CN115725570A

CN115725570A - Nanopore sequencing-based method for detecting drug-resistant/virulence genes of blood sample

Info

Publication number: CN115725570A
Application number: CN202211373132.XA
Authority: CN
Inventors: 苗卉; 王磊; 李杜衡; 李立锋; 田文佳; 涂浩波; 蒋智; 贾雪峰
Original assignee: Jinshi Zhizao Tianjin Medical Technology Co ltd; Tianjin Jinke Medical Technology Co ltd; Beijing Jinshao Medical Laboratory Co ltd
Current assignee: Beijing Jinshao Medical Laboratory Co ltd; Tianjin Huazhinuo Technology Co ltd; Tianjin Jinke Medical Technology Co ltd
Priority date: 2022-11-02
Filing date: 2022-11-02
Publication date: 2023-03-03
Anticipated expiration: 2042-11-02
Also published as: CN115725570B

Abstract

The application relates to the technical field of molecular diagnosis, in particular to a drug resistance/toxicity detection method for a blood infection sample.

Description

Nanopore sequencing-based method for detecting drug-resistant/virulence genes of blood sample

Technical Field

The application relates to the technical field of molecular diagnosis, in particular to a method for detecting drug resistance/virulence genes of a blood sample based on a nanopore sequencing technology.

Technical Field

Antibiotic abuse has become increasingly severe in recent years due to lack of awareness of antibiotic use, resulting in antibiotic resistance emerging at an alarming rate. From the discovery of penicillin in 1929, drug-resistant strains such as methicillin-resistant staphylococcus and vancomycin-resistant enterococcus gradually appear in the next 30 years. After 80 s, the drug resistance of bacteria is more gradually upgraded, and the beta-lactamase (enzyme which can make antibiotics ineffective) produced by bacteria is developed from common enzyme to broad-spectrum enzyme from gram-negative bacteria to gram-positive bacteria. Although the number of antibiotics currently used in clinical application is less than 200, and the number of antibiotics still increases at a rate of more than 10 per year, the research and development of antibiotics are far from keeping up with the drug resistance rate of bacteria. The increasing number of bacterial resistance, the continuous emergence of multi-drug resistant bacteria and the serious difficulty of treatment, which together lead to the prolongation of the course of the patient, the prolongation of the hospitalization and the higher mortality, especially in the Intensive Care Unit (ICU). According to statistics, about 1 million of patients suffering from infectious diseases need to be treated by assistance of a method for detecting pathogens in China every year, wherein the number of severely infected patients is more than ten million, and the conventional detection means is difficult to provide guidance for clinical application quickly for the more complicated critically infected patients. Besides, newly-developed pathogenic microorganisms and continuously-enhanced drug-resistant/virulence pathogenic bacteria are tested by conventional detection means to a great extent every year. Therefore, it is important to develop a detection method capable of rapidly identifying pathogenic bacteria and drug-resistant/virulence genes.

Currently, blood culture remains the "gold standard" for detection of pathogens in patients clinically suspected of having infectious disease. However, as is known, blood culture has quite obvious defects, 1) the culture time is long, 2) special pathogens cannot be cultured, and 3) antibiotics are used for treatment, so that the pathogen acquisition amount is insufficient; as for the detection of drug resistance/toxicity, the antibiotic drug sensitivity test is carried out after blood culture and yang reporting, so the detection time is further prolonged, and the patients who need immediate treatment are doubtless frosted.

The metagenome-based pathogenic microorganism detection technology (mNGS) is a high-throughput sequencing technology which does not depend on traditional microorganism culture and specific amplification, can directly perform indifference and nonselectivity on nucleic acid in a clinical sample, and can quickly judge the pathogenic microorganism species and drug-resistant/virulence genes in the clinical sample after sequencing data is compared and analyzed with a microorganism sequence database. Compared with the traditional detection mode, the mNGS has higher sensitivity and higher detection speed, but according to the guidance of applying the high-throughput metagenome sequencing technology to the consensus of Chinese experts in infectious disease pathogen detection in 2021, for the part without microorganism constant value, the mNGS can be considered for detection, but enough deep sequencing depth needs to be ensured, which inevitably causes the timeliness of the metagenome to be influenced. Meanwhile, the high cost is brought, and if the patient is not critically ill, the detection is generally difficult to accept.

