CN111518869A

CN111518869A - Nucleic acid enrichment method for fragile microorganisms on cell surface and application of nucleic acid enrichment method in high-throughput sequencing

Info

Publication number: CN111518869A
Application number: CN202010075837.8A
Authority: CN
Inventors: 盖伟; 边素莹
Original assignee: Micro Rock Medical Technology Beijing Co ltd
Current assignee: Micro Rock Medical Technology Beijing Co ltd
Priority date: 2020-01-22
Filing date: 2020-01-22
Publication date: 2020-08-11
Anticipated expiration: 2040-01-22
Also published as: CN111518869B

Abstract

The invention discloses a nucleic acid enrichment method of fragile microorganisms on the cell surface, which is characterized by comprising the following steps: step S1, detecting fragile microorganisms on the cell surface in a sample of the subject, step S2, sample preparation, step S3, performing library construction and sequencing on microorganism DNA, and step S4, analyzing data and giving a report. The invention also discloses application of the nucleic acid enrichment method of the fragile microorganism on the cell surface in high-throughput sequencing. The nucleic acid enrichment method of the fragile microorganisms on the cell surface can selectively protect the integrity of the fragile microorganisms, greatly improve the detection positive rate of the microorganisms in a sample, improve the type and range of pathogenic microorganisms detected by a high-throughput sequencing method, and improve the significance of auxiliary diagnosis by the technical means; the detection result has high detection efficiency, and is suitable for the fields of detection of fragile microorganisms in clinical samples, research on the microbial communities and the like.

Description

Nucleic acid enrichment method for fragile microorganisms on cell surface and application of nucleic acid enrichment method in high-throughput sequencing

Technical Field

The invention relates to the technical field of microbial detection, in particular to a method for enriching nucleic acid of a microbe with a fragile cell surface (thin/unstable/cell wall-free and the like) in a clinical sample based on high-throughput sequencing and application thereof.

Background

In recent years, with the application of next-generation sequencing technologies, metagenomic pathogen sequencing has made high-throughput, accurate genetic studies possible in the diagnostic field. However, in clinical samples, the content of pathogens is very small compared with that of host cells, and the detection sensitivity of pathogens is greatly reduced, so that the enrichment of pathogens plays an important role in the detection of microorganisms in clinical samples.

Currently, the enrichment of pathogens in the Extraction process is mainly divided into two forms of Pre-Extraction enrichment (Pre-Extraction) and Post-Extraction enrichment (Post-Extraction). Enrichment before extraction mainly occurs on a cell level, separation of a host and microbial cells is completed through cell level difference, microbial cells are obtained after host nucleic acid is removed, and microbial nucleic acid is enriched through a nucleic acid extraction step; enrichment after extraction mainly occurs at a nucleic acid level, microbial nucleic acid is captured in a targeting manner through a probe method after cells are broken, or enrichment is completed through methylation difference of host and microbial nucleic acid.

In the mode of enrichment after extraction, the enrichment targeting range after extraction by a probe method is small and the application is limited; methylation differences make it difficult to distinguish between host and eukaryotic microbial nucleic acids (and portions of prokaryotic nucleic acids). Before extraction, enrichment needs to consider that the common pathogens of clinical microorganisms have larger difference of cell wall surface structures, gram-positive bacteria (with more surface peptidoglycan layers) and fungi with stronger rigidity exist, and microorganisms with thin/unstable cell walls (such as mycoplasma, chlamydia, partial gram-negative bacteria and the like) exist, and the microorganisms are easily interfered and removed when host cells are broken, so that the pathogens are missed in sequencing results, and the disease condition is wrongly judged.

Based on the background, the invention aims to provide an enrichment mode aiming at fragile microorganisms on the cell surface in a metagenome-based high-throughput microorganism sequencing technology.

The invention constructs lysate proportioning combination for distinguishing host cells and fragile microorganisms (such as mycoplasma, chlamydia and the like) on the cell surface based on a high-throughput sequencing technology. The invention can distinguish host cells from the cell surfaces of pathogens (such as mycoplasma, chlamydia and the like). Based on the detection method disclosed by the invention, the detection sensitivity of the pathogens in high-throughput sequencing can be effectively improved, and the detection rate of the related pathogens in the high-throughput sequencing is improved.

