic microorganisms and their nucleic acid, protein and other markers in liquid samples is limited, affecting the accuracy of the test results. If the problem of low efficiency of pathogenic microorganism nucleic acid extraction can be solved, it will be beneficial to improve the accuracy of pathogenic microorganism sequencing. Therefore, obtaining high-purity and high-quality nucleic acids is very important for subsequent molecular detection of pathogenic microorganisms. Summary of the invention

[0004] In view of the defects of the prior art, the present invention designs a nucleic acid extraction kit based on the magnetic bead method, which can effectively improve the extraction efficiency of pathogenic microorganism nucleic acid in liquid samples and has good stability.

[0005] In order to achieve the above purpose, the present invention solves the technical problem by adopting the following technical solutions: In a first aspect, the present invention provides a liquid sample pathogenic microorganism nucleic acid extraction kit using a magnetic bead method, the kit comprising proteinase K, nucleic acid protection solution, lysis enhancement solution, lysis binding solution, magnetic beads MXVB, washing solution 1, washing solution 2, and elution solution; The lysis and binding solution includes 10~100mmol / L tris(hydroxymethyl)aminomethane, 2~3mol / L guanidine isothiocyanate, 0.2~0.7 mol / L ammonium thiocyanate, 10~30% (v / v) Tween-80, 0.1~1% (v / v) sodium dodecyl sulfate, 10~50mmol / L sodium chloride, and 20~30% (v / v) isopropanol.

[0006] In the lysis and binding solution, tris(hydroxymethyl)aminomethane is used for purification and storage of nucleic acids to protect them from degradation by nucleases; guanidine isothiocyanate and sodium dodecyl sulfate are used for denaturation and lysis of cells; sodium chloride can reduce the solubility of nucleic acids in water without destroying their structure, thereby extracting nucleic acids; Tween-80 can emulsify proteins and reduce the interaction between proteins; and isopropanol can precipitate nucleic acids, which is beneficial for the binding of nucleic acids to magnetic beads.

[0007] In some embodiments, the proteinase K concentration is 10 mg / mL.

[0008] In some embodiments, the nucleic acid protection solution includes 10-20 mmol / L tris(hydroxymethyl)aminomethane hydrochloride and pH 6.5-7.5, and 5-20% (v / v) formamide.

[0009] In some embodiments, the lysis enhancing solution is 5-20% (v / v) sodium dodecyl sulfate.

[0010] In some embodiments, the magnetic beads MXVB are prepared by mixing the washing solution 2 and the magnetic beads MXVB stock solution in a ratio of 19:1.

[0011] In some embodiments, the washing solution 1 includes 2-3 mol / L guanidine thiocyanate, 0.1-0.2 mol / L sodium chloride, 40-60% (v / v) ethanol, and a pH of 5.5-6.8.

[0012] In some embodiments, the washing solution 2 includes 10-100 mmol / L tris(hydroxymethyl)aminomethane, 0.6-0.68 mol / L sodium chloride, 70-90% ethanol, and a pH of 6.5-7.5.

[0013] In some embodiments, the eluent is 10-100 mmol / L tris(hydroxymethyl)aminomethane.

[0014] In a second aspect, the present invention provides a method for extracting nucleic acid, using the magnetic bead method liquid sample pathogenic microorganism nucleic acid extraction kit.