Nanopore sequencing is a single-molecule real-time sequencing technology, and the method does not depend on traditional microbial culture, and can quickly detect suspected pathogens in clinical samples. The method records the composition of basic groups according to the current change of single-molecule DNA or RNA in a sample when passing through a nanopore so as to complete the sequencing of the whole genome. The main advantages of nanopore sequencing are that on one hand, the nanopore has super-long read length, sequences with the length of 1kb to more than 100kb can be generated, and longer sequences have greater advantages in the aspects of analyzing pathogenic bacteria and drug resistance/virulence genes and the like. On the other hand, the method has the advantages that sequencing data can be obtained within a few seconds after the sample is added, and genome comparison can be carried out in real time, so that the species information of the microorganisms in the sample and the carried drug resistance/virulence gene information can be rapidly obtained. That is, when nanopore sequencing is combined with an efficient genome nucleic acid extraction and library construction method, a detection result can be obtained within 4-6 hours, and clinical diagnosis and treatment of critically ill patients are facilitated.

Disclosure of Invention

In order to solve the technical problems, the method is based on multiple PCR library construction and combines a high-throughput nanopore sequencing technology to detect bacteria and drug-resistant/virulence genes in blood samples.

Specifically, the application provides the following technical scheme:

the application firstly provides a targeting primer group for establishing a drug-resistant/virulence gene library of a blood infection metagenome sample based on a nanopore sequencing platform, wherein the primer aims at 11 beta lactam drug-resistant genes, 5 carbapenem drug-resistant genes, 1 polymyxin drug-resistant genes, 2 vancomycin drug-resistant genes and 4 virulence factor genes.

Further, the genes targeted by the primer group are specifically: mecA, CTX-M, OXA-24, OXA-23, blaTEM-bs, CTX-M-group1, CTX-M-group9, CTX-M-group2, blaTEM-esbl, SHV, orfx-SCC, VIM, KPC, OXA-48, IMP, NDM, MCR, vana, vanB, iuca, rmpA2, and PVL.

Further, the primer sequences in the primer group are shown as SEQ ID NO.1-38, or have at least 95% sequence identity with SEQ ID NO. 1-38.

Further, the concentration ratio of the primers is as follows: SEQ ID NO.1/2: SEQ ID NO.13/14: 3/25/26 SEQ ID No. 25/28 SEQ ID No.31/32 SEQ ID No.35/36 SEQ ID No.37/38 SEQ ID No. 39/40.

Preferably, the concentration ratio of the primers is: SEQ ID NO.1/2: SEQ ID NO.13/14: 3/25/26 SEQ ID No.29/30 SEQ ID No.35/36 SEQ ID No.37/38 SEQ ID No.39/40 SEQ ID No. 25/42.

Further preferably, the primer concentrations are respectively: the concentration of TEM primer was 0.1uM and the concentration of other drug/virulence primers was 0.15uM.

The application also provides a composition comprising any one of the primer sets described above.

The application also provides a kit for establishing a library aiming at drug resistance/virulence genes of a blood infection metagenome sample, which is used for a nanopore sequencing platform, and comprises any one of the primer sets.

The application also provides application of any one of the primer sets in the construction of a sequencing library of drug resistance/virulence genes of a blood infection metagenome sample.

The application also provides application of any one of the primer sets in detection of drug resistance/virulence genes of blood infection metagenome samples.

The application also provides application of any one of the primer sets in preparation of a detection kit for drug resistance/virulence genes of a blood infection metagenome sample.

The application also provides a library construction method of the blood infection sample, which comprises the following steps:

1) Treating the infected sample with host DNA;

2) Adding a DNA/RNA lysate for sample pretreatment;

3) Extracting nucleic acid from the treated sample;

4) Carrying out targeted amplification enrichment by using any one of the primer groups;

5) Purifying an amplification product;

6) Sequencing library construction was performed using an ONT commercial library construction kit.

The application also provides a method for detecting drug resistance/virulence genes of blood infection samples, which comprises the library construction method and further comprises the steps of sequencing and biological information analysis.

The application has the beneficial technical effects that:

the application establishes a set of effective targeted amplification system by exploring and establishing multiple targeted drug-resistant/virulent florae and gene combinations and designing, screening and optimizing a targeted primer system, can comprehensively amplify drug-resistant virulent genes in a blood infection metagenome sample and effectively establish a library, and ensures the detection comprehensiveness and accuracy in a third-generation nanopore sequencing platform.

The rapid detection method based on the targeted determination of the drug resistance/virulence gene can complete the detection of the drug resistance/virulence in the blood sample in the fastest 6 hours, and has the advantages of high sensitivity, high flux, high detection speed, accurate result and the like.