Disclosure of Invention

The invention mainly aims to provide a nucleic acid enrichment method of fragile microorganisms on the cell surface and application thereof in high-throughput sequencing, the method can selectively protect the integrity of the fragile microorganisms while cracking host cells by developing a novel lysate formula, greatly improves the detection positive rate of the microorganisms in a sample, perfects the type and range of pathogenic microorganisms detected by a high-throughput sequencing method, and improves the significance of auxiliary diagnosis by the technical means; the detection result has high detection efficiency, and is suitable for the fields of detection of fragile microorganisms in clinical samples, research on the microbial communities and the like.

In order to achieve the above purposes, the technical scheme adopted by the invention is as follows: a method for enriching nucleic acid of a fragile microorganism on the surface of a cell, which comprises the following steps:

step S1, detecting cell surface fragile microorganisms in the subject sample: detecting the fragile microorganisms on the cell surface in the sample of the subject by adopting a high-throughput sequencing detection method for detecting the fragile microorganisms on the cell surface in the sample of the subject;

step S2, sample preparation: selecting a nucleic acid extraction sample, and then sequentially carrying out host cell lysis, host nucleic acid removal, cell surface fragile microorganism lysis and nucleic acid extraction on a clinical sample by adopting a lysis reagent; wherein the host nucleic acid removing reagent component is TURBO^TM1-5 muL of DNase (2U/. mu.L); cell surface fragile microorganism lysis and nucleic acid extraction are completed by using QIAampUCP Pathologen Mini Kit or other kits with similar functions;

s3, constructing a library of the microbial DNA and sequencing the library;

and step S4, analyzing the data and issuing a report.

Further, the method for detecting fragile microorganisms on the cell surface in the sample of the subject by high throughput sequencing in step S1 is characterized by comprising the following steps:

step C1: extracting DNA of a cell surface fragile microorganism in a subject sample;

step C2: constructing a library of the microbial DNA obtained in the step S1 and sequencing the library;

step C3: the sequencing data of step S2 is subjected to bioinformatic analysis in a database to determine whether a sequence of microorganisms is present, thereby determining whether the patient is infected with the species of microorganism.

Further, the nucleic acid extraction sample type in step S2 includes at least one of peripheral blood, alveolar lavage fluid, cerebrospinal fluid, and sputum.

Further, in step S2, the lysis reagent components are: 0.5 to 2 percent of beta-octyl glucosinolate and 0.5 to 2 percent of beta-dodecyl maltoside, preferably 1 percent of beta-octyl glucosinolate and 1 percent of beta-dodecyl maltoside.

Further, in step S3, performing library construction and sequencing on the microbial DNA, specifically: constructing a library of the prepared nucleic acid; constructing a library, wherein the library construction comprises the procedures of nucleic acid fragmentation, linker addition, library amplification and the like; the library construction can be realized by an Illumina platform, an Ion torrent platform or other high-throughput sequencing platforms with the same/similar functions; and performing operational machine sequencing according to the instruction of an Illumina platform, an Ion torrent platform or other high-throughput sequencing platforms with the same/similar functions.

Further, the data analysis and reporting in step S4 includes evaluation of sequencing data quality, removal of host sequences, alignment with microbial databases, interpretation of positive microbial species, and reporting.

Another object of the present invention is to provide a method for enriching nucleic acids of the fragile microorganisms on the cell surface for application in high-throughput sequencing.

Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages:

(1) the invention relates to a nucleic acid enrichment method of a cell surface fragile microorganism and application thereof in high-throughput sequencing, wherein a detection object is the cell surface fragile microorganism in a clinical sample, and the cell surface fragile microorganism comprises mycoplasma, chlamydia, part of gram-negative bacteria, part of parasites and the like which are not supported by firm cell walls.

(2) The invention relates to a nucleic acid enrichment method of a fragile microorganism on the cell surface and application thereof in high-throughput sequencing, which constructs a lysate proportioning combination for distinguishing host cells and the fragile microorganism on the cell surface (such as mycoplasma, chlamydia and the like), can selectively protect the integrity of the fragile microorganism while cracking the host cells by developing a novel lysate formula, greatly improves the detection positive rate of the microorganism in a sample, perfects the type and range of the pathogenic microorganism detected by the high-throughput sequencing method, and improves the significance of auxiliary diagnosis by the technical means.

(3) The nucleic acid enrichment method of the fragile microorganisms on the cell surface and the application of the nucleic acid enrichment method in high-throughput sequencing have high detection efficiency of detection results, and are suitable for the fields of fragile microorganism detection in clinical samples, the research of microbial communities and the like.

