CN117126920A

CN117126920A - Central nervous infection pathogen library building and detecting method based on nanopore sequencing platform

Info

Publication number: CN117126920A
Application number: CN202310943705.6A
Authority: CN
Inventors: 周水莲; 丁然; 潘吾思; 程彪; 李诗濛; 任用
Original assignee: Jiangsu Xiansheng Medical Devices Co ltd; Nanjing Xiansheng Medical Laboratory Co ltd; Jiangsu Xiansheng Medical Diagnosis Co ltd
Current assignee: Jiangsu Xiansheng Medical Devices Co ltd; Nanjing Xiansheng Medical Laboratory Co ltd; Jiangsu Xiansheng Medical Diagnosis Co ltd
Priority date: 2023-07-31
Filing date: 2023-07-31
Publication date: 2023-11-28
Anticipated expiration: 2043-07-31
Also published as: CN117126920B

Abstract

The application belongs to the field of microorganism detection, and particularly provides a library construction and detection method based on a nanopore sequencing platform for a central nervous infected cerebrospinal fluid sample pathogen. The method combines the advantages of the nanopore sequencing platform, uses the rapid PCR bar code kit and the PCR-cDNA bar code kit together, mixes the two together, and puts on the machine, successfully builds the central nervous infection pathogen database and the detection method based on the nanopore sequencing platform, not only meets the requirement of macrogenome detection of a low initial quantity cerebrospinal fluid sample, but also obviously improves the sensitivity of detecting the pathogen difficult to detect of the cerebrospinal fluid sample.

Description

Central nervous infection pathogen library building and detecting method based on nanopore sequencing platform

Technical Field

The application belongs to the field of microorganism detection, and particularly relates to a central nervous infection pathogen library building and detection method based on a nanopore sequencing platform.

Background

Central nervous system infections (Central nervous systeminfection, CNSI) include acute or chronic encephalitis, meningitis, myelitis, etc., which are critical and fatal, with a high disability rate. The primary cause of infection is pathogenic microorganism infection. Pathogens causing central nervous infections include a large group of biological properties and taxonomic status-differentiating bacteria, viruses, cryptococcus and mold, parasites, and the like.

At present, diagnosis of central nervous system infection still highly depends on personal clinical experience of doctors, and the conventional detection technology method has low detection positive rate, limited speed and flux, difficult identification and low comprehensive detection efficiency. Rapid, broad-spectrum and high-throughput screening technology is required for diagnosis of central nervous infection, and meanwhile, the precise species identification and differential diagnosis capabilities are also required. Diagnostic methods based on metagenome sequencing technology (mNGS) meet the above requirements, but there are significant differences in the detection performance of pathogens of different biological characteristics, for example (Sheng-Yuan Yu et al 2020) with a diagnostic sensitivity of 42.6% for viral encephalitis or meningitis and 76.9% and 80.0% for cryptococcus encephalitis and aspergillosis, respectively; as the judgment is carried out only by 2-3 short-reading long sequences of 75bp, the mNGS has obvious false positive trouble, and especially the sensitivity for diagnosing tuberculosis is only 27.3 percent, which has larger difference from the expected performance index.

The Nanopore can start data analysis after a few seconds, so that valuable diagnosis time consumed by mNGS in the sequencing chemical reaction process is saved, and the requirement of rapid diagnosis of clinical infection can be met due to short turnover time. At present, the study of CNSI diagnosis method by Nanopore is limited, and bacterial and viral meningoepithymen encephalitis is evaluated mainly by using bacterial 16S amplicon and birtgun metagenome. Unlike sputum or alveolar lavage, cerebrospinal fluid is a sterile body fluid under physiological conditions, and the amount of cerebrospinal fluid obtained by a patient by puncturing is usually not more than 3mL. The sample type characteristics determine that the total amount of nucleic acid is extremely low, and have higher requirements on the sensitivity and yield of a library construction method.

In summary, the problem of difficulty in detecting pathogens with very low total amount of nucleic acid in samples of central nervous infection in the prior art still has a need for a more efficient and convenient database construction method capable of rapidly and accurately identifying central nervous infection pathogens. In view of this, the present application has been made.

Disclosure of Invention

The core problems to be solved by the application are small sample size and low total nucleic acid amount of cerebrospinal fluid for central nervous infection, and have higher requirements on the sensitivity and yield of a genome library establishment method. When the central nervous infection pathogen library construction and detection method of the nanopore sequencing platform is established, the rapid PCR bar code kit (SQK-RPB 004) and the PCR-cDNA bar code kit (SQK-PCB 111.24) are creatively combined to be used together and mixed and put on the machine, so that the detection of a metagenome of a cerebrospinal fluid sample with a low initial quantity is satisfied, the sensitivity of detecting a pathogen which is difficult to detect in the cerebrospinal fluid sample is remarkably improved, the simultaneous detection of a central nervous infection pathogen low initial quantity metagenome detection flow, a focus pathogen target flow and a DNA and RNA single flow is realized, the method has stronger targeting property and timeliness, and is suitable for clinical popularization and application.