Drawings

FIG. 1 shows the result of agarose gel electrophoresis, where M is Marker and the Marker interval is 200bp-2000bp;1 represents group 1; and 2 denotes group 2.

FIG. 2 shows the result of agarose gel electrophoresis, in which M is Marker with a Marker interval of 300bp-5000bp, and 1 represents that the concentration of drug-resistant/virulence primer in test protocol 1 is 0.15uM;2 represents test protocol 2 with a concentration of 0.25uM of drug/virulence primers; 3 represents that the concentration of TEM primer in the drug resistance/virulence primer of the test scheme 3 is 0.1uM;4 represents the test protocol 4 where the concentration of the TEM primer in the drug/virulence primers is 0.05uM;5 represents test protocol 5 with 0.25uM of drug/virulence primers and 0.1uM of TEM primer concentration; 6 represents test protocol 6 with 0.25uM drug/virulence primers and 0.05uM TEM primer concentration; 7 represents test protocol 7 with 0.15uM drug/virulence primers and 0.1uM TEM primer concentration; 8 represents test protocol 8 with 0.15uM drug/virulence primers and 0.05uM TEM primer concentration.

FIG. 3 shows the result of the detection performance of the drug-resistant virulence primer.

Detailed Description

Embodiments of the present application will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present application and should not be construed as limiting the scope of the present application. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by manufacturers, and are all conventional products available on the market.

Definition of partial terms

Unless defined otherwise below, all technical and scientific terms used in the detailed description of the present application are intended to have the same meaning as 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.

Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun.

The term "about" in the present application denotes an interval of accuracy that can be understood by a person skilled in the art, which still guarantees the technical effect of the feature in question. The term generally denotes a deviation of ± 10%, preferably ± 5%, from the indicated value.

As used in this application, 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 in the following a certain group is defined to comprise at least a certain number of embodiments, this should also be understood as disclosing a group which preferably only consists of these embodiments.

Furthermore, in the description and claims the terms first, second, third, (a), (b), (c) and the like are

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 described herein are capable of operation in other sequences than described or illustrated herein.

The method for detecting the drug resistance/toxicity of the blood infection metagenome sample is used for carrying out targeted library construction on the DNA of the blood metagenome sample based on the design of a targeted primer, realizes the rapid detection of the drug resistance/toxicity of the blood infection sample by combining nanopore sequencing, and has the advantages of high sensitivity, high flux, accurate result and the like.

The term "drug resistance" as used herein refers to the tolerance of microorganisms, parasites and tumor cells to the action of drugs, which is significantly reduced once drug resistance is developed. Preferred for this application is an in vivo infection that is resistant to antibiotic drugs.

The term "virulence" as used herein refers to the ability of bacteria, viruses, fungi to metabolize and produce virulence factors such as invasiveness and toxins. Virulence factors can be encoded on mobile genetic elements (e.g., plasmids, gene islands, phage, etc.) and undergo horizontal gene transfer, rendering harmless bacteria dangerous pathogens.

In some embodiments, the targeting primer is directed against 11 β lactam drug resistance genes, 5 carbapenem drug resistance genes, 1 polymyxin drug resistance gene, 2 vancomycin drug resistance genes, and 4 virulence factor genes.

In some embodiments, the primer set is specific for a gene that is: mecA, CTX-M, OXA-24, OXA-23, blaTEM-bs, CTX-M-group1, CTX-M-group9, CTX-M-group2, blaTEM-esbl, SHV, orfx-SCC, VIM, KPC, OXA-48, IMP, NDM, MCR, vana, vanB, iuca, rmpA2, and PVL.

In some embodiments, the primer sequences in the primer set are as set forth in SEQ ID nos. 1-38, or at least 95% sequence identity to SEQ ID nos. 1-38.

In some embodiments, the primer concentration ratio is: SEQ ID NO.1/2: SEQ ID NO.13/14: 3/25/26 SEQ ID No. 25/28 SEQ ID No.31/32 SEQ ID No.35/36 SEQ ID No.37/38 SEQ ID No. 39/40.

In some embodiments, the primer concentration ratio is: SEQ ID NO.1/2: SEQ ID NO.13/14: 3/25/26 SEQ ID No.29/30 SEQ ID No.35/36 SEQ ID No.37/38 SEQ ID No. 39/40.

The application is illustrated below with reference to specific examples.

Experimental example establishment of the basic method flow of the present application

1. Off-host process

Infected samples were treated for host DNA removal.