[0015] In some embodiments, the nucleic acid extraction method comprises the following steps: S1. Take 200~300 μL of liquid sample and 15~25 μL of proteinase K and add them to 500~700 μL of lysis binding buffer for lysis; S2. Take 200~300 μL of supernatant solution, add 600 μL of lysis binding solution, and then add 10~30μL magnetic beads MXVB mixed; S3. Add 700 μL of washing solution 1, vortex for 30 seconds to separate the magnetic beads, and discard the supernatant; S4. Add 700 μL of washing solution 2, vortex for 30 seconds to separate the magnetic beads, and discard the supernatant; S5. Add 700 μL of washing solution 2 again, vortex for 30 seconds to separate the magnetic beads, and discard the supernatant; S6. Add 30-100 μL of elution buffer DE, vortex at high speed for 2-3 minutes to break up the magnetic beads, and incubate at 60°C with shaking for 5 minutes to separate the magnetic beads and obtain the nucleic acid.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a liquid sample pathogenic microorganism nucleic acid extraction kit by magnetic bead method, characterized in that the kit comprises proteinase K, nucleic acid protection solution, lysis enhancement solution, lysis binding solution, magnetic beads MXVB, washing solution 1, washing solution 2, and eluent; wherein the lysis binding solution comprises tris(hydroxymethyl)aminomethane, guanidine isothiocyanate, ammonium thiocyanate, Tween-80, sodium dodecyl sulfate, sodium chloride, and isopropanol. The kit of the present invention can quickly extract and purify DNA of bacteria, fungi and viruses, improve the extraction efficiency of pathogenic microorganism nucleic acid, and after long-term storage, the components in the kit can still maintain their original activity and efficacy, thereby ensuring the accuracy and reliability of the experimental results. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solution of the present invention, the drawings required for use in the implementation mode will be briefly introduced below. Obviously, the drawings described below are only some implementation modes of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 The figure is a graph of nucleic acid detection results extracted by a kit with different guanidine thiocyanate concentrations. In the figure, (1) is 2 mol / L guanidine thiocyanate; (2) is 2.5 mol / L guanidine thiocyanate; and (3) is 3 mol / L guanidine thiocyanate.

[0019] Figure 2The following are the results of nucleic acid detection using kits with different Tween-80 concentrations. In the figure, (1) is 10% (V / V) Tween-80; (2) is 20% (V / V) Tween-80; and (3) is 30% (V / V) Tween-80.

[0020] Figure 3 The figure shows the nucleic acid test results extracted by the kit with different sodium chloride concentrations. In the figure, (1) is 10mmol / L sodium chloride; (2) is 30mmol / L sodium chloride; and (3) is 50mmol / L sodium chloride.

[0021] Figure 4 The following are the results of nucleic acid detection using kits with different isopropanol concentrations. In the figure, (1) is 20% (V / V) isopropanol; (2) is 25% (V / V) isopropanol; and (3) is 30% (V / V) isopropanol.

[0022] Figure 5 The figure is a graph of nucleic acid detection results extracted by a kit with different guanidine isothiocyanate concentrations in washing solution 1, in which: (1) is 2 mol / L guanidine isothiocyanate; (2) is 2.5 mol / L guanidine isothiocyanate; and (3) is 3 mol / L guanidine isothiocyanate.

[0023] Figure 6 The figure is a graph of nucleic acid detection results extracted by the kit with different ethanol concentrations in washing solution 1, in which: (1) is 40% ethanol; (2) is 50% ethanol; and (3) is 60% ethanol.

[0024] Figure 7 The figure is a graph of nucleic acid detection results extracted by the kit with different sodium chloride concentrations in washing solution 2, in which: (1) is 0.6 mol / L sodium chloride; (2) is 0.64 mol / L sodium chloride; and (3) is 0.68 mol / L sodium chloride.

[0025] Figure 8 The figure is a graph of nucleic acid detection results extracted by the kit with different ethanol concentrations in washing solution 2, in which: (1) is 70% ethanol; (2) is 80% ethanol; and (3) is 90% ethanol.

[0026] Fig. 9 The figures are the results of nucleic acid extraction using the kit with different amounts of MXVB magnetic beads. In the figure: (1) is 10 μL of MXVB magnetic beads; (2) is 20 μL of MXVB magnetic beads; and (3) is 30 μL of MXVB magnetic beads.

[0027] Fig.10 The figure is a graph of nucleic acid detection results extracted by a kit with different formamide concentrations in nucleic acid protection solutions, in which: (1) is 5% formamide; (2) is 10% formamide; and (3) is 20% formamide.

[0028] Fig.11 It is a linearity test result diagram of the kit of the present invention.

[0029] Fig.12 It is a diagram of the precision experiment results of the kit of the present invention.