(4) The invention relates to a nucleic acid enrichment method of fragile microorganisms on the cell surface and application thereof in high-throughput sequencing, wherein the method can distinguish host cells from the cell surface of pathogens (such as mycoplasma, chlamydia and the like). Based on the detection method disclosed by the invention, the detection sensitivity of the pathogens in high-throughput sequencing can be effectively improved, and the detection rate of the related pathogens in high-throughput sequencing is improved.

Detailed Description

The following description is presented to disclose the invention so as to enable any person skilled in the art to practice the invention. The preferred embodiments in the following description are given by way of example only, and other obvious variations will occur to those skilled in the art.

The experimental methods used in the following examples are all conventional methods unless otherwise specified; materials, reagents and the like used in the following examples are commercially available unless otherwise specified; unless otherwise defined, terms used herein have the ordinary meaning as understood by those of ordinary skill in the art. The following lists some explanations of terms used herein, and the explanations of these terms shall control the definitions herein, unless otherwise specified.

The term "cell surface fragile microorganism" refers to a class of microorganisms in which the cell wall is thin/unstable, including mycoplasma, chlamydia, part of gram-negative bacteria, part of parasites, and the like.

In the present invention, the clinical sample may be any clinical sample type, such as peripheral blood, alveolar lavage fluid, cerebrospinal fluid, sputum, etc. When the collected sample is a viscous substance, for example, the treatment process of the sputum comprises the steps of adding a liquefying agent with the volume of 1-3 times into the sample for liquefying for 15-30 min, then centrifuging at 12000rpm for 5min, collecting the precipitate, washing the precipitate for 2 times by using 1mL of physiological saline, collecting the precipitate and performing nucleic acid extraction. Collecting non-viscous substance such as peripheral blood and cerebrospinal fluid, directly centrifuging at 8000rpm for 5min, collecting precipitate, and extracting nucleic acid.

In the present invention, the extraction of the microorganism from which the host nucleic acid has been removed from the sample is carried out using QIAamp UCP pathway MiniKit or other kit having the same/similar function.

In the present invention, the sample Library construction kit can be implemented by using corresponding Library construction kit for different high throughput sequencing platforms, and the method provided in the embodiments herein is KAPA Library Preparation kit (Illumina platform).

In the present invention, sequencing can be performed by any high-throughput sequencing technology known in the art (library construction and on-machine sequencing kit equipped with corresponding platform, etc.), and the embodiments of the present invention provide methods such as Illumina sequencing platform (Nextseq550, etc.).

Since conventional differential lysis in the extraction of microorganisms from clinical specimens results in the massive lysis of fragile microorganisms and the digestion of nucleic acids, the detection rate of such microorganisms is severely limited. The invention provides a novel nucleic acid enrichment mode, which can avoid the cracking of microorganisms with fragile cell walls along with a host by selectively cracking the host cells through a novel lysate and improve the detection sensitivity.

The process in the method of the invention is as follows: extracting sample nucleic acid (including host cell lysis, host nucleic acid removal, microbial cell lysis, microbial nucleic acid enrichment and the like); constructing a library (using methods known in the art, including, for example, end-point repair, linker addition, PCR library enrichment, etc.); detecting and identifying the quality and concentration of the library through the Qubit and Agilent 2100; and (4) performing computer sequencing and completing data analysis.

Example 1

Cracking effect of different cracking components

Sample type and test microorganism

1. Sample preparation: fresh peripheral blood of adult

2. Testing of microorganisms:

a) gram-negative bacteria: pseudomonas aeruginosa (CGMCC 1.15148), Escherichia coli (CGMCC 1.12883);

b) mycoplasma: mycoplasma pneumoniae (ATCC 15531-TTR);

c) chlamydia: chlamydia pneumoniae (ATCC 53592).

Second, comparison method

Five tubes of 3mL samples (tubes divided after fresh peripheral blood was spiked with test microorganisms) were taken in parallel and numbered 1-A, 1-B, 1-C, 1-D and 1-E, and were compared by using different cell lysate components in the extraction.