2. Sample pretreatment

Adding DNA/RNA lysis buffer into the treated sample, fully mixing, centrifuging and collecting.

3. Nucleic acid extraction

And (3) extracting nucleic acid by adopting a commercial kit, wherein the extraction mode is according to the instruction specification of the kit.

4. Targeted amplification

Based on a designed drug resistance/virulence primer system, a2 x Phanta Flash M aster Mix (P510) kit of Novozan biotechnology limited is used for targeted amplification, and the specific system is as follows

And (3) mixing the prepared reaction system uniformly, performing instantaneous centrifugation, and performing amplification reaction on a PCR instrument.

As the drug/toxicity primers, 10uM stock solution was diluted to 2uM working solution using Low TE (10mM Tris,0.1mM EDTA, pH 8.0) from Biotechnology engineering (Shanghai) Ltd, and 2ul of each of the working solutions was taken to 20ul of PCR reaction system when the primers were mixed together, ensuring that the final concentration of the primers was 0.2uM per primer.

5. Amplification product purification

And selecting commercial purification magnetic beads for purification, wherein the purification mode is according to the requirements of magnetic bead purification instructions.

6. Library construction

Library construction was performed using an ONT commercial library construction kit.

7. Magnetic bead purification: and purifying by using a commercial kit.

8. Arranging a library on a machine: pre-machine preparation was performed using the ONT (Oxford Nanopore) SQK-PBK004 kit.

9. Sequencing and running: and (3) sequencing by adopting a third-generation nanopore sequencing platform GridION.

10. And (3) letter generation analysis: and performing letter generation analysis on the sequencing off-line data.

Example 1 selection of drug resistance/virulence genes

In view of the differences in microbial flora during human blood infections, the drug resistance and virulence reactions in blood after antibiotic treatment are also extremely complex. In practice, how to select the appropriate microbial flora and which specific drug resistance or virulence genes to detect is of crucial importance for clinical evaluation. The detection process not only needs to consider the detection comprehensiveness and avoid the problem of missed detection, but also needs to ensure that the selected target flora or genes can be detected practically and effectively and avoid the problems of false negative and the like.

In order to realize comprehensive and effective detection of drug resistance and toxicity of blood infection samples, on the basis of the existing theory, after a large amount of early-stage confidence prediction and wet experiment demonstration, beta lactamase-resistant gram-negative bacilli, carbapenem-resistant enterobacteriums/acinetobacter baumannii, vancomycin-resistant enterococci and the like are finally taken as research objects. The corresponding exploration logic is as follows:

the pathogenic bacteria containing VIM gene are generally selected from genera such as Pseudomonas aeruginosa, klebsiella pneumoniae, escherichia coli, and Enterobacter cloacae, and are resistant to carbapenem antibiotics;

the pathogenic bacterium containing the MecA gene is usually selected from the genera Staphylococcus aureus, etc., and can tolerate beta lactam antibiotics;

the pathogenic bacteria containing the CTX-M _ group8/25 gene are generally selected from the genera Escherichia coli, klebsiella pneumoniae, salmonella enterica and the like, and can tolerate cefotaxime antibiotics;

the pathogenic bacteria containing the OXA-24 gene are generally selected from Acinetobacter baumannii, acinetobacter pittii and other genera, and can tolerate beta lactam antibiotics;

the pathogenic bacteria containing the OXA-23 gene are generally selected from Acinetobacter baumannii, klebsiella pneumoniae and other genera, and can tolerate beta lactam antibiotics;

the pathogenic bacteria containing the blaTEM _ bs gene are typically selected from the genus Klebsiella pneumoniae, which are resistant to beta lactam antibiotics;

the pathogenic bacteria containing the CTX _ M _ group1 gene are typically selected from the genus Escherichia coli, and are resistant to cefotaxime antibiotics;

pathogenic bacteria containing the CTX _ M _ group9 gene are typically selected from the genus Escherichia coli, and are resistant to cefotaxime antibiotics;

the pathogenic bacteria containing KPC gene are usually selected from Klebsiella pneumoniae, escherichia coli, acinetobacter baumannii and other bacteria, and can tolerate beta lactam antibiotics;

pathogenic bacteria containing the CTX _ M _ group2 gene are typically selected from the genus Escherichia coli, and are resistant to cefotaxime antibiotics;

the pathogenic bacteria containing the OXA _48 gene are generally selected from the genera Klebsiella pneumoniae, which are resistant to beta lactam antibiotics;