[0030] Fig.13 1 is a graph showing the detection limit experimental results of the kit of the present invention, wherein: (1) is a concentration of 500 copies / mL; (2) is a concentration of 250 copies / mL.

[0031] Fig.14 It is a diagram showing the results of a cross-contamination experiment on nucleic acid extraction of lactobacillus liquid by the kit of the present invention.

[0032] Fig.15 It is a diagram showing the results of a cross-contamination experiment of nucleic acid extracted from a negative control group using the kit of the present invention. DETAILED DESCRIPTION

[0033] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] In the present invention, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are deemed to be continuous and include the minimum and maximum values ​​of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0035] In the present invention, there is no particular limitation on the specific dispersion and stirring treatment methods.

[0036] In the present invention, the test methods used are conventional methods unless otherwise specified; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0037] The sources of some of the raw materials involved in the following examples are shown in Table 1 below: Table 1 Raw material sources

[0038] Note: The source of raw materials is not limited to the manufacturers listed in the table. All qualified manufacturers can purchase the raw materials in the table.

[0039] Guanidine isothiocyanate: The label purity is ACS pure, analytical pure or ≥99%, the label molecular weight is 118.16, white powder, there may be a small amount of agglomerates, stored at room temperature.

[0040] Tween-80: Label purity is reagent grade (Reagent Grade), colorless, light yellow or dark yellow transparent viscous liquid, stored at room temperature.

[0041] MXVB magnetic beads: label concentration is 45~55 mg / mL, appearance is yellow, and storage is at room temperature; the magnetic beads are silicon hydroxy magnetic beads with a particle size of about 500nm.

[0042] Sodium chloride: The label purity is ACS pure, analytical pure or ≥99%, the label molecular weight is 58.44, white crystals, there may be a small amount of agglomerates, stored at room temperature.

[0043] Embodiment 1: 1. A magnetic bead method liquid sample pathogenic microorganism nucleic acid extraction kit, the kit comprising proteinase K, nucleic acid protection solution, lysis enhancement solution, lysis binding solution, magnetic beads MXVB, washing solution 1, washing solution 2, and elution solution.

[0044] Proteinase K: 20 mg / mL; Nucleic acid protection solution: 15 mmol / L tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl, pH=7.0), 10% (v / v) formamide; Lysis enhancement solution: 10% (v / v) sodium dodecyl sulfate; Lysis and binding solution: 50 mmol / L tris(hydroxymethyl)aminomethane, 2.5 mol / L guanidine isothiocyanate, 0.5 mol / L ammonium thiocyanate, 20% (v / v) Tween-80, 0.5% (v / v) sodium dodecyl sulfate, 30 mmol / L sodium chloride, 25% (v / v) isopropanol; MXVB magnetic beads: mixed with washing solution 2 and MXVB magnetic beads stock solution (purchased from Guangzhou Bay Area Biotechnology Co., Ltd.) in a ratio of 19:1; Washing solution 1: 2.5 mol / L guanidine thiocyanate, 0.17 mol / L sodium chloride, 50% (v / v) ethanol, pH 6.2; Washing solution 2: 50 mmol / L tris(hydroxymethyl)aminomethane, 0.64 mol / L sodium chloride, 80% (v / v) ethanol, pH 7.0; Eluent: 50 mmol / L tris(hydroxymethyl)aminomethane.

[0045] 2. A method for extracting nucleic acid of pathogenic microorganisms from a liquid sample, using the above kit, the specific method comprises the following steps: 1. Select pre-lysis treatment according to different sample types Virus, mycoplasma, and chlamydia nucleic acid extraction: Take 200-300 μL of liquid sample and 20 μL of proteinase K into a centrifuge tube, add 600 μL of lysis binding solution, and extract nucleic acid according to step 4 below. No pre-treatment is required.

[0046] Extraction of nucleic acid from non-fungi and easily lysable microorganisms: Take 350-400 μL of liquid sample into a grinding tube (ZB_M1), add 20 μL of nucleic acid protection solution, 40 μL of lysis enhancement solution, and 20 μL of proteinase K, and vortex at high speed for 1 minute to mix.

