CN118995383B - Microorganism genome extraction kit and microorganism genome extraction method by magnetic bead method - Google Patents
Microorganism genome extraction kit and microorganism genome extraction method by magnetic bead method Download PDFInfo
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Abstract
本发明属于生物医药技术领域，具体涉及一种磁珠法微生物基因组提取试剂盒及微生物基因组提取方法。本发明的试剂盒包括设置微生物破壁单元和微生物裂解单元；所述微生物破壁单元包括破壁试剂和破壁酶组合物试剂；所述微生物裂解单元包括蛋白酶试剂和裂解试剂。本发明还提供了一种基于所述磁珠法微生物基因组提取试剂盒来提取微生物基因组的方法。本发明的试剂盒能够对同时包含真菌（白色念珠菌）、革兰氏阳性菌（金黄色葡萄球菌、屎肠球菌）、革兰氏阴性菌（大肠埃希菌）的样本进行一次性基因组提取。
The present invention belongs to the field of biomedicine technology, and specifically relates to a magnetic bead method microbial genome extraction kit and a microbial genome extraction method. The kit of the present invention includes a microbial wall breaking unit and a microbial lysis unit; the microbial wall breaking unit includes a wall breaking reagent and a wall breaking enzyme composition reagent; the microbial lysis unit includes a protease reagent and a lysis reagent. The present invention also provides a method for extracting microbial genomes based on the magnetic bead method microbial genome extraction kit. The kit of the present invention can perform one-time genome extraction on samples containing fungi (Candida albicans), Gram-positive bacteria (Staphylococcus aureus, Enterococcus faecium), and Gram-negative bacteria (Escherichia coli) at the same time.
Description
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to a magnetic bead method microorganism genome extraction kit and a microorganism genome extraction method.
Background
Microbial infection is one of the common infectious diseases, and has become a global public health problem due to the unreasonable use of anti-infective drugs, the increasing aggravation of the problems of multi-drug resistance, pan-drug resistance and the like of anti-infective drugs, and the like. Therefore, early and accurate identification of pathogenic microorganisms causing infection can reduce blind empirical medication, reduce the use proportion of broad-spectrum anti-infection medicines, slow and inhibit the development of drug resistance of pathogenic microorganisms by reasonable medication, and play a certain role in controlling disease development and reducing infection hazard.
Nucleic acid detection is an important means of current pathogen screening. Nucleic acid extraction provides a vital raw material for research and application of nucleic acid detection (such as qRT-PCR and whole genome, second generation sequencing techniques in transcriptomes). In the process of nucleic acid extraction, it is often necessary to ensure sufficient nucleic acid extraction and to ensure the integrity of the primary structure of the nucleic acid, and it is also necessary to avoid contamination of biological macromolecules such as proteins, lipids, and polysaccharides. The existing extraction method and extraction reagent are required to achieve the technical purposes at the same time, often have complex extraction flow, or can not achieve the technical purposes at the same time when the extraction flow is simplified.
Taking the problem of complex operation of the commercial genome extraction kit commonly used at present as an example, ten steps are needed from the collection of bacteria to the acquisition of genome, even in some improper operations, the release of nucleic acid to pathogenic microorganisms is incomplete, so that the adsorption efficiency of the final target genome is lower. In addition, bacterial extraction and fungal extraction in the commonly used commercial genome extraction kit are separately set, and the types of pathogenic microorganisms in the sample need to be known first to select a proper extraction reagent for extraction. For samples with unknown types of pathogenic microorganisms and samples mixed with different types of pathogenic microorganisms (such as bacteria and fungi mixed samples), different extraction reagents are often required to be tried respectively, and the samples are tested for multiple times to be successfully extracted. In the current clinical practice, the type of the pathogen needs to be determined first and then the next sample pretreatment is carried out, and the pretreatment modes of different pathogens are different, so that the sample amount and the sample treatment time of the clinical sample are wasted greatly.
The publication No. CN112941066A, entitled "lysis binding solution for bacterial nucleic acid extraction and kit and application thereof", discloses a bacterial nucleic acid extraction method, which comprises the steps of treating a sample with lysozyme solution and proteinase K solution, adding the lysis binding solution, releasing nucleic acid from a biological sample, adding magnetic beads, obtaining a magnetic bead-nucleic acid complex under the action of an external magnetic field, and collecting and washing the magnetic bead-nucleic acid complex to obtain extracted nucleic acid. However, the bacterial nucleic acid extracted by the method of the patent needs to be amplified by PCR to detect the result, and the amplification by PCR has an amplifying effect on the original nucleic acid, presumably because the amount of the original nucleic acid extracted by the method is not large. Furthermore, the method of this patent does not allow for processing of samples containing mixed microorganisms of various kinds.
In view of the foregoing, there is a need for a more convenient and efficient means of extracting microbial nucleic acids.
Disclosure of Invention
In view of the above, the present invention aims to provide a magnetic bead method mixed microorganism genome extraction kit and a microorganism genome extraction method, so as to meet the demands of scientific research and clinical trials, and partially solve or alleviate the above-mentioned shortcomings in the prior art, and the specific technical scheme is as follows.
In one aspect, the invention provides a novel nucleic acid extraction kit, in particular to a mixed microorganism genome extraction kit.