The technical scheme adopted by the application is as follows:

the application firstly provides a central nervous infection pathogen library building and detecting method based on a nanopore sequencing platform, which comprises the following steps:

the method comprises the following steps of a metagenome process (macro process for short) and a targeting enrichment process (target process for short), wherein the specific steps are as follows:

(1) The metagenomic flow includes:

step 1) cerebrospinal fluid sample pretreatment;

step 2) metagenomic DNA extraction;

step 3) rapidly preparing a metagenomic library;

(2) The targeted enrichment procedure included:

step 1) DNA/RNA co-extraction;

step 2) PCR amplification of target pathogenic fragment;

step 3), connecting BP joint with PCR amplification and library establishment;

preferably, the method further comprises:

(3) The step of mixing the metagenome process and the targeting enrichment process

In the metagenomic flow of the step (1), the library rapid preparation of the step (3) is to use a rapid PCR bar code kit (SQK-RPB 004) for performing metagenomic library rapid preparation;

in the rapid library preparation of (2), the target procedure uses a PCR-cDNA barcode kit (SQK-PCB 111.24) for pathogen-targeted pooling.

In some embodiments, in the macroprocedural step, the pooling kit SQK-RPB004 and SQK-PBK004, preferably SQK-RPB004.

In some embodiments, due to the specificity of the cerebrospinal fluid sample, the pretreatment procedure avoids the operations of PBS washing, centrifugation to discard supernatant, etc., resulting in an absolute loss of subsequent nucleic acid yield. So the cerebrospinal fluid sample pretreatment in macro-flow step 1) is adjusted in 3 steps: 1) Cerebrospinal fluid samples were not concentrated by centrifugation: avoiding the loss of low-load pathogen caused by centrifugal supernatant discarding; 2) Adjusting the input concentration of DH: the sample input amount requirement is met, the host removal operation is not influenced, and the DH input concentration is preferably 3-5%; 3) Enzyme removal step, PBS rinsing, centrifugation and EDTA enzyme inactivation, preferably EDTA enzyme inactivation treatment.

In some specific embodiments, the step of pre-treating the cerebrospinal fluid sample comprises: the cerebrospinal fluid sample is delivered to a sterilization centrifuge tube; adding about 5% DH (saponin) and mixing; adding 0.5-1 mu L XX enzyme (HL-SAN), mixing, and incubating in metal bath under shaking; adding EDTA into the mixture, and mixing the mixture evenly by vortex.

In some embodiments, in the macroflow step, step 2) the metagenomic cerebrospinal fluid sample DNA extraction kit is preferably from Promega corporationRSC Whole Blood DNA Kit。

In some embodiments, the macroflow step, step 3) in the rapid preparation of metagenomic libraries, the reaction system for DNA fragmentation is 1-23 μl, preferably 23 μl; the fragmentation time is 1-3min, preferably 1min, at 30deg.C.

In some embodiments, in the macroflowsheet step, step 3) the metagenomic library rapidly prepares amplification cycles of 30-40cycles, preferably 35cycles.

In some embodiments, in the target flow step, step 2) PCR amplification of the pathogenic fragment of interest, the DNA multiplex enzyme is preferably Thermo Platinum ^TM Multiplex PCR Master Mix。

In some embodiments, in the target flow step, step 3) PCR amplification of the target pathogenic fragment; adaptor PCR primer efficacy optimization was determined by

An upstream primer: 5 '-TTTCTGTTGGTGCTGATATTGC-target pathogenic microorganism specific primer-3', downstream primer: 5 '-ACTTGCCTGTCGCTCTATCTTC-target pathogenic microorganism-specific primer-3'

Preferably is

An upstream primer: 5 '-TGGTGCTGATATTGC-target pathogenic microorganism-specific primer-3',

a downstream primer: 5 '-TGTCGCTCTATCTTC-target pathogenic microorganism specific primer-3'.

In some embodiments, in the target flow step, the RNA multiplex enzyme preferably comprises PrimeScript of Takara ^TM II RT Enzyme Mix and PrimeSTAR GXL for step RT-PCR.

In some embodiments, in the target flow step, step 2) the design principle of the microorganism-specific targeting primer for PCR amplification of the target pathogenic fragment (targeting primer1-4 in the example) is: the primer length is 18-21bp, the GC content is 40% -60%, the primer Tm is 50-70 ℃, the target fragment length is preferably 1-2K, and the conserved region and the variable region are covered.

In some embodiments, the steps of the metagenomic and targeted enrichment process pooling are: the DNA obtained by the metagenome process and the targeting enrichment process is independently mixed according to the total library amount; and mixing the metagenome process mixed library sample and the targeting enrichment process mixed library sample, and keeping the total mixed library amount to be more than or equal to 900ng.

In some embodiments, in the macroflow step, a metagenomic library is created using a rapid PCR barcode kit (SQK-RPB 004) and the target flow uses a PCR-cDNA barcode kit (SQK-PCB 111.24) for pathogen-targeted library creation. Although different library building kits are used in the macro-target process, the RAP connector can be connected with the RAP connector together in a mixed manner for sequencing and letter generation analysis, so that the macro-target process and the DNA-RNA process can be detected simultaneously.

In some aspects, the metagenomic identification methods of the present application may be applied to fields including, but not limited to, clinical research and scientific research.

Compared with the prior art, the application has at least the following advantages:

1) Based on a nanopore sequencing platform, the application creatively combines a rapid PCR bar code kit (SQK-RPB 004) and a PCR-cDNA bar code kit (SQK-PCB 111.24) to be put on a mixed library machine, thereby skillfully solving the problems of low initial quantity library establishment flow construction and pathogen detection of central nervous system samples.

2) Through innovation of the library establishment flow, the application not only satisfies metagenomic detection of the cerebrospinal fluid sample with low initial quantity, but also obviously improves the sensitivity of detecting the pathogen which is difficult to detect in the cerebrospinal fluid sample, realizes simultaneous detection of the central nervous infection pathogen with low initial quantity metaflow and the pathogen target flow which focuses on and DNA and RNA single flow, has stronger targeting and timeliness, and is suitable for clinical popularization and application.