[0047] Easily lysable bacteria: Gram-negative bacteria such as Escherichia coli, Haemophilus influenzae, Legionella pneumophila, Bordetella pertussis, Vibrio cholerae, etc.

[0048] Nucleic acid extraction of fungi and difficult-to-lyse microorganisms: Take 350-400 μL of liquid sample into a grinding tube (ZB_M1), then add 20 μL of nucleic acid protection solution, 40 μL of lysis enhancement solution, and 20 μL of proteinase K. Vortex at the maximum speed of a vortexer (Tian Gen, OSE-VX-01) or transfer to an oscillating disruptor for high-speed grinding of the sample for 10 minutes.

[0049] Fungi: Candida albicans, Candida tropicalis, Aspergillus flavus, Penicillium.

[0050] Difficult to lyse bacteria: Gram-positive bacteria such as Staphylococcus, mold, Streptococcus pneumoniae, etc.

[0051] 2. After the treatment in step 1, transfer the liquid sample to a water bath and warm it at 70°C for 20 minutes to further lyse the sample. After lysis, take it out and vortex mix it for 30 seconds.

[0052] If the solution is turbid after lysis, centrifuge at 10,000×g for 1 minute; if the solution is clear after lysis, centrifuge for 30 seconds.

[0053] 3. Pipette 200-300 μL of supernatant solution into a new centrifuge tube, add 600 μL of lysis and binding solution, and pipette carefully to avoid aspirating precipitate or glass beads.

[0054] 4. Add 20 μL of MXVB magnetic beads, vortex mix for 30 seconds, shake or vortex for 5-7 minutes to allow the magnetic beads to adsorb nucleic acids, then transfer to a magnetic stand for magnetic separation until the solution is clear, and discard the solution.

[0055] 5. Add 700 μL of washing solution 1, vortex for 30 seconds, transfer to a magnetic rack and adsorb until the solution is clear, then discard the solution.

[0056] 6. Add 700 μL of washing solution 2, vortex for 30 seconds to break up the magnetic beads, then transfer to a magnetic stand for magnetic separation until the solution is clear, and discard the solution.

[0057] 7. Add 700 μL of washing solution 2 again, vortex for 30 seconds to break up the magnetic beads, then transfer to the magnetic stand for magnetic separation until the solution is clear, and discard the solution.

[0058] 8. After instant centrifugation in a handheld centrifuge, transfer to a magnetic rack and adsorb until the solution is clear. Discard all the solution, open the tube cap, and air dry for 3 to 5 minutes.

[0059] 9. Add 30~100 μL of elution buffer DE and vortex at high speed for 2~3 minutes to break up the magnetic beads.

[0060] 10. Incubate at 60℃ with shaking for 5 minutes. If the thermostat does not have an shaking function, take it out during the incubation period and vortex at high speed to break up the magnetic beads 2 to 3 times, each time for 10 to 30 seconds.

[0061] 11. Collect the liquid droplets from the tube cap by instant centrifugation and transfer them to the magnetic rack for magnetic separation.

[0062] 12. Transfer the nucleic acid solution to a new centrifuge tube and store it at -15~-25℃ for future use (for long-term storage, store at -70℃ or lower).

[0063] Embodiment 2: The effects of different concentrations or dosages of the components in the magnetic bead method liquid sample pathogenic microorganism nucleic acid extraction kit in Example 1 on the extraction of pathogenic microorganism nucleic acid from liquid samples were analyzed.

[0064] 1. Lysis and Binding Solution 1. Guanidine thiocyanate concentration According to the kit of Example 1, other conditions remain unchanged, the concentration of guanidine isothiocyanate in the lysis and binding solution is set to 2 mol / L, 2.5 mol / L, and 3 mol / L respectively; the counted lactobacillus solution (Gram-positive bacteria) is diluted with TE buffer as the sample to be tested, the concentration is 500 copies / mL, and 8 samples; Nucleic acid extraction was performed on a fully automatic nucleic acid extraction and purification instrument (Guangzhou Bay Area Biotechnology Co., Ltd.: BBEX-32), and after nucleic acid extraction, it was detected using a Lactobacillus (LB) nucleic acid detection kit (fluorescence quantitative PCR method, from Guangzhou Bay Area Biotechnology Co., Ltd.).