TABLE 1 cell lysate formulation

Numbering	Cell lysate formula
		1-A	2％Tween-20
1-B	2％Saponin
		1-C	2％Triton X-100
1-D	2% β -octyl thioglucoside
		1-E	2% β -dodecyl maltoside

Third, the experimental process

Nucleic acid extraction

(1) Centrifuging at 8000rpm for 5min, and discarding the supernatant;

(2) add 550 μ L PBS to the pellet to resuspend;

(3) the different cracking components were added as in table 1;

(4) incubating at room temperature for 10 min;

(5) centrifuging at 12000rpm for 5min, and discarding the supernatant (residual volume about 50 μ L);

(6) adding 5 mu L of 10 Xbuffer and 2 mu L of Turbo DNase into the precipitate;

(7) incubating at 37 ℃ for 15 min;

(8) centrifuging at 12000rpm for 5min, and discarding the supernatant;

(9)200 μ L PBS suspension precipitation;

(10) centrifuging at 12000rpm for 5min, and collecting precipitate.

The following steps were performed with the QIAamp UCP Pathologen Mini Kit.

(11) Adding 500 μ L Buffer ATL (containing DX) to the centrifuge tube, and resuspending the precipitate;

(12) swirling the centrifuge tube at maximum speed for 10 min;

(13) centrifuge tube 8000x g for 5s to remove water drop in the tube

(14) Carefully transfer 400. mu.l of supernatant into a fresh 2ml new centrifuge tube

(15) Continuously adding 40 mul of proteinase K, vortex mixing for 10s, and instantaneous centrifuging for 3s

(16) Incubating at 56 deg.C for 10min

(17) 200. mu.l of buffer APL2 was added to the centrifuge tube. Vortex mix for 30s with a lid and centrifuge instantaneously for 3 s.

(18) Incubating at 70 deg.C for 10 min;

(19) instantaneous centrifugation to ensure no residual liquid on the tube cover

(20) And continuously adding 300 mu l of ethanol into the centrifuge tube, covering a cover, vortex, uniformly mixing for 15-30s, and carrying out instantaneous centrifugation for 3 s.

(21) Carefully taking the mixed liquid after mixing, placing the mixed liquid in a 2mL collecting tube, centrifuging at 8000rpm for 1min, placing an adsorption column in a clean 2mL collecting tube, and discarding the test tube containing the filtrate

(22) Mixing the rest liquid in the step (20) and repeating the operation according to the step (21)

(23) Carefully open the lid of the adsorption column and add 600. mu.l of Buffer APW1, centrifuge at 8000rpm for 1min with the lid closed, place the adsorption column in a clean 2ml collection tube, discard the tube containing the filtrate

(24) The adsorption column was carefully uncapped and supplemented with 750. mu.l of Buffer APW2 and the lid was closed. Centrifuging at 14,000rpm for 3min

(25) It is recommended that the adsorption column be transferred to a new collection tube of 2ml and the tube containing the filtrate discarded, centrifuged at 14,000rpm for 1min

(26) Place the adsorption column in a fresh 2ml collection tube, discard the tube containing the filtrate, open the lid and incubate at 56 ℃ for 3min to ensure complete drying of the column membrane

(27) Placing the adsorption column in a new 1.5ml elution tube, discarding the tube containing the filtrate, carefully adding 20-100. mu.l Buffer AVE on the column membrane, covering with a cover, and standing at room temperature for 1min

(28) The mixture was centrifuged at 14,000rpm for 1min to collect nucleic acids.

Library construction

The kit used was KAPA Library Preparation Kits. The library construction process comprises four steps of end repair, end adding A, adding a joint and library amplification.

2.1 end repair

(1) Taking 50 mu L of the enriched and extracted free nucleic acid DNA, adding 8 mu L of nuclease-free water, 7 mu L of end repairing buffer solution and 5 mu L of end repairing enzyme, and uniformly mixing;

(2) the reaction tube was placed in a PCR apparatus and the reaction was carried out according to the following procedure: 20 ℃ for 30 min;

(3) adding 120 μ L (1.7X) KAPA purified magnetic beads into the reaction tube, mixing with vortex, and standing at room temperature for 5 min;

(4) placing the reaction tube on a magnetic plate, standing for 3min until the solution is clear, and removing the supernatant;

(5) add 200 μ L of freshly prepared 80% ethanol to the tube, incubate at room temperature for 30s, remove supernatant;

(6) repeating the step 5 once; drying at room temperature for 3-5min, and taking off the tube from the magnetic plate;

2.2 addition of A at the end

(1) Preparing a mixed solution A at the tail end: taking a 200 mu L PCR tube, adding 42 mu L nuclease-free water, 5 mu L end buffer solution A and 3 mu L end enzyme A in sequence, mixing uniformly, and performing instantaneous centrifugation;