3) The application also optimizes the parameters of the whole process, thereby establishing an optimal system. For example, the efficiency of the linker primer in the target process is optimized, and the linker primer is adjusted from 22bp to 15bp, so that the adjustment obviously improves the detection sensitivity of low-load pathogens; the pretreatment of the cerebrospinal fluid sample with low initial quantity is very important to the whole system, and the pretreatment of the sample in the macro flow is optimized, so that the yield of the low-load pathogen of the cerebrospinal fluid sample is improved; in addition, the application also has compatible double-flow system on the basis of ensuring the extraction yield through the optimized selection of the DNA extraction kit.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present application, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1, an overall flow chart of the present application;

FIG. 2 shows comparison of bacterial and fungal nucleic acid extraction effects against Acinetobacter baumannii (G-), listeria monocytogenes (G+), staphylococcus aureus (G+), and Cryptococcus neoformans (fungi);

FIG. 3, comparison of the extraction effect of different extraction kits against cytomegalovirus (DNA virus) and Coxsackie virus (RNA virus);

FIG. 4, SQK-RPB004 and SQK-PBK004 were compared for library construction performance, A being the total library amount; b is the total ready number of the on-line; c is the host proportion; d is microorganism detection;

FIG. 5, results of different fragmentation times and purification multipliers are compared in the process of metagenomic flow library construction;

FIG. 6, 3 comparison of the amplification effect of DNA amplicons on a target;

FIG. 7, comparison of the efficacy of the adaptor primer before and after optimization.

Detailed Description

The following description of the embodiments of the present application will be made apparent and fully in view of the accompanying drawings, in which some, but not all embodiments of the application are shown. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.

The following terms or definitions are provided solely to aid in the understanding of the application. These definitions should not be construed to have a scope less than understood by those skilled in the art.

Unless defined otherwise hereinafter, all technical and scientific terms used in the detailed description of the application are intended to be identical to what is commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present application.

As used herein, the terms "comprising," "including," "having," "containing," or "involving" are inclusive or open-ended and do not exclude additional unrecited elements or method steps. The term "consisting of …" is considered to be a preferred embodiment of the term "comprising". If a certain group is defined below to contain at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists of only these embodiments.

The indefinite or definite article "a" or "an" when used in reference to a singular noun includes a plural of that noun.

The terms "about" and "substantially" in this application mean the range of accuracy that one skilled in the art can understand yet still guarantee the technical effect of the features in question. The term generally means a deviation of + -10%, preferably + -5%, from the indicated value.

Furthermore, the terms first, second, third, (a), (b), (c), and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the application described herein are capable of operation in other sequences than described or illustrated herein.

The following are specific embodiments of the present application.

The following examples and experimental examples relate to instruments comprising: biological safety cabinet, oscillating metal bath, pipettor, centrifuge, breaking instrument, super clean bench, PCR instrument, magnetic rack, gridION, qubit 4.0.0, refrigerator, etc.

The reagents involved include:

sample preprocessing module: host removal and microbiome DNA isolation kit (pioneering diagnosis); nucleic acid extraction module: MP Biomedicals Lysing Matrix E (E-tube for short), promegaRSC Whole Blood DNA Kit (cat No. AS 1520), root micro sample genome DNA extraction kit (cat No. DP 316), root magnetic bead method pathogenMicroorganism DNA/RNA extraction kits (accession number NG 550), QIAGEN QIAamp Viral RNA Mini Kit (accession number 52904), QIAGEN AllPrep DNA/RNA Mini Kit (accession number 80204), QIAGEN MagAttract HMW DNA Kit (accession number 67563), zymoBIOMICS Quick-DNA/RNA visual Kit (accession number D7020), kaisha virus nucleic acid extraction reagent bulk (accession number RC 1016); standard/reference: zymoBIOMICS ^TM Microbial Community DNA Standard (accession number: D6305), pondenson herpes simplex virus type I deoxyribonucleic acid (HSVI DNA) liquid indoor quality control (accession number BDS-IQC-031), pondenson Cheng Ren cytomegalovirus deoxyribonucleic acid (HCMV DNA) liquid indoor quality control (accession number BDS-IQC-025), pondenson EBV virus deoxyribonucleic acid (EBV DNA) liquid indoor quality control (accession number BDS-IQC-050), pondenson Cheng Jiehe mycobacteria deoxyribonucleic acid (TB DNA) liquid indoor quality control (accession number BDS-IQC-060), pondenson Cheng Kesa Qivirus CA16 ribonucleic acid (CA 16 RNA) liquid indoor quality control (accession number BDS-IQC-067), pondenson Cheng Feiyan mycoplasma deoxyribonucleic acid (MP DNA) liquid indoor quality control (accession number BDS-IQC-009); qPCR kit: the kit comprises a quantitative detection kit (PCR-fluorescent probe method) for EB virus nucleic acid of Saint Hunan organisms, a quantitative detection kit (PCR-fluorescent probe method) for human cytomegalovirus nucleic acid of Saint Hunan organisms, a detection kit (fluorescent PCR method) for Mycoplasma Pneumoniae (MP) nucleic acid of Saint Hunan organisms, a detection kit (fluorescent PCR method) for Acinetobacter baumannii nucleic acid of Shanghai Bijie medical treatment, a detection kit (fluorescent PCR method) for Cryptococcus celer nucleic acid of Hunan organisms, a detection kit (multiple fluorescent PCR method) for six respiratory tract pathogens, a detection kit (PCR-fluorescent probe method) for Coxsackie virus A16 type nucleic acid of Saint Hunan organisms, a detection kit (fluorescent PCR method) for Mycobacterium tuberculosis nucleic acid of Saint organisms, and a detection kit (PCR-fluorescent probe method) for general type nucleic acid of herpes simplex virus of Saint Hunan organisms; nucleic acid detection kit (fluorescence PCR method) of Hangzhou associated blue organism Aspergillus fumigatus, aspergillus flavus and Aspergillus niger; nucleic acid purification reagent: AMPure XP purified beads (cat# A63881); qubit fluorescence quantitative instrument DNA detection kit: qubit 1X dsDNA HS Assay Kit (cat number: Q33231); library-building PCR amplification enzyme: takara->GXL DNA Polymer (cat# R050A), toyobo KOD OneTM PCR Master Mix (cat# KMM 101), thermo Platinum ^TM Multiplex PCR Master Mix (goods No. 4464270), takara PrimeScript ^TM II High Fidelity One Step RT-PCR Kit (cat# R026A); ONT library building kit (SQK-RPB 004, SQK-PBK004, SQK-PCB 111.24); ONT sequencing chips, and the like.