[0065] like Figure 1 As shown, when the concentration of guanidine thiocyanate was 2.5 mol / L, all eight samples were detected and an S-shaped curve was amplified.

[0066] 2. Tween-80 concentration According to the kit of Example 1, other conditions remained unchanged, the concentration of Tween-80 in the lysis and binding solution was set to 10%, 20%, and 30%, respectively, and other operations were the same as the above-mentioned guanidine thiocyanate concentration test.

[0067] like Figure 2 As shown, when the concentration of Tween-80 was 20%, all eight samples were detected and an S-shaped curve was amplified.

[0068] 3. Sodium chloride concentration According to the kit of Example 1, other conditions remained unchanged, the concentration of sodium chloride in the lysis and binding solution was set to 10 mmol / L, 30 mmol / L, and 50 mmol / L, respectively, and other operations were the same as the above-mentioned guanidine thiocyanate concentration test.

[0069] like Figure 3 As shown, when the sodium chloride concentration was 30mmol / L, all 8 samples were detected and an S-shaped curve was amplified.

[0070] 4. Isopropyl alcohol concentration According to the kit of Example 1, other conditions remained unchanged, the isopropanol concentrations in the lysis and binding solution were set to 20%, 25%, and 30%, respectively, and other operations were the same as the above-mentioned guanidine thiocyanate concentration test.

[0071] like Figure 4 As shown, when the isopropanol concentration was 20% and 25%, all 8 samples were detected and amplified to form an S-shaped curve, among which the amplification curve when the isopropanol concentration was 20% was the best.

[0072] 2. Washing liquid 1 1. Guanidine thiocyanate concentration According to the kit of Example 1, other conditions remain unchanged, the concentration of guanidine thiocyanate in washing solution 1 is set to 2 mol / L, 2.5 mol / L, and 3 mol / L, respectively, and other operations are the same as the guanidine thiocyanate concentration test of the above lysis and binding solution.

[0073] like Figure 5 As shown, when the sodium chloride concentration was 0.64 mol / L, all eight samples were detected and an S-shaped curve was amplified.

[0074] 2. Ethanol concentration According to the kit of Example 1, other conditions remain unchanged, the ethanol concentration in the washing solution 1 is set to 40%, 50%, and 60%, respectively, and other operations are the same as the guanidine thiocyanate concentration test of the above-mentioned lysis and binding solution.

[0075] like Figure 6 As shown, when the ethanol concentration was 50%, all eight samples were detected and an S-shaped curve was amplified.

[0076] 3. Washing liquid 2 1. Sodium chloride concentration According to the kit of Example 1, other conditions remain unchanged, the concentration of sodium chloride in the washing solution 2 is set to 0.6 mol / L, 0.64 mol / L, and 0.68 mol / L, respectively, and other operations are the same as the guanidine thiocyanate concentration test of the above lysis and binding solution.

[0077] like Figure 7 As shown, when the sodium chloride concentration was 0.64 mol / L, all eight samples were detected and an S-shaped curve was amplified.

[0078] 2. Ethanol concentration According to the kit of Example 1, other conditions remain unchanged, the ethanol concentration in the washing solution 2 is set to 70%, 80%, and 90%, respectively, and other operations are the same as the guanidine thiocyanate concentration test of the above lysis and binding solution.

[0079] like Figure 8 As shown, when the ethanol concentration was 80%, all eight samples were detected and an S-shaped curve was amplified.

[0080] 4. Dosage of MXVB beads According to the kit of Example 1, other conditions remain unchanged, the amount of magnetic beads MXVB is set to 10 μL, 20 μL, and 30 μL respectively, and other operations are the same as the guanidine thiocyanate concentration test of the above lysis binding solution.

[0081] like Fig. 9 As shown, when the amount of magnetic beads MXVB was 20 μL, all 8 samples to be tested were detected and an S-shaped curve was amplified.