A magnetic bead method (mixed) microorganism genome extraction kit comprises a microorganism wall breaking unit and a microorganism cracking unit, wherein the microorganism wall breaking unit comprises a wall breaking reagent and a wall breaking enzyme composition reagent, the microorganism cracking unit comprises a protease reagent and a cracking reagent,
The wall breaking reagent comprises 5-10 mM dithiothreitol, 10-20mM tris (hydroxymethyl) aminomethane hydrochloride, 1-5 mM ethylenediamine tetraacetic acid and 0.1-0.5% polyethylene glycol octyl phenyl ether, wherein the pH value of the wall breaking reagent is in the weak alkalinity of 7.5-8.5, and the unit dosage of each group of wall breaking reagent in the magnetic bead mixed microorganism genome extraction kit is not less than 80 mu L;
the wall-breaking enzyme composition reagent comprises muramidase, lysozyme and lysostaphin, wherein the unit dosage of each group of wall-breaking enzyme reagent in the magnetic bead method mixed microorganism genome extraction kit is not less than 5 mu L;
The protease reagent is set as single protease and/or composite protease, and the protease in the protease reagent has biological activity suitable for pH range 4-12.5 and temperature range 50-70 ℃;
The cracking reagent comprises 200-400 mM of tris hydrochloride, 20-50 mM of ethylenediamine tetraacetic acid, 1-5% of polyethylene glycol octyl phenyl ether, 500-1000 of mM sodium chloride, 0.1-0.5 of mM of sodium methylparaben and 1-5% of tween, wherein the pH value of the cracking reagent is in weak alkaline state of 7.5-8.5;
The unit dosage of each group of protease reagent in the magnetic bead method mixed microorganism genome extraction kit is not less than 5 mu L, and the unit dosage of each group of lysis reagent is not less than 30 mu L;
The magnetic bead method mixed microorganism genome extraction kit is also provided with magnetic beads with carboxyl and/or hydroxyl modified surfaces, a high-salt magnetic bead binding reagent and a low-salt magnetic bead eluting reagent.
In some preferred embodiments, a plurality of sets of functionally equivalent reagents may be provided in the magnetic bead hybrid microbial genome extraction kit.
In some preferred embodiments, the magnetic bead method mixed microorganism genome extraction kit is further provided with a reagent for assisting sedimentation of magnetic beads.
In some preferred embodiments, the ratio of enzyme activities of muramidase, lysozyme and lysostaphin is 100:100:3 (20000:20000:1200) and the ratio of concentrations is 40:100:1.
Further, the wall breaking reagent comprises 5 mM dithiothreitol, 20mM tris (hydroxymethyl) aminomethane hydrochloride, 2 mM ethylenediamine tetraacetic acid and 0.1% polyethylene glycol octyl phenyl ether, and the pH value of the wall breaking reagent is 8.
It is understood that the wall breaking reagent is used for providing a weak alkaline reduction environment for the wall breaking enzyme composition, so as to ensure the enzyme activity. For example, the wall breaking agent may include dithiothreitol as a reducing agent, and the wall breaking agent may include more than one reducing agent.
Further, the protease is a single protease, and the concentration is 20-50 mg/mL.
In some preferred embodiments, the protease is proteinase K. Proteinase K is chosen, firstly because it is a powerful proteolytic enzyme, which has strong activity in decomposing protein, and secondly because it has strong stability, which maintains activity in a wide pH range (4-12.5) and at high temperatures (50-70 ℃). Further, the cracking reagent comprises 400 mM of tris hydrochloride, 40 mM of ethylenediamine tetraacetic acid, 5% of polyethylene glycol octyl phenyl ether, 800 mM of sodium chloride, 0.1 mM of sodium methylparaben and 5% of tween, and the pH value of the cracking reagent is 8.
In some preferred embodiments, the magnetic beads comprise one or more selected from silica hydroxyl magnetic beads having a particle size of 1 μm, silica carboxyl magnetic beads having a particle size of 300 nm, and/or silica carboxyl magnetic beads having a particle size of 600 nm.
Further, the high-salt magnetic bead binding reagent comprises 30-50 mM 4-hydroxyethyl piperazine ethane sulfonic acid, 1-4M sodium chloride and 5% polyethylene glycol 8000, and the low-salt magnetic bead eluting reagent is nuclease-free water.
It will be appreciated that the terms "high salt" and "low salt" as used herein are a relative set of concepts in the present kit setting.
Further, the mixed microorganism includes one or more selected from fungi, gram positive bacteria and/or gram negative bacteria.
In another aspect, the invention also provides a method for extracting the genome of a mixed microorganism based on the kit.
The method for extracting the microbial genome based on the mixed microbial genome extraction kit by the magnetic bead method comprises the following steps of:
S01, collecting a sample containing pathogenic microorganisms, wherein the sample comprises a body fluid sample, a urine sample and/or a blood sample;
S02, respectively adding a wall breaking reagent and a wall breaking enzyme composition reagent in a kit into the sample containing the pathogenic microorganism, and reacting for a short time under the condition of the 1 st temperature;
S03, continuously adding a protease reagent and a lysis reagent in a kit into the sample containing the pathogenic microorganisms respectively, and reacting for a short time under the condition of the 2 nd temperature, wherein the 2 nd temperature is higher than the 1 st temperature and the 2 nd temperature inactivates the wall breaking enzyme composition;
S04, continuously adding magnetic beads and magnetic bead binding reagents in a kit into the reacted sample containing the pathogenic microorganisms, uniformly mixing by vortex, and carrying out adsorption extraction on nucleic acid of the pathogenic microorganisms;
s05, eluting the magnetic beads adsorbed with the nucleic acid by using a magnetic bead eluting reagent in the kit, and releasing to obtain a nucleic acid sample.
Further, the 1 st temperature condition is 30-40 ℃, and the 2 nd temperature condition is 70-80 ℃.
Further, the short-time reaction is a reaction time of not more than 30 minutes.