the pathogenic bacteria containing the blaTEM _ esbl gene are typically selected from the genus Klebsiella pneumoconiae, which are resistant to beta lactam antibiotics;

the pathogenic bacteria containing the SHV gene are generally selected from genera such as Klebsiella pneumoniae, klebsiella oxytoca and Escherichia coli, and can tolerate beta lactam antibiotics;

pathogenic bacteria containing the orfX-SCCmec gene are generally selected from the genus Coagulase negative staphylococcus, and are resistant to beta lactam antibiotics;

the pathogen containing the mcr gene is generally selected from the genera Escherichia coli, klebsiella pneumoniae, salmonella sp, and the like, and is resistant to polymyxin antibiotics;

the pathogenic bacteria containing IMP gene are generally selected from Acinetobacter baumannii, pseudomonas putida, klebsiella oxytoca and other genera, and can tolerate carbapenem antibiotics;

the pathogenic bacteria containing NDM gene are usually selected from Escherichia coli, klebsiella pneumoniae, providencia stuartii and other genera, and can tolerate carbapenem antibiotics;

pathogenic bacteria containing the vanA gene are typically selected from the genus Enterococcus faecalium, which are resistant to vancomycin antibiotics;

the pathogenic bacteria containing the vanB gene are typically selected from the genus Enterococcus faecalis, which is resistant to vancomycin antibiotics.

The CTX-M gene-containing pathogenic bacteria are generally selected from the genera Escherichia coli, klebsiella pneumoniae, enterobacteriaceae, and the like, and can tolerate beta lactam antibiotics;

pathogenic bacteria containing the orfx-SCC gene are usually selected from the genera Staphylococcus and the like, and can tolerate beta lactam antibiotics;

the pathogen containing the iucA gene is typically selected from the genus Klebsiella pneumoniae, which is resistant to carbapenem antibiotics;

the pathogenic bacteria containing the rmpA gene are typically selected from the genera Klebsiella pneumoniae, which are resistant to carbapenem antibiotics;

the pathogenic bacteria containing the rmpA2 gene are generally selected from the genus Klebsiella pneumoniae, which are resistant to carbapenem antibiotics;

the pathogenic bacteria containing the PVL gene are generally selected from the genus Staphylococcus aureus and are resistant to beta lactam antibiotics.

Finally, the application establishes that drug/virulence genes specifically include the following: 11 beta lactam drug resistance genes, 5 carbapenem drug resistance genes, 1 polymyxin drug resistance gene, 2 vancomycin drug resistance genes and 4 virulence factor genes, and further analyzing and evaluating to finally determine the specific genes to comprise: mecA, CTX-M, OXA-24, OXA-23, blaTEM-bs, CTX-M-group1, CTX-M-group9, CTX-M-group2, blaTEM-esbl, SHV, orfx-SCC, VIM, KPC, OXA-48, IMP, NDM, MCR, vana, vanB, iuca, rmpA2, and PVL.

Example 2 primer design and screening

The drug-resistant/virulence amplification system of the system is used as a high-multiplex amplification system, and not only the amplification efficiency of a single primer but also the compatibility of each primer in the system need to be considered in the design process of the primers, and simultaneously a sequencing platform needs to be matched. In this example, for the gene determined in example 1, an optimal target amplification region is screened according to the gene sequence, and simultaneously, a target primer is designed, screened and filtered based on the characteristic requirements of a nanopore sequencing platform, and primer sequence optimization is performed comprehensively.

1. Optimization of primer sequence design

For reasons of space, this example is exemplified by the optimization of primer design for the MecA gene (including the optimization of the messenger and wet test ends, respectively).

1. Collecting the DNA sequence of the target gene

MecA resistance gene sequences were downloaded from the NDARO database.

2. Multiple sequence alignment of target genes and selection of target sequences

Performing multiple sequence alignment on the MecA gene sequence by using MAFFT algorithm, setting more than 50% of bases of species at the same position in a continuous region of more than 20bp by integrating company self-research algorithm in a relatively conservative window of the MecA gene, and selecting the region with the highest consistency as a target.

3. Design of primers

Using the sequence of the MecA gene conserved region determined in the previous step of Primer-blast as a target template sequence candidate Primer; the amplicon length reading is limited to 1500bp upper limit and 500bp lower limit.

4. Examination of the melting temperature of MecA primers

The melting temperature of MecA was predicted using Primer-blast, and the difference between the melting temperature and the average melting temperature (melting temperature deviation) was calculated.