(2) adding the prepared reaction solution into the first step, drying the reaction solution in a tube containing magnetic beads at room temperature, and mixing the reaction solution uniformly in a vortex manner;

(3) the reaction tube was placed in a PCR instrument and the following procedure was run: 30min at 30 ℃;

(4) after the reaction is finished, adding 90 mu L of PEG/NaCl solution into the reaction solution, mixing uniformly by vortex, and incubating for 10-15min at room temperature;

(5) placing the reaction tube on a magnetic plate, standing for 3min until the solution is clear, and removing the supernatant;

(6) add 200 μ L of freshly prepared 80% ethanol to the tube, incubate at room temperature for 30s, remove supernatant;

(7) repeating the step 6 once; drying at room temperature for 3-5min, and taking off the tube from the magnetic plate;

add the piecing

(1) Preparing a joint connection mixed solution: adding 30 μ L of nuclease-free water, 10 μ L of ligation buffer solution and 5 μ L of DNA ligase into a 200 μ L PCR tube in sequence, and mixing by vortex

(2) Adding the prepared joint connection mixed solution into the magnetic beads dried in the second step, and adding 5 mu L of corresponding joint storage solution;

(3) putting the reaction tube into a PCR instrument for gathering, and reacting according to the following procedure at 20 ℃ for 15 min;

(4) after the reaction is finished, adding 50 mu L of PEG/NaCl solution into the reaction solution, mixing uniformly by vortex, and incubating for 10-15min at room temperature;

(7) repeating the step 6 once;

(8) drying at room temperature for 3-5min, and taking off the tube from the magnetic plate;

(9) resuspend the beads with 50. mu.L of elution buffer and incubate at room temperature for 2min

(10) Add 50. mu.L of PEG/NaCl solution to the above bead solution pool

(11) Mixing uniformly by vortex, incubating for 5-15min at room temperature,

(12) performing instantaneous centrifugation, placing the tube on a magnetic frame, standing for 2min until the solution is clear, and removing the supernatant;

(13) add 200 μ L of freshly prepared 80% ethanol to the tube, incubate at room temperature for 30s, remove supernatant;

(14) repeat step 13 once

(15) Placing the tube on a magnetic frame, drying at room temperature for 3-5min, adding 25 μ L of elution buffer solution into the manifold, resuspending magnetic beads, and incubating at room temperature for 2 min;

(16) placing the tube on a magnetic frame, standing for 2min until the solution is clear, transferring 20 μ L of the supernatant to a new 200 μ L tube, and keeping

2.4 library amplification

(1) Adding 25 mul of amplification buffer solution and 5 mul of primer mixed solution into the 20 mul of nucleic acid solution prepared in the third step, and uniformly mixing;

(2) placing the reaction tube in a PCR instrument, and reacting for 1 cycle at the temperature of 98 ℃ for 45s according to the following procedure; 98 ℃, 15s → 60 ℃, 30s → 72 ℃, 1min, 8-10 cycles; 72 ℃ for 1min, 1 cycle, 4 ℃ hold

(3) After the reaction is finished, adding 50 μ L (1 ×) KAPA purified magnetic beads into the reaction tube, mixing by vortex, and standing at room temperature for 5 min;

(6) repeating the step 5) once;

(7) drying at room temperature for 3-5min, and taking off the tube from the magnetic plate;

(8) adding 30 μ L of elution buffer solution, mixing, and incubating at room temperature for 2 min;

(9) the tube was placed on a magnetic frame and allowed to stand for 2min until the solution became clear, and the supernatant was transferred to a 200 μ L PCR tube for Qubit quantification and Agilent2100 library quality identification.

3. Sequencing on machine

Sequencing can be performed by using an Illumina platform instrument such as Nextseq550 and the like according to commercial instructions.

4. Data analysis and reporting

Fourth, experimental results

On-machine sequencing result statistics, as shown in table 2, the five methods have no significant difference on the host cell lysis effect.

However, as shown in Table 3, the specific reads numbers of mycoplasma, chlamydia and two gram-negative bacteria were compared, and the specific reads number ratio of the four microorganisms detected in the two D/E lysis methods (i.e., β -octylthioglucoside and β -dodecylmaltoside as lysates) was the highest, and the A/B/C effect was weak and no significant difference was observed. The mycoplasma class has the most obvious effect (the lifting effect is about 5-10 times) by the D method; chlamydia, the most significant (about 10 times) improvement effect is achieved by the E method; the specific reads number of gram-negative bacteria (Escherichia coli and pseudomonas aeruginosa) is similar to that of D/E method, and is improved by about 50 percent.