Advantageous effects
(1) The invention provides a novel microorganism genome extraction kit which can extract a sample containing a plurality of unknown microorganisms rapidly (DNA+RNA) without distinguishing microorganism types. Specifically, the kit can perform one-time genome extraction on a sample containing fungi (candida albicans), gram-positive bacteria (staphylococcus aureus, enterococcus faecium) and gram-negative bacteria (escherichia coli) at the same time.
(2) The kit realizes the full release of nucleic acid under mild reaction conditions through the optimal configuration of each reagent in the microorganism wall breaking unit and the microorganism cracking unit. Meanwhile, the whole reaction condition is mild, and the nucleic acid structure is not damaged, so that the total quantity of the finally extracted genes is large and the quality is high. For example, in the method of extracting genome using the present kit, two reaction temperatures were designed, and the 2 nd temperature was higher than the 1 st temperature. The enzyme at temperature 1 is inactivated during the reaction because the activity of the enzyme affects the nucleic acid. In addition, the activity of the reagent at the 2 nd temperature is not affected, so that the whole extraction is not required to be purified, the whole extraction time is short, and the efficiency is high. For another example, the present invention provides a complex enzyme composition comprising a low enzyme activity and a high concentration of lysostaphin to achieve a gentle but sufficient wall breaking release of microbial nucleic acids.
(3) The magnetic beads arranged on the kit provided by the invention are modified with a large number of silanol groups (hydroxyl groups), can be specifically combined with nucleic acid in the solution under the conditions of high salt and low pH through hydrophobic effect, hydrogen bond effect, electrostatic effect and the like, and are not combined with other impurities (such as proteins), so that the nucleic acid is rapidly separated from a biological sample, and then the nucleic acid is eluted by using a low-salt solution.
In conclusion, the invention provides the kit which can realize synergistic effect and mutual coordination among various reagents, and has good extraction effect and high extraction efficiency.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. Like elements or portions are generally identified by like reference numerals throughout the several figures. In the drawings, elements or portions thereof are not necessarily drawn to scale. It will be apparent to those of ordinary skill in the art that the drawings in the following description are of some embodiments of the invention and that other drawings may be derived from these drawings without inventive faculty.
FIG. 1 is a graph showing agarose gel electrophoresis results of genome extraction in one example;
FIG. 2 is a graph showing agarose gel electrophoresis results of genomic PCR products extracted in one example;
FIG. 3 is a schematic diagram of a magnetic bead method mixed microorganism genome extraction kit of the invention;
FIG. 4 is a schematic flow chart of the extraction of samples by using the magnetic bead method mixed microorganism genome extraction kit of the invention.
The reference numerals summarize that the mixed microorganism genome extraction kit 10, the biological wall breaking unit 20, the biological lysis unit 30, the other 40, the wall breaking reagent 21, the wall breaking enzyme composition reagent 22, the protease reagent 31, the lysis reagent 32, the magnetic beads 41, the magnetic bead binding reagent 42 and the magnetic bead eluting reagent 43.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.
Herein, "and/or" includes any and all combinations of one or more of the associated listed items.
Herein, "plurality" means two or more, i.e., it includes two, three, four, five, etc.
As used in this specification, the term "about" is typically expressed as +/-5% of the value, more typically +/-4% of the value, more typically +/-3% of the value, more typically +/-2% of the value, even more typically +/-1% of the value, and even more typically +/-0.5% of the value.
In this specification, certain embodiments may be disclosed in a format that is within a certain range. It should be appreciated that such a description of "within a certain range" is merely for convenience and brevity and should not be construed as a inflexible limitation on the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all possible sub-ranges and individual numerical values within that range. For example, the description of ranges 1-6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, e.g., 1,2,3,4,5, and 6. The above rule applies regardless of the breadth of the range.
Noun paraphrasing:
The "genome performance" of the present invention includes genome concentration and genome quality, and it should be specifically noted that the kit of the present invention involves co-extraction of DNA and RNA. The genome quality refers to the detection value of a 260/A280.
A 260/A280 judging index refers to that the ratio of A 260/A280 is about 2.0, the quality of the genome is good, more than 2.2 represents the degradation of partial RNA, and less than 1.8 represents the pollution of protein (aromatic) or phenols, and the quality of the genome is poor.
Example 1
The present embodiment provides an example of a magnetic bead method mixed microorganism genome extraction kit (abbreviated as mixed microorganism genome extraction kit), and a schematic diagram of the magnetic bead method mixed microorganism genome extraction kit is shown in fig. 3.
The mixed microorganism genome extraction kit 10 provided in this embodiment includes a biological wall breaking unit 20, a biological lysis unit 30 and other 40, specifically, the biological wall breaking unit 20 includes a wall breaking reagent 21 and a wall breaking enzyme composition reagent 22, the biological lysis unit 30 includes a protease reagent 31 and a lysis reagent 32, and the other 40 may include a magnetic bead 41, a magnetic bead binding reagent 42 and a magnetic bead eluting reagent 43.
Other 40 may include nucleic acid precipitation aids, washing solutions, and the like, as described in detail below.
(1) The wall breaking reagent comprises 5mM Dithiothreitol (DTT), 20mM pH Tris-HCl of 8.0, 2 mM ethylenediamine tetraacetic acid (EDTA) and 0.1% polyethylene glycol octyl phenyl ether (Triton X-100), and the unit dosage of the wall breaking reagent is 80 μl.
(2) The wall-breaking enzyme composition reagent comprises wall-breaking enzyme, lysozyme and lysostaphin, wherein the specific activity of the wall-breaking enzyme is more than or equal to 20U/mg and the concentration is 20 mg/mL, the specific activity of the lysozyme is more than or equal to 20000U/mg and the concentration is 50 mg/mL, the specific activity of the lysostaphin is more than or equal to 1200U/mg and the concentration is 0.5mg/mL, and the unit dosage of the wall-breaking enzyme composition reagent is 5 mu L.