5. Prediction of non-specific amplification of host DNA by MecA primer

Primer-blast was used to predict the non-specific amplification of host DNA between MecA pairs themselves and different primer pairs, where the host DNA has the human hg38 genome as the reference sequence, and then the number of complementary bases for non-specific amplification of primer pairs was counted.

6. Predicting the propensity of MecA to form dimers

Primer-pooler software was used to check the complementarity between the Primer pairs themselves and the different Primer pairs and to predict the dimer formation tendency. The lowest Δ G values in dimers that the MecA primer can form were counted.

7. Primer selection and pooling

And comprehensively evaluating and selecting a primer pair according to the identification degree, the coverage surface, the melting temperature deviation degree, the number of non-specific amplification complementary bases and the delta G value of a dimer of the primer.

Multiple groups of primer sequences aiming at MecA are preliminarily designed through the letter optimization, and partial results are as follows;

as proved by amplification verification of a wet experiment, the group2 is superior to the group1 in amplification effect, and is specifically shown in figure 1, wherein M is Marker, and the Marker interval is 200bp-2000bp;1 represents group 1;2 represents group2, the theoretical amplification length of group2 should be 574bp, and the theoretical amplification length of group1 should be 1489bp.

As can be seen from the results, the amplification length of the pair of primers in group2 is about 500bp, which is consistent with the theoretical amplification length of 574bp, while the amplification length of the pair of primers in group1 is below 1400bp, which is not consistent with the theoretical amplification length, and the primers in group2 are selected.

2. Primer combination optimization

Aiming at the whole multiplex primer system, the application adopts a plurality of alternative schemes for designing each different type of gene in an initial system when designing the primer. Then, a single primer amplification experiment is carried out on all the alternative primers, the aim is to check the amplification capacity and the amplification efficiency of the designed primers and eliminate partial primers. In this example, 44 pairs of drug-resistant/virulence primers were tested, and 10 pairs of primers that could not be amplified were eliminated in the first single-primer amplification experiment; and then, building a multiple system of the tested primers, and testing 30 pairs of primers in total, wherein 7 pairs of primers cannot be amplified and eliminated, and 4 pairs of primers cannot be amplified and eliminated due to the failure of optimal amplification effect. And subsequently, the primer sequence is further adjusted, and the rest 23 pairs of primers are amplified normally and successfully to construct a multiple system.

Through the optimization, the application finally determines the following target primer sequences:

wherein R and Y are degenerate primers, R = A or G; y = C or T.

Example 3 primer concentration optimization

In the process of carrying out equal proportion mixed amplification based on the primer sequences, the actual amplification effect is always not as expected, and the analysis reason can cause amplification deviation of other sequences due to relatively high efficiency or relatively low efficiency amplification of part of the primers. Therefore, the concentration of the primers in the primer system is further optimized in the embodiment, so that the amplification balance of each primer in the system is improved, the real abundance of each drug-resistant or virulence gene in the sample is ensured as much as possible, and the comprehensiveness, accuracy and the like of the subsequent nanopore sequencing are ensured.

Analysis shows that the amplification efficiency of 2 pairs of amplification primers aiming at TEM in 23 pairs of drug-resistant/virulence primers is obviously higher than that of other drug-resistant/virulence primers, so that in order to ensure balanced amplification among the drug-resistant/virulence primers and reduce deviation value, the proper adjustment of the input concentration and proportion of the primers is tried, and the specific adjustment concept comprises the following steps: 1) independently adjusting the concentration of 21 pairs of drug-resistant/virulence primers, 2) independently adjusting the concentration of 2 pairs of blaTEM primers, and 3) integrally adjusting the concentration of 23 pairs of drug-resistant/virulence primers.

a) Preparation of Mixed pure microbial nucleic acid

Mixed pure bacterial nucleic acid: commercial ATCC species were used. Selecting 4 ATCC strains with drug-resistant genes for activation culture, and extracting by using a micro sample genome DNA extraction kit (DP 316) of Tiangen Biochemical technology (Beijing) Ltd to obtain pure bacterial nucleic acid after culture. The 4 selected important pathogenic microorganisms are all known to have drug resistance genes, as shown in the following table.