Experimental example, basic method system establishment of the application

The specific experimental flow of the central nervous infection pathogen library building and detecting method based on the nanopore sequencing platform is shown in figure 1, and the specific steps are as follows:

the detection technical flow is divided into a metagenome flow and a targeted enrichment operation flow (see an experimental flow schematic diagram)

1. Metagenomic procedure (SQK-RPB 004)

1. Cerebrospinal fluid pretreatment: (host removal and microbiome DNA isolation)

(1) Taking 800 mu L of cerebrospinal fluid sample into a sterilized 2ml centrifuge tube;

(2) Add 15. Mu.L 5% DH (saponine), 90. Mu.L SS, mix upside down;

(3) Adding 0.5-1 μl XX enzyme (HL-SAN), mixing, standing on metal bath at 1000rpm and shaking at 37deg.C for 10min;

(4) 100 μ L0.5M (pH 8.0) EDTA was added and vortexed to terminate the reaction.

2. Metagenomic DNA extraction: (refer to PromegaRSC Whole Blood DNA Kit AS1520)

The reaction product of the previous cerebrospinal fluid pretreatment (about 1000 μl) was transferred in its entirety to a Lysing Matrix E disruption tube and milling conditions were set on a fastprep-5G (MP Biomedicals) instrument: 6m/s,40 s. The E tube 14000RCF was centrifuged for 5min, and 650. Mu.L of the ground suspension was transferred to a fresh sterile EP tube for nucleic acid extraction. Specific nucleic acid extraction procedure is referred to PromegaRSC Whole Blood DNA Kit AS1520。

3. Rapid construction of metagenomic library (SQK-RPB 004)

(1) DNA fragmentation

A fragmentation reaction system was prepared in a 0.2mL PCR thin-walled amplification tube as described in the following table, and the tube cap was capped. Vortex mixing, briefly centrifuge and collect the reaction mixture at the bottom of the tube. And placing the PCR tube on a thermal cycler to start a reaction PCR tube to prepare the following system:

component (A)	Volume of
		Mixed liquid of fragment enzyme	1μL
Sample DNA (total amount is less than or equal to 20 ng)	23μL
		Totalizing	24μL

The reaction procedure was as follows:

temperature (temperature)	Time
		30℃	1min
80℃	1min
		4℃	Hold

(2) Library amplification

After the reaction of the previous step is finished, adding a connector into the PCR tube for library amplification, wherein the system is as follows:

component (A)	Volume of
		Fragmenting the reaction product in the last step	24μL
5×PrimeSTAR GXL Buffer(Mg2+plus)	10μL
		dNTP Mixture(2.5mM each)	4μL
PrimeStar GXL DNA Polymerase(1.25U/μL)	2μL
		Nuclease-free water	9μL
Barcode-RLB(01-12A)	1μL

The reaction procedure was as follows:

(3) Purification

1) Adding 0.5 XAMPure XP beads, rotating at room temperature, mixing, incubating for 5min, centrifuging, and discarding supernatant;

2) Washing with 80% alcohol for 2 times;

3) Adding Nuclear-free water, rotating at room temperature, mixing uniformly, incubating for 2min, and measuring the concentration by Qubit;

2. targeted enrichment process flow

1. DNA/RNA nucleic acid co-extraction: (refer to PromegaRSC Whole Blood DNA Kit AS1520)

500 μl cerebrospinal fluid sample specific nucleic acid extraction procedure reference PromegaRSC Whole Blood DNA Kit AS1520。

2. Targeted amplification forward enrichment product

(1) PCR amplification of target pathogenic fragments

Type(s)	Template
		Fungi, bacteria	Metagenomic process for extracting DNA nucleic acid
DNA virus	DNA+RNA nucleic acid extracted by targeting procedure
		RNA virus	DNA+RNA nucleic acid extracted by targeting procedure

1) Fungus, bacteria and DNA virus reaction tube configuration:

component (A)	Volume of
		Platinum ^TM Multiplex PCR Master Mix，2X	25μL
Targeting Primer1	0.1-0.5μM each
		Targeting Primer 2	0.1-0.5μM each
Template	10μL
		GC Enhancer	2μL
Nuclease-free water	Up to 50μL

2) PCR amplification reaction procedure for fungi, bacteria and DNA viruses:

3) RNA virus reaction tube arrangement

Component (A)	Volume of
		2×One Step High Fidelity Buffer	25μL
PrimeScript II RT Enzyme Mix	1μL
		PrimeSTAR GXL for 1 step RT-PCR	4μL
Targeting Primer 3	0.1-0.4μM each
		Targeting Primer 4	0.1-0.4μM each
Template	10μL
		Nuclease-free water	Up to 50μL

4) RNA virus reaction procedure

(2) Purification

Mixing the special pathogen and the virus PCR product, adding 0.5 XAMPure XP beads, mixing at room temperature, incubating for 5min, centrifuging, and discarding the supernatant;

washing with 80% alcohol for 2 times;

nuclease-free water was added and incubated for 2min at room temperature with mixing.