(3) The specific activity of the proteinase K is more than or equal to 30 units/mg protein, the concentration is 30 mg/mL, and the unit dosage is 5 mu L.
(4) The lysis reagent comprises Tris-HCl with the concentration of 400 mM pH being 8.0, 40 mM ethylenediamine tetraacetic acid (EDTA), 5% polyethylene glycol octyl phenyl ether (Triton X-100), 800 mM sodium chloride (NaCl), 0.1 mM sodium methylparaben and 5% Tween 20, and the unit dosage of the lysis reagent is set to be 30 mu L.
(5) The nucleic acid sedimentation aid is absolute ethyl alcohol, the volume ratio of the absolute ethyl alcohol to the sample is 1.5:1, and the dosage is 180 mu L.
(6) The magnetic bead binding reagent comprises 50mM 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES), 4M sodium chloride and 5% polyethylene glycol PEG8000, wherein the volume ratio of the magnetic bead binding reagent to a sample is 1:5 when the magnetic bead binding reagent is used, and the dosage is 75 mu L.
(7) The washing liquid comprises 80% ethanol, and the unit dosage is 500 mu L. The washing liquid is used for cleaning impurities on the magnetic beads.
(8) The magnetic bead eluting reagent comprises water without nuclease.
Further, the embodiment also discloses a preparation method of each reagent in the microbial genome extraction kit.
(1) The preparation of the wall breaking reagent comprises taking 10 mg Triton X-100 into a 15 mL centrifuge tube, adding 200 μL of 1M Tris-HCl (pH 8.0) and 40 μL of 0.5M EDTA, and adding sterilized water to constant volume to 10 mL.
The muramidase preparation in (2-1) is that a 1/15 phosphate buffer solution is used for preparing the muramidase with the concentration of 40 mg/mL, and then an equal volume of 100% glycerol is added to prepare a 20 mg/mL muramidase storage solution, and the storage solution is preserved at-20 ℃.
(2-2) Preparation of lysozyme 100 mg/mL of lysozyme was prepared using 10mM Tris-HCl (pH 8.0), and then an equal volume of 100% glycerol was added to prepare 50 mg/mL of lysozyme stock solution, which was stored at-20 ℃.
(2-3) The lysostaphin was prepared by preparing 6000U/mL of lysostaphin using 50 mM Tris-HCl (pH 8.0), then adding an equal proportion of 100% glycerol to prepare 3000U/mL of lysostaphin stock solution, and preserving at-20℃and diluting at 600U/mL when in use.
(3) The proteinase K is prepared by preparing proteinase K of 60 mg/mL by using 10mM Tris-HCl (pH8.0), then adding 100% glycerol of equal volume to prepare proteinase K stock solution of 30 mg/mL, and preserving at-20 ℃.
(4) The preparation of the lysis reagent comprises taking 400 mg Triton X-100, 400 mg Tween 20, 468 mg NaCl in a 15 mL centrifuge tube, adding 4 mL 1M Tris-HCl (pH 8.0), 800 μL 0.5M EDTA, 10 μL 100 mM sodium methylparaben, and adding sterilized water to constant volume to 10 mL.
(6) The preparation of the magnetic bead binding reagent comprises the steps of taking 500 mg PEG8000 into a 15 mL centrifuge tube, then adding 8 mL 5M NaCl and 500 mu L1M HEPES, and adding sterilized water to fix the volume to 10 mL.
Example 2
The embodiment provides a method for extracting a microbial genome based on the magnetic bead method mixed microbial genome extraction kit of the embodiment 1, the flow chart of the extraction method is shown in fig. 4, and it should be noted that the method of the embodiment is mainly used for scientific research and clinical test, but not for diagnosis purposes, and the specific steps are as follows.
S01, placing a sample containing unknown pathogenic microorganisms (short for test sample) into a centrifuge tube, wherein the test sample generally comprises 1X 10 5-1×108 bacteria. The sample sources comprise body fluid samples, urine samples and/or blood samples, the samples to be tested are centrifuged in a centrifuge for 2 minutes at 12000 rpm, the supernatant is discarded, and the sediment is collected and is thalli.
S02, adding 80 mu L of wall breaking reagent in the microbial genome extraction kit into the thalli, mixing uniformly by vortex, then adding 5 mu L of wall breaking enzyme composition reagent, mixing uniformly by vortex, and fully breaking the walls of the thalli at 37 ℃ in a 400 rpm-temperature bath for 30 minutes.
S03, adding 5 mu L of proteinase K and 30 mu L of lysis reagent into the bacterial cells after the reaction, and then heating at 70 ℃ for 400 rpm min to fully lyse the bacterial cells.
S04, continuously adding 20 mu L of magnetic beads, 180 mu L of nucleic acid sedimentation aid and 75 mu L of magnetic bead binding reagent into the lysed thalli, and uniformly mixing by vortex.
S05, continuously adding 30-200 mu L of magnetic bead eluting reagent into a sample to be detected containing magnetic beads, uniformly mixing by vortex, and standing for 5 minutes to obtain the gene to be detected.
Further, the specific steps of S04-S05 include:
1) And (3) adding 20 mu L of magnetic beads into the sample to be tested after the S04 treatment, uniformly mixing by vortex, and standing for 5 minutes at room temperature. The magnetic beads comprise silica hydroxyl magnetic beads with the particle size of 1 μm, silica carboxyl magnetic beads with the particle size of 300 nm, silica carboxyl magnetic beads with the particle size of 600 nm and silica hydroxyl magnetic beads with the particle size of 500 nm.