[0082] 5. Formamide concentration in nucleic acid protection solution According to the kit of Example 1, other conditions remain unchanged, the formamide concentrations are set to 5%, 10%, and 20%, respectively, and other operations are the same as the guanidine thiocyanate concentration test of the above lysis binding solution.

[0083] like Fig.10 As shown, when the formamide concentration was 10%, all eight samples were detected and an S-shaped curve was amplified.

[0084] Example 3 1. Linearity Experiment The lactobacillus solution (Gram-positive bacteria) was diluted with sample diluent (Guangzhou Bay Area Biotechnology Co., Ltd., sample diluent NPG), and the concentrations after dilution were E1: 50000 copies / mL, E2: 50000 copies / mL, E3: 5000 copies / mL, and E4: 500 copies / mL. The nucleic acid was extracted using the kit of Example 1 on a fully automatic nucleic acid extraction and purification instrument (Guangzhou Bay Area Biotechnology Co., Ltd., BBEX-32), and the nucleic acid was detected using a Lactobacillus (LB) nucleic acid detection kit (fluorescence quantitative PCR method) after nucleic acid extraction.

[0085] like Fig.11 As shown in the figure, E1, E2, and E3 have one data, and E4 has three data. The concentration of the extracted counted lactobacillus solution (Gram-positive bacteria) is 500,000 copies / mL to 5,000 copies / mL, and there is a good linear relationship (R 2 ≥0.99), all detected at a concentration of 500 copies / mL.

[0086] 2. Precision Experiment Dilute the lactobacillus solution (Gram-positive bacteria) with sample diluent to a concentration of 5×10 3 copies / mL, 5×10 2 8 replicates / mL were used for each concentration, and other operations were the same as those in the linear experiment.

[0087] like Fig.12 As shown, the S-shaped curve was amplified when the concentration of the extracted and counted lactobacillus solution (Gram-positive bacteria) was 5000 copies / mL and 500 copies / mL, indicating that the nucleic acid of lactobacillus with a concentration of 5000 copies / mL and 500 copies / mL extracted by the kit of the present invention can all be detected, and the Ct value deviation does not exceed 1, with good precision.

[0088] 3. Detection limit experiment The lactobacillus solution (Gram-positive bacteria) was diluted with sample diluent to a concentration of 500 copies / mL and 250 copies / mL, with 20 replicates for each concentration. Other operations were the same as those in the linear experiment.

[0089] Table 2 Detection limit experimental results

[0090] As shown in Table 2, when the concentration of the lactobacillus solution was 500 copies / mL, the positive rate of nucleic acid detection extracted by the kit of Example 1 was 100%, while when the concentration of the lactobacillus solution was 250 copies / mL, the positive rate of nucleic acid detection extracted was only 80%, which did not meet the requirements; Fig.13 As shown, all 20 samples of 500 copies / mL lactobacillus solution (Gram-positive bacteria) extracted and counted were detected and amplified with an S-shaped curve. Therefore, the minimum concentration of lactobacillus nucleic acid extracted by the kit of Example 1 is 500 copies / mL.

[0091] 4. Cross-contamination experiment The lactobacillus solution (Gram-positive bacteria) was diluted with sample diluent to a concentration of 5000 copies / mL, which was used as the experimental group. A negative control group without the addition of lactobacillus solution was set up. There were 16 replicate samples in each of the experimental group and the negative control group, and other operations were the same as the linear experiment.

[0092] In the same experiment, the negative sample had no amplification curve in the FAM detection channel, and the extracted and counted lactobacillus liquid (Gram-positive bacteria) quality control sample 5000 copies / ml had an amplification curve in the FAM detection channel, which determined that there was no cross-contamination in the nucleic acid extraction and purification reagents extracted in the instrument.

[0093] like Fig.14 , 15 As shown, the nucleic acid extraction and purification reagents will not cause cross contamination when extracting nucleic acids on the BBEX-32 instrument, and the nucleic acid extraction and purification reagents are compatible with the BBEX-32 instrument.