3. BP joint connection PCR amplification (SQK-PCB 111.24 library)

(1) The configuration system is as follows:

component (A)	Volume of
		Platinum ^TM Multiplex PCR Master Mix，2X	25μL
DNA	22μL
		BP joint primer	1μL
GC Enhancer	2μL
		Nuclease-free water	Is added to 50 mu L

(2) The reaction procedure was as follows:

(3) Purification

Adding 0.5 XAMPure XP beads, mixing at room temperature, incubating for 5min, centrifuging, and discarding supernatant;

washing with 80% alcohol for 2 times;

3. Double-flow mixed library of metagenome flow and targeting enrichment flow

1. Metagenome flow mixed library

The pool was performed according to the total amount of DNA library (200 ng per sample, less than 200ng total input).

2. Targeting enrichment process mixed library

The pool was performed according to the total amount of DNA library (40 ng per sample, less than 40ng total input).

3. Keeping the total amount of the metagenome library and the targeted enrichment library to be more than or equal to 900ng.

4. Double-flow mixed warehouse purifying and adding joint

1. Mixing the metagenome process and the target enrichment process library samples, adding 0.5 Xbeans, rotating at room temperature, mixing uniformly, incubating for 5min, centrifuging briefly, standing on a magnetic rack, and removing the supernatant;

2. 2 times beads with 200 μl of freshly prepared 80% wine;

3. adding 12 mu L of 10mM Tris-HCl (50 mM NaCl) pH 8.0 eluent, rotating at room temperature, uniformly mixing, incubating for 2min, centrifuging briefly, placing on a magnetic rack until the mixture is clear, and carefully sucking 11 mu L of supernatant into a 1.5mL low-adsorption centrifuge tube;

4. taking 1 mu LQubit for detection, and recording library concentration;

5. and (3) adding a joint: to the 10. Mu.L sample, 1. Mu.L of RAP was added and the mixture was reacted at room temperature for 5 minutes.

5. And (5) performing on-machine sequencing according to a standard nanopore sequencing on-machine flow.

The establishment of the method system is based on optimization examples including but not limited to the following:

optimization example 1, macroflow step 1) cerebrospinal fluid sample pretreatment optimization

The application is based on an optimization experiment carried out by the specific steps of tissue sample metagenomic library construction and detection method patent (CN 113249440A, publication date 20210813) of applicant's earlier-stage nanopore sequencing platform reference specification sample pretreatment.

Unlike sputum or alveolar lavage, cerebrospinal fluid is a sterile body fluid under physiological conditions, and the amount of cerebrospinal fluid obtained by a patient by puncturing is usually not more than 3mL. The sample type characteristics determine that the total amount of nucleic acid is extremely low, and higher requirements are imposed on the sensitivity and yield of each step of the method. Therefore, in the sample pretreatment link, aiming at the cerebrospinal fluid sample, the experimental procedure is adjusted: (1) without centrifugation concentration: avoiding the loss of low-load pathogen caused by centrifugal supernatant discarding; (2) adjusting the concentration of DH: the sample input amount requirement is met, the host removing operation is not influenced, the concentration is enlarged through conversion, and the input volume is reduced; (3) enzyme removal link adjustment: after rinsing with PBS, centrifugation and EDTA enzyme inactivation are carried out, and EDTA enzyme inactivation treatment is preferable.

The purpose of the experiment is as follows: optimizing pretreatment flow and improving yield of low-load pathogenic nucleic acid of cerebrospinal fluid sample

The experimental steps are as follows:

qPCR pathogen quantification results:

sample name (pathogen)	Scheme 1 (Ct)	Scheme 2 (Ct)	Flow 3 (Ct)
				JP201(EBV)	31.46	28.91	29.20
JP204(HCMV)	28.31	25.87	26.95
				JN233(MP)	34.62	30.43	30.45

EBV: EB virus (Human gammaherpesvirus 4); HCMV: cytomegalovirus (Human betaherpesvirus 5); MP: mycoplasma pneumoniae (Mycoplasma pneumoniae).

Conclusion: as shown in the table qPCR results, the quantitative evaluation effect is carried out on the extracted pathogen by adjusting the experimental flow, the flow 2 is more than the flow 3 is more than the flow 1, and the pretreatment flow 2 can obviously improve the yield of the cerebrospinal fluid sample low-load pathogen.

Optimization example 2, step 2) metagenomic DNA extraction in the macroflow, selection of different extraction kits

Unlike sputum or alveolar lavage fluid, cerebrospinal fluid is a sterile body fluid in physiological state, the total amount of nucleic acid is extremely low, and the sensitivity and the yield of each step of the method are more required. So that the adjustment is needed to make the optimal selection in each step. In the extraction link of the macro flow, the experiment tests different brands of extraction rates of bacterial and fungal pathogens aiming at cerebrospinal fluid samples.