Sample number	Extraction of pathogenic microorganism nucleic acid	Strain numbering	Drug resistance gene
				1	Klebsiella pneumoniae	ATCC BAA-1705	KPC
2	Enterococcus faecium	ATCC 700221	vanA
				3	Enterococcus faecalis	ATCC 700802	vanB
4	Staphylococcus aureus	ATCC BAA1747	mecA

b) Primer concentration adjustment

1) Independently adjusting the concentration of 21 pairs of drug resistance/virulence primers

Test protocol 1 the concentration of the drug/virulence primer was 0.15uM;

test protocol 2 the concentration of the drug/virulence primer was 0.25uM;

2) Individual adjustment of 2 pairs of blaTEM primer concentrations

Test protocol 3 TEM primer concentration in the drug resistant/virulence primers was 0.1uM;

test protocol 4 TEM primer concentration in the drug/virulence primers was 0.05uM;

3) Overall adjustment of 23 for drug/virulence primer concentrations

Test protocol 5 other drug/virulence primers were 0.25uM, while TEM primer concentration was 0.1uM;

test protocol 6 other drug/virulence primers were 0.25uM, while TEM primer concentration was 0.05uM;

test protocol 7 other drug/virulence primers were 0.15uM and TEM primer concentration was 0.1uM;

test protocol 8 other drug/virulence primers were 0.15uM, while TEM primer concentration was 0.05uM. '

548

c) Agarose gel electrophoresis detection

Using 1% agarose gel, voltage 120V, running for 30min, and carrying out electrophoresis detection with sample loading of 5ul, wherein the detection results are shown in figure 2, and 8 groups of test results are tested in total.

As for the electrophoresis results, the bands were single and concentrated at 500-800bp because the amplification lengths of the primers were substantially the same for the drug resistance of the above four species of bacteria, the KPC amplification length was 798bp, the vana amplification length was 805bp, the vanB amplification length was 805bp, and the mecA amplification length was 574bp, so the bands were mixed together and could not be distinguished, and they fell in the range of 500-800 bp.

1) Results of adjusting the concentration of 21 pairs of drug/virulence primers alone: test protocol 1 the amplified band was more bright than test protocol 2, so that the concentration of 21 pairs of drug/virulence primers, 0.15uM, was initially taken as the concentration of the pending primer. Protocol 1 amplifies the bands more upwardly than protocol 2, i.e., more toward KPC, vanA, vanB, and protocol 2 more toward mecA, both alternatives.

2) Results of adjusting the concentration of 2 pairs of blaTEM primers individually: in test scheme 3 and test scheme 4, the concentration of the TEM-resistant primers is adjusted individually, so that it is obvious that the amplification efficiency is stronger than the sum of other resistant primers and also stronger than that in test scheme 1 and test scheme 2, but the difference between test scheme 3 and test scheme 4 is not significant in terms of the amplified fragment size or the amplified brightness, and thus, the method can be used as an alternative.

3) Results for global adjustment of 23 versus drug/virulence primer concentrations: test protocol 5 did not differ much in band size and band brightness compared to test protocol 6, whereas test protocol 7 was stronger in band brightness than test protocol 8, with more pronounced amplification. If the 4 protocols are compared simultaneously, it can be seen that the bands of

tests

7 and 8 are slightly higher than those of 5 and 6, while the upper half of the band is brighter, so tests 7 and 8 are more biased toward amplifying the TEM gene.

In conclusion, the amplification efficiency of test scheme 7 (0.15 uM for other drug/virulence primers and 0.1uM for TEM primer) was significantly higher than that of

test schemes

5 and 6, while it was more efficient to amplify TEM genes than test scheme 8.

Through system test optimization, the primer proportion of the application is determined as follows:

SEQ ID NO.1/2: SEQ ID NO.13/14: 3, SEQ ID No. 25/26; 3.

Example 4 evaluation of sequencing detection Performance

This example evaluates the system through a full flow of sequencing analysis.

a) Preparation of Mixed pure microbial nucleic acid

Mixed pure bacterial nucleic acid: commercial ATCC species were used. Selecting a plurality of ATCC strains with drug-resistant genes for activation culture, and extracting by using a micro sample genome DNA extraction kit (DP 316) of Tiangen Biochemical technology (Beijing) Co., ltd after culture to obtain pure bacterial nucleic acid. Several selected pathogenic microorganisms are known to have drug resistance genes, as shown in the following table.

b) Sample pretreatment and library establishment and computer utilization

Treating mixed pure bacterial nucleic acid according to steps 3) -9) of the experimental example scheme of the application, and constructing all samples into qualified sequencing libraries

c) Detection on machine

And (3) performing on-machine detection on the library constructed by the mixed pure bacterial nucleic acid, and adopting the same sequencing depth.