The reason is presumed to be that the difference exists on the surfaces of mycoplasma and chlamydia cells, so that the method is suitable for different types of lysates; and 2-3 layers of cell walls composed of peptidoglycan exist in gram-negative bacteria, so that the gram-negative bacteria have stronger protection effect on microbial cells than mycoplasma and chlamydia.

TABLE 2 comparison of host lysis results for different lysis methods

	Total reads number	Host reads number (ratio)
			1-A	23615220	18585178(78.7％)
1-B	21589472	15630778(72.4％)
			1-C	20568432	16516451(80.3％)
1-D	19582368	14569282(74.4％)
			1-E	22356284	17661464(79.0％)

TABLE 3 comparison of the enrichment effect of microorganisms by different lysis methods

Example 2

Testing of different ratios of cracking components

Sample type and test microorganism

1. Sample preparation: fresh peripheral blood of adult

2. Testing of microorganisms:

b) mycoplasma: mycoplasma pneumoniae (ATCC 15531-TTR);

c) chlamydia: chlamydia pneumoniae (ATCC 53592);

second, comparison method

12 parallel 3mL samples (tube division after fresh peripheral blood is spiked with test microorganisms) were taken, numbered according to Table 4, and compared by different concentrations of β -octyl thioglucoside and β -dodecyl maltoside in the extraction.

Third, the experimental process

The four-vessel peripheral blood was subjected to nucleic acid extraction, library construction, on-machine sequencing and data analysis as described in example 1, wherein the concentrations were as follows.

TABLE 4 cell lysate recipe

Numbering	Cell lysate formula
		2-1A	0.5% β -octyl thioglucoside
2-1B	1% β -octyl thioglucoside
		2-1C	2% β -octyl thioglucoside
2-1D	5% β -octyl thioglucoside
		2-2A	0.5% β -dodecyl maltoside
2-2B	1% β -dodecyl maltoside
		2-2C	2% β -dodecyl maltoside
2-2D	5% β -dodecyl Maltoside
		2-3A	0.5% β -octyl thioglucoside + 0.5% β -dodecyl maltoside
2-3B	1% β -octyl thioglucoside + 1% β -dodecyl maltoside
		2-3C	2% β -octyl thioglucoside + 2% β -dodecyl maltoside
2-3D	5% β -octyl thioglucoside + 5% β -dodecyl maltoside

Fourth, experimental results

The results of the measurements are shown in tables 5 and 6 below.

As shown in Table 5, the lysis effect of the host cells is enhanced with the increase of the concentrations of the two lysates, but the host cell rejection is not obviously enhanced when the concentration is increased to more than 2%.

As shown in Table 6, the numbers of microorganism-specific reads detected in both lysates increased with increasing concentration, and the peak value was 2% of the total concentration of the lysate. Analyzing the reason, when the concentration of the lysate is low, the host elimination effect is poor, and the relative quantity of the microorganisms measured in the similar total quantity of the reads is small; reaching a microorganism detection peak value when the concentration of the lysis solution is increased to have stronger host cell lysis capacity but weaker microorganism lysis capacity which is unstable on the outer wall of the cell; as the lysate concentration is further increased, a large number of microorganisms are lysed simultaneously with the host cells and digested by the nuclease, and thus the detection amount decreases. Therefore, a total concentration of 2% was determined to be optimal during host lysis.

The specific reads number and the proportion of the microorganisms are analyzed, and the result shows that the detection effect on representative mycoplasma bacteria, chlamydia bacteria and gram-negative bacteria is better after the two lysates are mixed by 1% + 1%.

Example 3

Application in actual clinical samples

Firstly, sample type: 3 cases of adult fresh peripheral blood, 3 cases of alveolar lavage fluid, 3 cases of cerebrospinal fluid and 3 cases of sputum. All clinical samples were obtained, and the results of the institutional testing are shown in Table 7.

Second, experimental process

1. Nucleic acid extraction

1.1 the method of the invention: nucleic acid extraction was accomplished by the method of example 1, where the host cell lysate was 1% β -octyl thioglucoside + 1% β -dodecyl maltoside.