2) And placing the sample to be tested, into which the magnetic beads are added, on a magnetic rack, and sucking out supernatant after the magnetic beads are completely gathered.
3) Adding 200 mu L of washing liquid into a sample to be detected containing magnetic beads, rotating and washing on a magnetic rack, standing, and sucking out the supernatant as much as possible.
4) The above steps are repeated.
5) And taking the sample off the magnetic rack, standing for 5-10 minutes, and completely volatilizing the residual liquid.
6) And continuously adding 30-200 mu L of magnetic bead eluting reagent into the sample to be detected containing the magnetic beads, uniformly mixing by vortex, and standing for 5 minutes.
7) And placing a sample to be detected containing magnetic beads on a magnetic rack, and sucking out supernatant after the magnetic beads are completely gathered to obtain a purified genome.
The magnetic beads need to adsorb nucleic acid under certain conditions, such as a high-salt and low-pH solution, and then the solution is replaced by a low-salt solution to release the nucleic acid on the magnetic beads.
Example 3
This example compares the performance of genomes extracted by different kits. The sample to be extracted comprises (i) a candida albicans sample, (ii) an escherichia coli sample, (iii) a staphylococcus aureus sample, and (iv) a mixed bacterial liquid sample of candida albicans, escherichia coli, staphylococcus aureus and enterococcus faecium.
Extraction reagent the mixed microorganism genome extraction kit, the Tiangen bacterium genome DNA extraction kit (control 1) and the Takara high-efficiency extraction and refining yeast genome DNA kit (control 2) provided in the example 1.
The method steps of (I) extraction using different kits are as follows.
1. The mixed microbial genome extraction kit provided in example 1 was used for extraction.
1) Taking 1 mL of candida albicans with the concentration of 2 OD/mL as a test sample 1, 1 mL of escherichia coli with the concentration of 1 OD/mL as a test sample 2, 1 mL of staphylococcus aureus with the concentration of 1 OD/mL as a test sample 3, and 1 mL of mixed bacterial liquid containing 0.5 OD/mL of candida albicans, 0.25 OD/mL of escherichia coli, 0.25 OD/mL of staphylococcus aureus and 0.25 OD/mL of enterococcus faecium as a test sample 4.
2) The above sample was treated by the method of example 2
2. Extraction was performed using the Tiangen bacterial genomic DNA extraction kit (control 1).
(1) Extraction of Escherichia coli genome
1) An Escherichia coli sample having a concentration of 1 mL at 1 OD/mL was taken as test sample 5.
2) 10000 Rpm centrifuging for 1 min, absorbing supernatant as much as possible, adding 200 μl buffer GA into thallus, vortex mixing until thallus is thoroughly suspended, adding 20 μl proteinase K solution, vortex mixing, adding 220 μl buffer GB, shaking for 15s,70 ℃ for 10min, adding 220 μl absolute ethanol, vortex mixing for 15s, instantaneous centrifuging, adding all solutions into an adsorption column CB3, 12000 rpm centrifuging for 30s, pouring out waste liquid, placing the adsorption column CB3 into a collecting tube, adding 500 μl buffer GD into the adsorption column, 12000 rpm centrifuging for 30s, pouring out waste liquid, placing the adsorption column CB3 into the collecting tube, adding 600 μl buffer PW into the adsorption column, 12000 rpm centrifuging for 30s, pouring out waste liquid, placing the adsorption column CB3 into the collecting tube, repeating the previous step, 12000 rpm centrifuging for 2 min, placing the adsorption column CB3 into room temperature for several min to thoroughly dry residual rinse in the adsorption material, transferring the adsorption column into a new centrifuge tube, centrifuging for 120 min, adding the membrane into the centrifuge tube for 120 min, and standing for 4980 min.
(2) Extraction of staphylococcus aureus genome
1) A sample of 1 mL Staphylococcus aureus at a concentration of 1 OD/mL was taken as test sample 6.
2) 10000 Rpm centrifuging for 1 min, sucking the supernatant as much as possible, adding 110 mu L G + buffer (20 mM Tris,pH 8.0;2 mM Na2-EDTA;1.2% Triton) and 70 mu L lysozyme solution into the thallus, treating for 30min at 37 ℃, adding 20 mu L proteinase K solution, vortex mixing, adding 220 mu L buffer GB, shaking for 15s, reacting for 10 min at 70 ℃, adding 220 mu L absolute ethanol, vortex mixing for 15s, transient centrifuging, adding all the solutions into an adsorption column CB3, centrifuging for 30s 12000 rpm, pouring out the waste liquid, placing the adsorption column CB3 into a collecting tube, adding 500 mu L buffer GD into the adsorption column, 12000 rpm centrifuging for 30s, pouring out the waste liquid, placing the adsorption column CB3 into the collecting tube, adding 600 mu L buffer into the adsorption column, pouring out the waste liquid, placing the adsorption column CB3 into the collecting tube, repeating the previous steps, centrifuging for 2 min 12000, pouring out the adsorption column CB3 into a room temperature, placing the adsorption column in the air-dried for 5min, standing for 5min, eluting the residual solution into the hollow space of the hollow centrifuge tube, centrifuging for 5min, and completely eluting the residual solution in the hollow space of the hollow tube, and standing for 120 min, and pouring the residual solution into the hollow space of the hollow tube, namely, cooling for 5min, and eluting the residual solution.
3. The extract was extracted using Takara high-efficiency extraction and purification yeast genomic DNA kit (control 2).
1) A sample of Candida albicans at a concentration of 1.5 OD/mL at 1 mL was taken as test sample 7.
