阅读说明：本技术 一种细胞表面脆弱微生物的核酸富集方法及在高通量测序中的应用 (Nucleic acid enrichment method for fragile microorganisms on cell surface and application of nucleic acid enrichment method in high-throughput sequencing ) 是由 盖伟 边素莹 于 2020-01-22 设计创作，主要内容包括：本发明公开了一种细胞表面脆弱微生物的核酸富集方法,其特征在于,包括如下步骤：步骤S1、检测受试者样品中细胞表面脆弱微生物,步骤S2、样本制备,步骤S3、微生物DNA进行文库构建并进行测序,步骤S4、数据分析和出具报告。本发明还公开了所述细胞表面脆弱微生物的核酸富集方法在高通量测序中的应用。本发明公开的细胞表面脆弱微生物的核酸富集方法可以选择性地保护脆弱微生物的完整性,大大提高了样本中该类微生物的检测阳性率,完善了高通量测序方法检测病原微生物的种类和范围,提升了通过此类技术手段辅助诊断的意义；检测结果检测效率高,适用于临床样本中脆弱微生物检测及该类微生物群落研究等领域。(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.)

1. 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 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:

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.

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.

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.