The purpose of the experiment is as follows: testing the extraction effect of different brands of extraction kits on bacterial and fungal nucleic acid in cerebrospinal fluid samples

Sample type: cerebrospinal fluid negative sample spike in

The detection method comprises the following steps: qPCR

The test kit as determined by the preliminary screening is as follows:

the cerebrospinal fluid clinical negative samples were spiked with the following 4 strains including Acinetobacter baumannii (G-), listeria monocytogenes (G+), staphylococcus aureus (G+), cryptococcus neoformans (fungi) 4 consecutive times with 10-fold dilutions.

Extracting by using extraction kits of different manufacturers, and qPCR quantification of extracted nucleic acid: (Ct value of 0 represents underwermined).

As shown in FIG. 2, in samples of cerebrospinal fluid clinical negative spike in bacteria/fungi, AS1520 and DP316 are two better kits in the 7-set extraction kit alignment, followed by NG550. Considering the purity of extracted nucleic acid and the influence on downstream library construction, the magnetic bead method Promega is selected comprehensivelyRSC Whole Blood DNA Kit AS1520。

Optimization example 3, target procedure step 1) DNA/RNA Co-extraction, selection optimization of different nucleic acid extraction kits

The purpose of the experiment is as follows: and testing the extraction effect of different brands of extraction kits on DNA and RNA virus nucleic acid in the cerebrospinal fluid sample.

Sample type: a cerebrospinal fluid negative sample spike in;

the detection method comprises the following steps: qPCR;

the test kit as determined by the preliminary screening is as follows:

the clinical negative samples of cerebrospinal fluid were spiked with BDS-IQC-025 human cytomegalovirus deoxyribonucleic acid (HCMV DNA) liquid indoor quality control, BDS-IQC-067 coxsackievirus ribonucleic acid (CA 16 RNA) liquid indoor quality control, and 10-fold dilutions were performed 3 times in succession.

Numbering device	HCMV(copies/mL)	CA16(copies/mL)
			S1	10000	10000
S2	1000	1000
			S3	100	100
S4	10	10

Extracting by using extraction kits of different manufacturers, and qPCR quantification of extracted nucleic acid: (Ct value of 0 represents unesterified):

as shown in FIG. 3, it can be seen that in the cerebrospinal fluid clinical negative spike in virus sample, the nucleic acids extracted from the three kits have no obvious difference in Ct value, and the magnetic bead method Promega is considered to be selectedRSC Whole Blood DNA Kit AS1520 has obvious advantages in macro flow extraction, and unified use of AS1520 extraction is more convenient.

Optimization example 4, selection of metagenomic flow library building kit: SQK-RPB004 and SQK-PBK004 library construction performance comparison

The purpose of the experiment is as follows: aiming at the initial input of a cerebrospinal fluid low template, a kit with high success rate of metagenomic library establishment is searched.

Experimental samples: clinical cerebrospinal fluid samples.

Evaluation index: library total amount, total reads on-line, host ratio, and microbial detection.

As shown in the experimental results in FIG. 4, the total amount SQK-RPB004 of the 10 clinical cerebrospinal fluid sample libraries in 4A is obviously higher than SQK-PBK004; SQK-RPB004 60% samples were able to reach 200ng of stock out, SQK-PBK004 0 cases reached 200ng of stock out. SQK-RPB004 sensitivity is higher. 10 cases of clinical cerebrospinal fluid samples in 4B are on-machine total ready SQK-RPB004 higher than SQK-PBK004; SQK-RPB004 70% of the sample reads can reach 10k, and SQK-PBK004 only 10% of the sample reads can reach 10k. The host proportion of 10 clinical cerebrospinal fluid samples in 4C is generally SQK-RPB004 lower than SQK-PBK004. The number of the dominant microorganisms read SQK-RPB004 detected in 10 clinical cerebrospinal fluid samples in 4D is obviously higher than SQK-PBK004.

To sum up, for the cerebrospinal fluid low-template initial input sample, the metagenomic flow library construction method is better than SQK-RPB004 kit.

Optimization example 5, metagenomic procedure library construction fragmentation time and purification multiplier determination

The purpose of the experiment is as follows: testing different breaking times and different purification multipliers of the fragmenting enzyme;

experimental samples: zymoBIOMICS ^TM Microbial Community DNA Standard；

TABLE 1 zymoBIOMICS ^TM Microbial Community DNA Standard Strain information

The application selects a DNA nucleic acid standard product zymoBIOMICS produced by Zymo Research company ^TM Microbial Community DNA Standard (Catalog Nos. D6305) as input to the validation, which is a clinically common composition of 8 bacteria and 2 fungi, the overall GC content distribution is very broad (15% -85%, as shown in the above table, DNA standard composition (Catalog Nos. D6305 (Zymo Research, product specification)) which is a good standard for studying microbiome.

Evaluation index: library concentration, 4200 glue panel distribution

The experimental results are shown in the following table and in fig. 5:

it can be seen that the different break times and different purification multipliers of the fragmenting enzyme have no significant effect on the library concentration and fragment size distribution, optimally 1min, and the purification multiplier is selected to be 0.5X.

Optimization example 6 optimization of metagenomic flow library construction and amplification cycle number

The purpose of the experiment is as follows: simulating a cerebrospinal fluid low-input sample, and determining the number of metagenomic flow amplification cycles;

experimental samples: zymoBIOMICS ^TM Microbial Community DNA Standard；

Evaluation index: concentration measurement of Qubit in warehouse

From the results, the number of amplification cycles was optimized from 30 to 40, with the 35cycles being the best for template amplification at low initial amounts, and not leading to higher background amplification.