[0094] Example 4 1. Real-time stability test According to the "Basic Principles and Methods of Determining the Validity Period of Medical Devices by Accelerated Aging Experiments" in "China Medical Device Information" Vol. 14, No. 5, 2008, the accelerated aging test was designed using the simplified 10-degree principle. The calculation formula is as follows: TimeT1=TimeRT / Q10 (T1-RT) / 10 (1); Wherein, TimeT1 is the accelerated aging time, TimeRT is the room temperature time (lifespan), T1 is the accelerated aging temperature (50°C), RT is the room temperature (22°C), and Q10 is the reaction rate coefficient (taken as 2).

[0095] The kit was stored at 50°C for accelerated aging test. When the accelerated aging time of the kit was 6 weeks, 9 weeks, 10 weeks, 11 weeks and 12 weeks, the counted lactobacillus solution (Gram-positive bacteria) was extracted on BBEX-32 and diluted with sample diluent. The lactobacillus solution diluted to 500 copies / mL was used as the positive sample, and the sample diluent was the negative sample. Ten samples were set for each sample, and then tested with the Lactobacillus (LB) nucleic acid detection kit (fluorescence quantitative PCR method).

[0096] Table 3 Results of real-time stability test of the kit

[0097] As shown in Table 3, after accelerated aging, the CV% of the kit of Example 1 was no more than 5%. Even after 12 weeks of accelerated aging, the test results of various indicators of the kit were qualified. In addition, except for the lysate of wells 1 and 7, which was light yellow, and the magnetic bead MXVB solution of wells 2 and 8, which was brown after shaking, the liquid component solutions of other wells were clear, without precipitate, and the appearance was not damaged, indicating that the kit of the present invention has long-term stability when stored under accelerated aging conditions.

[0098] 2. Low temperature stability Place the test kit in a 4°C refrigerator, and take out the extracted lactobacillus fluid (Gram-positive bacteria) nucleic acid for testing after 4, 6, and 8 weeks (the specific operation is the same as the real-time stability test).

[0099] Table 4 Test results of low temperature stability of the test kit

[0100] As can be seen from Table 4, the results of the stability test of the kit of Example 1 after being stored at 4°C for 8 weeks show that the test results of various indicators are qualified, and except for the lysate in wells 1 and 7 which is light yellow, the magnetic bead MXVB solution in wells 2 and 8 is brown after shaking, the liquid component solutions in other wells are clear, without precipitate, and the appearance is not damaged, indicating that the kit of the present invention has good low-temperature stability, and even if it is stored at 4°C for 8 weeks, it does not affect the nucleic acid extraction of pathogenic microorganisms.

[0101] 3. 37℃ Stability Place the test kit in a 37°C constant temperature refrigerator, and take out the extracted lactobacillus liquid (Gram-positive bacteria) nucleic acid for testing after 48, 72, and 96 hours (the specific operation is the same as the real-time stability test).

[0102] Table 5 Stability test results of the kit at 37°C

[0103] As can be seen from Table 5, the results of the stability test of the kit of Example 1 after being stored at 37°C for 96 hours are all qualified, and except for the lysate in wells 1 and 7 which is light yellow, the magnetic bead MXVB solution in wells 2 and 8 is brown after shaking, the liquid component solutions in other wells are clear, without precipitate, and the appearance is not damaged, indicating that the kit of the present invention has good low-temperature stability, and even if it is stored at 37°C for 96 hours, it does not affect the nucleic acid extraction of pathogenic microorganisms.

[0104] 4. Freeze-thaw stability Place the kit in a 37°C constant temperature refrigerator, freeze and thaw 5, 10, and 15 times, extract the lactobacillus solution (Gram-positive bacteria) nucleic acid and test it (the specific operation is the same as the real-time stability test). Freeze and thaw once every half an hour, and return the kit to room temperature for nucleic acid extraction.