2) 12000 Rpm min, removing supernatant with micropipette to remove liquid as much as possible, adding 500 μl GENTLE YEAST Solution A to the pellet, shaking slightly and suspending the pellet sufficiently, bathing at 37deg.C for 1 hr, shaking gently the centrifuge tube several times during which time 100 μl GENTLE YEAST Solution B is added, shaking slightly and mixing slightly, heating at 70deg.C for 10 min, adding 200 μl GENTLE YEAST Solution C, shaking slightly and mixing slightly, cooling on ice for 5 min, centrifuging at 12000 rpm, 4deg.C for 5 min, carefully transferring supernatant into new microtubes, adding 400 μl isopropanol, mixing thoroughly upside down the centrifuge tube, centrifuging at 12000 rpm, 4deg.C for 5 min, discarding supernatant, adding 500 μl pre-cooled 70% ethanol to the pellet, washing the pellet upside down, centrifuging at 12000 rpm, 4deg.C for 5 min, removing supernatant with micropipette, naturally drying the pellet at room temperature until there is no ethanol smell, and adding 80 μl of eluting reagent to dissolve genome.
And (II) using different kits to extract genome experimental results.
(1) Genome concentration detection, namely 1 mu L of genome of different microorganism samples extracted by different kits is taken, 199 mu L Qubit working solution is added, vortex mixing is carried out, instantaneous centrifugation is carried out, and then the detection of genome concentration and total genome amount is carried out by using Qubit4.0, and the results are shown in tables 1 and 2.
(2) Genome quality test 2. Mu.L of each of the genomes extracted from test samples 1 to 7 was subjected to genome quality test using Nanodrop, and the results are shown in tables 1 and 2.
TABLE 1 microbial sample detection grouping
TABLE 2 genome detection results
As can be seen from the above results, the genome concentration of the microbiome extracted by the kit of example 1 (i.e., the kit of the present invention) was higher, the total amount was higher, and the genome quality was better (the value of A 260/A280 was substantially about 2) at the same bacterial load. Furthermore, only the kit of example 1 can extract an unknown mixed bacterial liquid sample. Further, agarose gel electrophoresis of the extracted genome of the above sample was confirmed by taking 100 ng genome and electrophoresis using 1% agarose gel at 140: 140V voltage for 25 minutes, and the results are shown in FIG. 1.
Lane 1 is the candida albicans genome extracted by the kit, lane 2 is the escherichia coli genome extracted by the kit, lane 3 is the staphylococcus aureus genome extracted by the kit, lane 4 is the mixed bacterium genome extracted by the kit, lane 5 is the candida albicans genome extracted by the Takara kit, lane 6 is the escherichia coli genome extracted by the root kit, and lane 7 is the staphylococcus aureus genome extracted by the root kit.
The result shows that compared with the genome extracted by the kit, the agarose gel electrophoresis of the genome extracted by the root kit has more obvious dispersion phenomenon, namely the degradation degree is higher, which also proves that the quality of the genome extracted by the kit is better than that of the genome extracted by the root kit.
And (III) PCR validation of genomes extracted using different kits.
1) The primer design comprises the steps of searching the 16s region sequences of enterococcus faecium and staphylococcus aureus by using NCBI database and designing the primer, referring to the primers of escherichia coli in patent CN110499378A (a method for detecting escherichia coli pathogenic bacteria from clinical blood) and the primers of candida albicans in CN102417931A (a candida albicans PCR fluorescent detection kit and a detection method). The primer sequences are shown in Table 3.
Table 3 PCR validates primers
2) And (3) performing PCR reaction, namely verifying the extracted genome according to a system, wherein each bacterium uses a primer corresponding to the bacterium to perform PCR, and the mixed bacterium uses a candida albicans primer, a staphylococcus aureus primer, an enterococcus faecium primer and an escherichia coli primer to perform PCR respectively.
TABLE 4 PCR reaction system
Table 5 PCR reaction procedure
3) Agarose gel electrophoresis was performed to verify that 10. Mu.L of the PCR product was subjected to electrophoresis using 3.5% agarose gel at 140V voltage for 30 minutes, and the results are shown in FIG. 2.
The method comprises the steps of extracting escherichia coli genome PCR products from a kit in lane 1, extracting candida albicans genome PCR products from a kit in lane 2, extracting escherichia coli genome PCR products from a kit in lane 3, extracting staphylococcus aureus genome PCR products from a kit in lane 4, extracting mixed bacterium genome escherichia coli primer PCR products from a kit in lane 5, extracting mixed bacterium genome candida albicans primer PCR products from a kit in lane 6, extracting mixed bacterium genome staphylococcus faecium primer PCR products from a kit in lane 7, extracting mixed bacterium genome staphylococcus aureus primer PCR products from a kit in lane 8, extracting escherichia coli genome PCR products from a kit in lane 9, extracting candida albicans genome PCR products from a kit in lane 10, and extracting staphylococcus aureus genome PCR products from a kit in lane 11.
The results show that the kit can normally extract single bacteria and mixed bacteria. The primer PCR of the four bacteria is used for mixed bacteria genome, and the primer PCR of the four bacteria has single bright strip at the corresponding primer fragment size, so that the kit can extract the genome of a sample containing fungi (candida albicans), gram positive bacteria (staphylococcus aureus, enterococcus faecium) and gram negative bacteria (escherichia coli) at one time.
Example 4
This example was based on the magnetic bead method mixed microorganism genome extraction kit provided in example 1 for performance comparison of genome extraction.
Genome extraction.
1. The genome was extracted using the mixed microorganism genome extraction kit provided based on example 1.
1) 1 ML Candida albicans with a concentration of 2 OD/mL is taken as a sample A, and staphylococcus aureus with a concentration of 1 mL of 1 OD/mL is taken as a sample B.