1.2 comparative methods: a comparison experiment is carried out by using a commercial Kit QIAamp DNAmicrobiome Kit, MolYsis Kit and Pureprove Kit which are used for carrying out differential lysis by adopting a similar method.

2. Library construction: library construction was accomplished using the method of example 1.

3. Computer sequencing and data analysis: the method of example 1 was used to complete the on-machine sequencing and data analysis.

Third, experimental results

As shown in Table 7, compared with several conventional commercial kits, the enrichment effect of the method of the invention on mycoplasma, chlamydia and part of gram-negative bacteria is obviously improved (by 10-50 times), and the detection effect on gram-positive bacteria is equivalent. Supposing that the host cell lysate of the conventional commercial kit has strong effect, so that part of microorganisms with fragile cell wall structures are cracked and then digested in the step, and the detection efficiency is reduced; gram-positive bacteria with harder cell walls are tolerated, so that there is no significant difference in detection efficiency.

The result shows that the method can effectively improve the detection efficiency of the cell wall fragile microorganisms in various clinical samples.

TABLE 5 comparison of host lysis effects of different lysates

	Total reads number	Host reads number (ratio)
			2-1A	18369852	17543209(95.5％)
2-1B	20658463	17601010(85.2％)
			2-1C	18635945	13697420(73.5％)
2-1D	25396784	18336478(72.2％)
			2-2A	22363968	21782505(97.4％)
2-2B	27585235	23144012(83.9％)
			2-2C	23369685	18158245(77.7％)
2-2D	20363652	15476376(76.0％)
			2-3A	17596523	14798676(84.1％)
2-3B	22365521	16885968(75.5％)
			2-3C	20954223	15317537(73.1％)
2-3D	20036987	14166150(70.7％)

TABLE 6 comparison of the enrichment effect of different lysates on microorganisms

TABLE 7 comparison of the results of enrichment of microorganisms by different methods

The foregoing is directed to embodiments of the present invention and, more particularly, to a method and apparatus for controlling a power converter in a power converter, including a power converter, a power.

Claims

1. A method for enriching nucleic acid of a fragile microorganism on the surface of a cell, which comprises the following steps:

step S2, sample preparation: selecting a nucleic acid extraction sample, and then sequentially carrying out host cell lysis, host nucleic acid removal, cell surface fragile microorganism lysis and nucleic acid extraction on a clinical sample by adopting a lysis reagent; wherein the host nucleic acid removing reagent component is TURBO^TM1-5 muL of DNase (2U/. mu.L); cell surface fragile microorganism lysis and nucleic acid extraction are completed by using QIAamp UCPPathogen Mini Kit or other kits with similar functions;

s3, constructing a library of the microbial DNA and sequencing the library;

and step S4, analyzing the data and issuing a report.

2. The method of claim 1, wherein the nucleic acid of the fragile microorganism on the cell surface is enriched,

the high throughput sequencing detection method for detecting the fragile microorganisms on the cell surface in the sample of the subject in the step S1 comprises the following steps:

3. The method of claim 1, wherein the nucleic acid sample extracted in step S2 comprises at least one of peripheral blood, alveolar lavage fluid, cerebrospinal fluid, and sputum.

4. The method of claim 1, wherein the lysis reagent of step S2 comprises: 0.5 to 2 percent of beta-octyl glucosinolate and 0.5 to 2 percent of beta-dodecyl maltoside.

5. The method of claim 1, wherein the lysis reagent of step S2 comprises: 1% beta-octylthioglucoside + 1% beta-dodecylmaltoside.

6. The method for enriching nucleic acid of fragile microorganisms on cell surface according to claim 1, wherein the DNA of said microorganisms is library-constructed and sequenced in step S3, specifically: constructing a library of the prepared nucleic acid; constructing a library, wherein the library construction comprises the procedures of nucleic acid fragmentation, linker addition, library amplification and the like; the library construction can be carried out by an Illumina platform, an Iontorrent platform or other high-throughput sequencing platforms with the same/similar functions; and performing operational sequencing according to the instruction of an Illumina platform, an Iontorrent platform or other high-throughput sequencing platforms with the same/similar functions.

7. The method of claim 1, wherein the data analysis and reporting in step S4 includes evaluating the quality of sequencing data, removing host sequences, aligning with a microbial database, identifying positive microbial species, and reporting.

8. Use of a method of enriching nucleic acids of a fragile microorganism on the cell surface according to any one of claims 1 to 7 for high throughput sequencing.