Optimization example 7 optimization selection of target enrichment Process pool-building DNA multiple enzymes

The purpose of the experiment is as follows: screening multiple amplification enzyme to cover central nervous pathogen list on the premise of ensuring performance;

experimental samples: a cerebrospinal fluid negative sample spike in;

the three DNA multiplex enzymes determined by the preliminary screening were as follows:

the typical pathogens of the cerebrospinal fluid negative sample spike in are mycobacterium tuberculosis, aspergillus, EB virus, mycoplasma pneumoniae, human herpesvirus and cytomegalovirus. Wherein the bacterial fungus spike in pathogen amount is 100CFU, the virus spike in pathogen amount is 100copies, and the High concentration (High) is defined; the bacterial fungus spike in pathogen amount is 10CFU, the viral spike in pathogen amount is 10copies, and a Low concentration (Low) is defined. And carrying out full-flow warehouse building and on-line on samples with two concentrations.

Evaluation index: the ONT sequencing was run off the machine and the Reads were log.

As shown in FIG. 6, it can be seen that the three types of amplification enzymes after screening can amplify the target under the condition of 2 templates of the representative pathogen. In ONT sequencing results, thermo (4464270) > Toyobo (KMM 101) > Takara (R050A) was selected on the basis of the number of representative pathogen reads, taking into consideration the selection of Thermo Platinum ^TM Multiplex PCR Master Mix。

Optimization example 8, targeted enrichment procedure library construction SQK-PCB111.24 adapter primer Performance optimization

The purpose of the experiment is as follows: optimizing the efficiency of the adaptor primer;

experimental samples: a cerebrospinal fluid negative sample spike in;

evaluation index: library concentration, 4200 gel plot;

the clinical negative sample of cerebrospinal fluid is doped with BDS-IQC-050EB virus deoxyribonucleic acid (EBV DNA) liquid indoor quality control material and BDS-IQC-025 human cytomegalovirus deoxyribonucleic acid (HCMV DNA) liquid indoor quality control material.

The specific optimization scheme is as follows:

original adaptor primer sequence:

Optimized street sequence:

The results before and after the optimization are shown in the following table and fig. 7.

From the concentration and fragment size distribution of the 22bp and 15bp linker library, the preferred linker primer is 15bp, and the optimized primer improves the detection sensitivity of the pathogen with low load in the cerebrospinal fluid sample.

Verification example 1, run-on-machine verification based on SQK-RPB004 and SQK-PCB111.24

The purpose of the experiment is as follows: the test SQK-RPB004 and SQK-PCB111.24 are mixed for use, and the dry and wet experiment is smoothly carried out on the machine.

Experimental samples: cerebrospinal fluid clinical positive sample

Therefore, after the samples (CSF 654, CSF093 and CSF 564) of three clinical diagnosis results selected by the application are mixed and used, the samples can be successfully put on the machine after the test SQK-RPB004 and the test SQK-PCB111.24 are mixed and used, and the raw information is normally split, and the detected pathogen is consistent with the clinical diagnosis result, so that the advantages of the method are demonstrated.

In addition, two clinical diagnosis results of sample numbers CSF654 and CSF093 were cryptococcosis meningitis (cryptococcosis culture positive, ink staining positive, cryptococcosis antigen positive), and the extracted nucleic acid was also positive by verification of a third-party cryptococcosis qPCR kit. The process constructed by the method can detect the novel cryptococcus pathogen in both the macro process and the target process, and the number of detected Reads in the target process is higher than that in the macro process, which indicates that the detection sensitivity of the target process is better than that in the macro process. The effectiveness of the flow on the pathogen detection of clinical samples is fully described.

The clinical diagnosis result of sample number CSF564 is cryptococcoid meningitis (cryptococcus culture positive, ink dyeing positive, cryptococcus antigen positive), the cryptococcus garter pathogen can not be detected in the macro process, the cryptococcus garter pathogen can be detected in the target process, the clinical sample extracted nucleic acid is verified to have Ct of 34.76 (cut off 35) by a third-party cryptococcus qPCR kit, the pathogen load is relatively low, the sensitivity of the cryptococcus garter pathogen with low load in cerebrospinal fluid sample is obviously improved in the targeted enrichment process, and the omission of the macro process is compensated.

It is specifically noted that the method of the present application can directly distinguish between Cryptococcus neoformans and Cryptococcus gatus (e.g., sample CSF 564) in clinical specimens at the species level, whereas clinical culture, ink staining, cryptococcus antigen detection, qPCR validation can only reach Cryptococcus levels. This sample was also cryptococcus garteus as verified by mNGS, but the second generation full-flow report period (TURN-AROUND TIME, TAT) was much longer than the method of the present application, with poor timeliness.

Verification example 2, clinical sample spike in sensitivity test

The purpose of the experiment is as follows: the sensitivity of SQK-RPB004 and SQK-PCB111.24 pooling procedures was evaluated.

Experimental samples: cerebrospinal fluid clinical negative samples spike in representative species were pooled and gradient diluted.

The evaluation method comprises the following steps: representative species near the limit of detection.

For evaluating the sensitivity of the analysis according to the method of the present application, staphylococcus aureus, klebsiella pneumoniae, mycobacterium tuberculosis, herpes simplex type 1 virus, coxsackievirus, and cryptococcus neoformans are used as representative species of gram-positive bacteria, gram-negative bacteria, mycobacteria, DNA viruses, RNA viruses, and yeast fungi, respectively. To prepare serial reference products with different microorganism loading levels, culturing and increasing bacteria and yeast fungi, continuously diluting the freshly prepared bacterial suspension for 10 times, and performing colony count (CFU/mL) on the diluted bacterial suspension; mixing the mixed bacterial liquid into a clinical cerebrospinal fluid sample with priori negative; for mycobacteria and viruses, nucleic acid is extracted, serially diluted and then mixed into a priori negative cerebrospinal fluid total nucleic acid sample. The samples are subjected to mixed warehouse and then are subjected to on-machine sequencing based on the method.