2) The above samples were treated in the same manner as in example 2 to obtain purified genomes.
2. The cell wall breaking methods of the Tiangen and Takara kits were changed, respectively, and the mixed microorganism genome extraction kit provided in example 1 was used to extract the genome.
1) 1ML Candida albicans with a concentration of 2 OD/mL is taken as sample C, and 1mL Staphylococcus aureus with a concentration of 1 OD/mL is taken as sample D.
2) The microbial samples were centrifuged in a centrifuge at 12000 rpm for 2 minutes, the supernatant was discarded and the pellet was collected. To Candida albicans cells, 85 mu L GENTLE YEAST Solution A was added, vortexed and mixed well, incubated at 37℃and 400: 400 rpm for 60 minutes, and to Staphylococcus aureus cells, 52 mu L G + buffer (20 mM Tris,pH 8.0;2 mM Na2-EDTA;1.2% Triton) and 70. Mu.L lysozyme were added, vortexed and mixed well, incubated at 37℃and 400: 400 rpm for 30 minutes.
3) Adding 5 mu L of proteinase K and 30 mu L of cell lysis reagent into the sample after wall breaking, heating at 70 ℃ for 20 minutes, adding 180 mu L of nucleic acid sedimentation aid and 75 mu L of magnetic bead binding reagent into the sample after wall breaking, mixing uniformly by vortex, adding 20 mu L of magnetic beads, standing at room temperature for 5 minutes, placing the sample on a magnetic rack, sucking out supernatant after the magnetic beads are completely aggregated, adding 200 mu L of washing liquid into the sample, spin-washing on the magnetic rack, standing for 1 minute, sucking out supernatant as much as possible, repeating the previous step, taking down the sample, standing for 5-10 minutes, until the residual liquid is completely volatilized, adding 80 mu L of magnetic bead eluting reagent into the sample, mixing uniformly by vortex, standing for 5 minutes, placing the sample on the magnetic rack, sucking out the supernatant after the magnetic beads are completely aggregated, and obtaining the purified genome.
3. The genome was extracted by using other reagents of the mixed microorganism genome extraction kit provided in example 1, respectively, in the manner of lysis of the Tiangen and Takara kits.
1) 1ML Candida albicans with a concentration of 2 OD/mL was taken as sample E, and Staphylococcus aureus with a concentration of 1mL at 1 OD/mL was taken as sample F.
2) Adding 80 μL of wall breaking reagent into thallus, mixing by vortex, adding 5 μL of wall breaking enzyme composition reagent, mixing by vortex, and bathing at 37deg.C and 400 rpm for 30min.
3) Adding 35 mu L GENTLE YEAST Solution B into a sample E after wall breaking, adding 5 mu L of proteinase K Solution and 30 mu L of buffer Solution GB into a sample F after wall breaking, carrying out vortex mixing uniformly, then heating at 70 ℃ for 20 minutes, adding 180 mu L of nucleic acid sedimentation aid and 75 mu L of magnetic bead binding reagent into a sample after cracking, carrying out vortex mixing uniformly, adding 20 mu L of magnetic beads, carrying out vortex mixing uniformly, standing at room temperature for 5 minutes, placing the sample on a magnetic frame, sucking out supernatant after the magnetic beads are completely aggregated, adding 200 mu L of washing liquid into the sample, carrying out rotary washing on the magnetic frame, standing for 1 minute, sucking out supernatant as much as possible, repeating the previous step, taking down the sample, standing for 5-10 minutes, adding 80 mu L of magnetic bead eluting reagent into the sample, carrying out vortex mixing uniformly, standing for 5 minutes, placing the sample on the magnetic frame, and sucking out supernatant after the magnetic beads are completely aggregated, thus obtaining purified genome.
TABLE 6 reagent components and contents of different extraction methods used for extracting genome by the kit
And (II) genome concentration and quality detection.
(1) Genome concentration detection 1. Mu.L of the extracted genome was taken, 199. Mu L Qubit working solution was added, vortexed and mixed well, and after transient centrifugation, the genome concentration was detected using Qubit4.0, and the results are shown in Table 7.
(2) Genome quality test 2. Mu.L of genome was taken and genome quality test was performed using Nanodrop, and the results are shown in Table 7.
TABLE 7 test results of kit Performance
The above results show that the extraction efficiency of the genome is significantly reduced after the wall breaking method of the kit of example 1 is replaced by the radix et rhizoma zingiberis and Takara wall breaking method, which is manifested in less total amount of the extracted genome and poorer quality of the extracted genome.
The genome extraction efficiency was also significantly reduced after the cleavage method of the kit of example 1 was changed to the radix et rhizoma Nardostachyos, takara cleavage method, which was shown to be less in total amount of extracted genome and poorer in quality of extracted genome.
Example 5
The embodiment provides verification of wall breaking effect of lysozyme composition.
The main component of bacterial cell walls is peptidoglycan (peptidoglycan), also known as mucin (mucopetide).
Bacterial cell walls can be classified into g+ bacteria (i.e., gram-positive bacteria) and G-bacteria (i.e., gram-negative bacteria) according to their structure and chemical composition. G+ bacteria have a relatively thick cell wall (20-80 nm), but have a relatively single chemical composition, and contain only 90% peptidoglycan and 10% teichoic acid, and G-bacteria have a relatively thin cell wall (10-15 nm), but have a multi-layer structure (peptidoglycan, lipopolysaccharide layer, etc.), and the chemical composition contains a certain amount of lipid, protein, etc. in addition to peptidoglycan. The yeast cell wall is rich in various bioactive substances such as beta-glucan, mannan (MOS) and the like.