[0105] Table 6 Freeze-thaw stability test results of the kit

[0106] As can be seen from Table 6, after repeated freezing and thawing, the test results of various indicators of the kit of Example 1 are qualified, and except for the lysate of wells 1 and 7 which is light yellow, and the magnetic bead MXVB solution of wells 2 and 8 which is brown after shaking, the liquid component solutions of other wells are clear, without precipitate, and the appearance is not damaged, indicating that the kit of the present invention has good freeze-thaw stability.

[0107] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A kit for extracting nucleic acid from pathogenic microorganisms in liquid samples by magnetic bead method, characterized in that: The kit includes proteinase K, nucleic acid protection solution, lysis enhancement solution, lysis binding solution, magnetic beads MXVB, washing solution 1, washing solution 2, and elution solution; The lysis and binding solution includes 10~100mmol / L tris(hydroxymethyl)aminomethane, 2~3mol / L guanidine isothiocyanate, 0.2~0.7 mol / L ammonium thiocyanate, 10~30% (v / v) Tween-80, 0.1~1% (v / v) sodium dodecyl sulfate, 10~50mmol / L sodium chloride, and 20~30% (v / v) isopropanol.

2. The kit for extracting nucleic acid from pathogenic microorganisms from liquid samples using magnetic beads according to claim 1, characterized in that: The proteinase K concentration is 10 mg / mL.

3. The kit for extracting nucleic acid from pathogenic microorganisms from liquid samples by magnetic bead method according to claim 1, characterized in that: The nucleic acid protection solution comprises 10-20 mmol / L tris(hydroxymethyl)aminomethane hydrochloride with a pH of 6.5-7.5 and 5-20% (v / v) formamide.

4. The kit for extracting nucleic acid from pathogenic microorganisms from liquid samples using magnetic beads according to claim 1, characterized in that: The lysis enhancement solution is 5-20% (v / v) sodium dodecyl sulfate.

5. The kit for extracting nucleic acid from pathogenic microorganisms from liquid samples using magnetic beads according to claim 1, characterized in that: The magnetic beads MXVB are prepared by mixing the washing solution 2 and the magnetic beads MXVB stock solution in a ratio of 19:

1.

6. The kit for extracting nucleic acid from pathogenic microorganisms from liquid samples using magnetic beads according to claim 1, characterized in that: The washing solution 1 includes 2-3 mol / L guanidine thiocyanate, 0.1-0.2 mol / L sodium chloride, 40-60% (v / v) ethanol, and a pH of 5.5-6.

8.

7. The kit for extracting nucleic acid from pathogenic microorganisms from liquid samples using a magnetic bead method according to any one of claims 1 or 5, characterized in that: The washing solution 2 includes 10-100 mmol / L tris(hydroxymethyl)aminomethane, 0.6-0.68 mol / L sodium chloride, 70-90% ethanol, and a pH of 6.5-7.

5.

8. The kit for extracting nucleic acid from pathogenic microorganisms from liquid samples using a magnetic bead method according to claim 1, characterized in that: The eluent is 10-100 mmol / L tris(hydroxymethyl)aminomethane.

9. A method for extracting nucleic acid, characterized in that: A liquid sample pathogenic microorganism nucleic acid extraction kit using the magnetic bead method as described in any one of claims 1 to 8.

10. The method for extracting nucleic acid according to claim 1, characterized in that: The nucleic acid extraction method comprises the following steps: S1. Take 200~300 μL of liquid sample and 15~25 μL of proteinase K and add them to 500~700 μL of lysis binding buffer for lysis; S2. Take 200~300 μL of supernatant solution, add 600 μL of lysis binding solution, and then add 10~30μL magnetic beads MXVB mixed; S3. Add 700 μL of washing solution 1, vortex for 30 seconds to separate the magnetic beads, and discard the supernatant; S4. Add 700 μL of washing solution 2, vortex for 30 seconds to separate the magnetic beads, and discard the supernatant; S5. Add 700 μL of washing solution 2 again, vortex for 30 seconds to separate the magnetic beads, and discard the supernatant; S6. Add 30-100 μL of elution buffer DE, vortex at high speed for 2-3 minutes to break up the magnetic beads, and incubate at 60°C with shaking for 5 minutes to separate the magnetic beads and obtain the nucleic acid.

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