After mixed library sequencing, different detection methods are adopted for different microorganism types: performing metagenome flow detection and targeted enrichment flow detection on mycobacterium and yeast fungi in parallel; performing only metagenomic flow detection for gram positive and negative bacteria; only carrying out targeted enrichment process detection on viruses; and finally, obtaining target species specificity comparison reads in sequencing data, carrying out counting analysis, independently repeating detection for 3 times on each dilution level, and considering the lowest bacterial or viral genome copy count (or genome copy number equivalent) of the specificity comparison reads in all the repetitions, wherein the lowest detection limit of the current method is considered.

The detection analysis sensitivity of the targeting enrichment flow of the representative species shows that the detection sensitivity of the mycobacterium tuberculosis is 5.83 multiplied by 10 ² cobies/mL; novel cryptococcus detection sensitivity is 1.72×10 ² CFU/mL; herpes simplex type 1 virus and coxsackievirus have a sensitivity equivalent to 50 copies of the viral genome.

The metagenome flow detection analysis sensitivity of the representative species shows that the detection sensitivity of the mycobacterium tuberculosis is 5.83 multiplied by 10 ³ cobies/mL; the detection sensitivity of the novel cryptococcus is 1.72 multiplied by 10 ³ CFU/mL; the sensitivities of Klebsiella pneumoniae and staphylococcus aureus are respectively 4.4x10 ¹ 、2.73×10 ² CFU/mL。

As can be seen from the detection results of the targeting enrichment process and the metagenome process, after the mixed use of the test SQK-RPB004 and SQK-PCB111.24, the machine can be successfully started, the biological information is normally split, the detected pathogen is consistent with the mixed pathogen result, the sensitivity is high, and the detection method is 10 ² And 10 ³ In between, the detection level is generally required to be 10 according to the quality evaluation of the metagenome high throughput sequencing among rooms of the respiratory tract infection under the country of 2022 ⁴ The detection limit of the application is at least 1-2 orders of magnitude below this standard.

In conclusion, the mixed library and the on-machine method for the central nervous infection pathogens are built based on the application, so that the pathogens can be detected rapidly, comprehensively and accurately, the metagenome detection of a low initial quantity cerebrospinal fluid sample is satisfied, the sensitivity of detecting the pathogens difficult to detect in the cerebrospinal fluid sample is obviously improved, and the possibility is provided for improving the diagnosis rate of CNS infection and pathogen research.

The foregoing descriptions of specific exemplary embodiments of the present application are presented for purposes of illustration and description. It is not intended to limit the application to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the application and its practical application to thereby enable one skilled in the art to make and utilize the application in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the application be defined by the claims and their equivalents.

Claims

1. A library building method of central nervous infection pathogens based on a nanopore sequencing platform, which is characterized by comprising a metagenome flow and a targeting enrichment flow;

preferably, the method further comprises the step of carrying out database mixing on the metagenome process and the targeting enrichment process.

2. The method of claim 1, wherein the metagenomic procedure comprises:

1) Pretreatment of cerebrospinal fluid samples; 2) Metagenomic DNA extraction; 3) Metagenomic libraries are rapidly prepared.

3. The method of claim 2, wherein 3) the rapid metagenomic library preparation is performed using a rapid PCR barcode kit SQK-RPB004 or SQK-PBK004; preferably, the rapid PCR barcode kit is SQK-RPB004.

4. The pooling method of claim 1, wherein the targeted enrichment procedure comprises:

a) Co-extracting DNA/RNA; b) PCR amplification of target pathogenic fragment; c) The BP joint is connected with PCR amplification and library establishment.

5. The method of claim 4, wherein the step 3) BP linker ligation PCR amplification pooling uses PCR-cDNA barcode kit SQK-PCB111.24 for pathogen targeted pooling.

6. The method of constructing a library according to any one of claims 4 to 5, wherein said c) BP adaptor is ligated to PCR amplified library as follows:

7. A method of pooling according to any of claims 2-3, wherein the step of 1) pre-treating the cerebrospinal fluid sample is: adding the cerebrospinal fluid sample into a sterilization centrifuge tube, adding 3-5% of saponine by mass concentration, and mixing uniformly; adding HL-SAN enzyme, mixing uniformly, and incubating by metal bath oscillation; and adding EDTA (ethylene diamine tetraacetic acid) into the mixture and uniformly mixing the mixture by vortex.

8. A method of constructing a library according to any one of claims 2 to 3, wherein 2) metagenomic DNA extraction is performed using Promega corporationRSC Whole Blood DNA Kit the extraction is performed.

9. The method of claim 4-5, wherein the DNA multiplex enzyme used in PCR amplification of the target pathogenic fragment of b) is Thermo Platinum ^TM Multiplex PCR Master Mix。

10. The method for constructing a library according to any one of claims 1 to 9, wherein the steps of mixing the metagenomic process and the targeted enrichment process are as follows: the DNA obtained by the metagenome process and the targeting enrichment process is independently mixed according to the total library amount; and mixing the metagenome process mixed library sample and the targeting enrichment process mixed library sample, and keeping the total mixed library amount to be more than or equal to 900ng.