The wall breaking treatment of the mixed microorganism sample is difficult to realize by single lysozyme. Complex enzymes can be realized, but the combination mode is diversified and has higher cost.
Lysostaphin (Lysostaphin) is a Zn 2+ -containing metalloprotease, has endopeptidase activity, and can specifically hydrolyze peptidoglycan pentaglycine peptide bond bridge (peptide bond formed by Gly at 2 nd and 3 rd positions) of bacterial cell wall, so that bacterial cell wall is rapidly dissolved to generate wall-breaking and lysozyme effects. The invention realizes the mild treatment of the mixed sample of the multiple microorganisms by adding the lysostaphin with a proper proportion.
TABLE 8 verification of the Gene quantity extracted after cleavage by different Complex enzymes
The embodiments of the present invention have been described above with reference to the accompanying drawings, but the present invention is not limited to the above-described embodiments, which are merely illustrative and not restrictive, and many forms may be made by those having ordinary skill in the art without departing from the spirit of the present invention and the scope of the claims, which are to be protected by the present invention.
Claims (7)
1. A magnetic bead method microorganism genome extraction kit is characterized by comprising a microorganism wall breaking unit and a microorganism cracking unit, wherein the microorganism wall breaking unit comprises a wall breaking reagent and a wall breaking enzyme composition reagent, the microorganism cracking unit comprises a protease reagent and a cracking reagent,
The wall breaking reagent consists of 5-10 mM dithiothreitol, 10-20 mM tris (hydroxymethyl) aminomethane hydrochloride, 1-5 mM ethylenediamine tetraacetic acid and 0.1-0.5% polyethylene glycol octyl phenyl ether, wherein the pH value of the wall breaking reagent is in the weak alkalinity of 7.5-8.5, and the unit dosage of each group of wall breaking reagent in the magnetic bead method microorganism genome extraction kit is not less than 80 mu L;
The wall-breaking enzyme composition reagent consists of muramidase, lysozyme and lysostaphin, wherein the enzyme activity ratio of the muramidase to the lysozyme to the lysostaphin is 100:100:3, and the unit dosage of each group of wall-breaking enzyme reagent arranged in the magnetic bead method microorganism genome extraction kit is not less than 5 mu L;
the protease reagent is set as proteinase K, and the concentration is 20-50 mg/mL;
The cracking reagent consists of 200-400 mM of tris hydrochloride, 20-50 mM of ethylenediamine tetraacetic acid, 1-5% of polyethylene glycol octyl phenyl ether, 500-1000 mM of sodium chloride, 0.1-0.5mM of sodium methylparaben and 1-5% of tween, wherein the pH value of the cracking reagent is in weak alkaline state of 7.5-8.5;
The unit dosage of each group of protease reagent in the magnetic bead method microorganism genome extraction kit is not less than 5 mu L, and the unit dosage of each group of lysis reagent is not less than 30 mu L;
The magnetic bead method microorganism genome extraction kit is also provided with magnetic beads with carboxyl and/or hydroxyl modified on the surfaces, a high-salt magnetic bead binding reagent and a low-salt magnetic bead eluting reagent, wherein the high-salt magnetic bead binding reagent consists of 30-50 mM 4-hydroxyethyl piperazine ethane sulfonic acid, 1-4M sodium chloride and 5% polyethylene glycol 8000, and the low-salt magnetic bead eluting reagent is nuclease-free water.
2. The kit for extracting genome of microorganism according to claim 1, wherein the wall breaking reagent comprises 5 mM dithiothreitol, 20 mM tris hydrochloride, 2 mM ethylenediamine tetraacetic acid and 0.1% polyethylene glycol octyl phenyl ether, and the pH value of the wall breaking reagent is 8.
3. The kit for extracting genome of microorganism according to claim 1, wherein the lysis reagent comprises 400 mM of tris hydrochloride, 40 mM of ethylenediamine tetraacetic acid, 5% of polyethylene glycol octylphenyl ether, 800 of mM of sodium chloride, 0.1 of mM of sodium methylparaben and 5% of tween, and the pH value of the lysis reagent is 8.
4. A method for extracting a genome of a microorganism based on the magnetic bead method microorganism genome extraction kit of any one of claims 1 to 3, comprising the steps of:
s01, collecting a sample containing pathogenic microorganisms, wherein the sample comprises a body fluid sample or a urine sample, and the microorganisms comprise one or more of fungi, gram positive bacteria and/or gram negative bacteria;
S02, respectively adding a wall breaking reagent and a wall breaking enzyme composition reagent in a kit into the sample containing the pathogenic microorganism, and reacting for a short time under the condition of the 1 st temperature;
S03, continuously adding a protease reagent and a lysis reagent in a kit into the sample containing the pathogenic microorganisms respectively, and reacting for a short time under the condition of the 2 nd temperature, wherein the 2 nd temperature is higher than the 1 st temperature and the 2 nd temperature inactivates the wall breaking enzyme composition;
S04, continuously adding magnetic beads and magnetic bead binding reagents in a kit into the reacted sample containing the pathogenic microorganisms, uniformly mixing by vortex, and carrying out adsorption extraction on nucleic acid of the pathogenic microorganisms;
s05, eluting the magnetic beads adsorbed with the nucleic acid by using a magnetic bead eluting reagent in the kit, and releasing to obtain a nucleic acid sample.
5. The method of claim 4, wherein the body fluid sample comprises a blood sample.
6. The method of claim 4, wherein the 1 st temperature condition is set to 30-40 ℃ and the 2 nd temperature condition is set to 70-80 ℃.
7. The method of claim 4, wherein the short-time reaction is set to a reaction time of no more than 30 minutes.
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