The results are shown in FIG. 3, in which Vancomycin resistance gene includes VanA and VanB. Therefore, the method can effectively detect beta-lactam antibiotic resistance genes such as KPC, SHV and the like and glycopeptide antibiotic resistance genes such as vanA, vanB and the like carried by bacteria in clinical samples, and can provide a basis for drug sensitive phenotype prediction. Wherein the detection sensitivity of the glycopeptide antibiotic drug-resistant gene is slightly better than that of the beta-lactam antibiotic drug-resistant gene (related to pathogen load).

Example 5 clinical sample validation and detection Limit evaluation

Since part of drug-resistant and virulence genes are not clinically common like KPC/SHV, verification of part of drug-resistant virulence genes is carried out by using plasmids. In the embodiment, the effectiveness and the accuracy of the method are verified by detecting and verifying real clinical samples, laboratory plasmid samples or laboratory microorganism culture samples and comparing the detection results with the detection results and the culture results of the traditional mNGS.

And (3) completing all library construction and sequencing through steps 1-9) of the experimental example, wherein the time for machine sequencing is 2 hours. And (4) normally establishing a library for the samples and obtaining sequencing data, wherein the depth of the sequencing data is kept consistent.

The specific comparative test results are shown in the following table:

in the aspect of detection accuracy, the detection result obtained by the method is consistent with the identification result obtained by the traditional method. Wherein, for 6 clinical samples (2 sputum/1 alveolar lavage fluid/3 blood samples), the consistency of the detection result and the detection result of the mNGS reaches 100% (3/3), the consistency of the detection result of the multiple targeted drug resistance/toxicity of the blood sample and the detection result of the blood culture reaches 100% (4/4), and the consistency of the detection result and the detection result of the mNGS reaches 100% (4/4). Similarly, the method can detect 100% of other plasmids and pure bacteria.

In terms of detection sensitivity, the method of the present application has a detection sensitivity at least an order of magnitude higher than that of the mNGS, as can be seen by comparing the lowest detected concentrations of mNGS in the above table with the method of the present application.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit the same; although the present application has been described in detail with reference to the foregoing embodiments, it should be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A targeting primer group for blood infection metagenome sample drug resistance/virulence gene library construction based on a nanopore sequencing platform is characterized in that the primer aims at 11 beta lactam drug resistance genes, 5 carbapenem drug resistance genes, 1 polymyxin drug resistance gene, 2 vancomycin drug resistance genes and 4 virulence factor genes.

2. The primer set according to claim 2, wherein the genes targeted by the primer set are specifically: mecA, CTX-M, OXA-24, OXA-23, blaTEM-bs, CTX-M-group1, CTX-M-group9, CTX-M-group2, blaTEM-esbl, SHV, orfx-SCC, VIM, KPC, OXA-48, IMP, NDM, MCR, vana, vanB, iuca, rmpA, rmpA2, and PVL.

3. The primer set of any one of claims 1-2, wherein the primer sequences in the primer set are as shown in SEQ ID No.1-38, or have at least 95% sequence identity with SEQ ID No. 1-46.

4. The primer set according to claim 3, wherein the primer concentration ratio is: SEQ ID NO.1/2: SEQ ID NO.13/14: 3/25/26 SEQ ID No. 25/28 SEQ ID No.31/32 SEQ ID No.35/36 SEQ ID No.37/38 SEQ ID No. 39/40.

5. A composition comprising the primer set of any one of claims 1 to 4.

6. A kit for establishing a drug/virulence gene library for a blood infection metagenomic sample for a nanopore sequencing platform, comprising the primer set of any one of claims 1-4.

7. Use of the primer set of any one of claims 1-4 in the construction of a sequencing library of drug/virulence genes from a blood infection metagenomic sample.

8. The use of the primer set according to any one of claims 1 to 4 in the detection of drug-resistant/virulence genes in blood infection metagenomic samples, or in the preparation of a kit for detecting drug-resistant/virulence genes in blood infection metagenomic samples.

9. A library construction method of a blood infection sample is characterized by comprising the following steps:

1) Treating the infected sample with host DNA;

2) Adding a DNA/RNA lysate for sample pretreatment;

3) Extracting nucleic acid from the treated sample;

4) Performing targeted amplification enrichment by using the primer set of any one of claims 1-4;

5) Purifying the amplification product;

10. A method for detecting a drug/virulence gene in a blood infection sample, comprising the method of claim 9, and further comprising the steps of sequencing and biological analysis.