CN115725570B - Nanopore sequencing-based blood sample drug resistance/virulence gene detection method - Google Patents

Abstract

The application relates to the technical field of molecular diagnosis, in particular to a drug resistance/toxicity detection method of a blood infection sample, which is used for carrying out targeted library construction on DNA of a blood metagenome sample based on a targeted primer design and realizing 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.

Description

Nanopore sequencing-based blood sample drug resistance/virulence gene detection method

Technical Field

The application relates to the technical field of molecular diagnosis, in particular to a blood sample drug resistance/toxicity gene detection method based on a nanopore sequencing technology.

Technical Field

Due to the lack of awareness of the use of antibiotics, antibiotic abuse has increased in recent years, resulting in the emergence of antibiotic resistance at a surprising rate. From the discovery of penicillin in 1929, drug resistant strains such as methicillin-resistant Lin Putao cocci and vancomycin-resistant enterococci developed gradually over the last 30 years. Bacterial resistance is more gradually upgraded after the 80 s, from gram negative bacteria to gram positive bacteria, and beta-lactamase (an enzyme that can deactivate antibiotics) produced by bacteria is developed from common enzymes to ultra-broad spectrum enzymes. Although there are no more than 200 antibiotics currently used in clinic, and more than 10 antibiotics are growing each year, the research and development of antibiotics are far from the drug resistance of bacteria. The increasing emergence of bacterial resistance, multiple resistant bacteria, and the difficulty in treatment has become serious, which together lead to prolonged patient duration, prolonged hospital stay, and higher mortality rates, particularly in Intensive Care Units (ICU). It is counted that about 1 million patients with infectious diseases need to be treated in an auxiliary way by using a pathogen detection method every year, wherein the number of severe infections is more than ten million, and for the more complex critically infected patients, the conventional detection means are difficult to provide guidance for clinic rapidly. In addition to this, every year new pathogenic microorganisms and increasingly resistant/virulent pathogenic bacteria are largely challenged with conventional detection means. Therefore, it is important to develop a detection method capable of rapidly determining pathogenic bacteria and drug resistance/virulence genes.

Currently, blood culture remains the "gold standard" for detecting pathogens in patients clinically suspected of having infectious disease. However, it is known that blood culture has very obvious defects, 1) long culture time 2) failure of specific pathogens to culture 3) treatment with antibiotics, and the conditions of insufficient pathogen acquisition exist; for drug resistance/toxicity detection, the antibiotic drug sensitivity experiment needs to be carried out after the blood culture and the positive reporting, so that the detection time is further prolonged, and the patients needing immediate treatment can be undoubtedly frosted on snow.

The metagenome-based pathogenic microorganism detection technology (mNSS) is a high-throughput sequencing technology which does not depend on traditional microorganism culture, does not need specific amplification, can directly carry out indiscriminate and non-selective nucleic acid in clinical samples, and can rapidly judge the pathogenic microorganism types and drug resistance/virulence genes in the clinical samples after comparing and analyzing sequencing data 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 the expert consensus of China for detecting infectious disease pathogens by applying the 2021 metagenome high-throughput sequencing technology, for the part without a microbial constant value, the detection by adopting the mNGS can be considered, but enough deep sequencing depth needs to be ensured, which can possibly cause the timeliness of the metagenome to be influenced. At the same time, high costs, such as non-critically ill patients, are generally unacceptable for detection.

Nanopore sequencing is a single-molecule real-time sequencing technology, and the method is also independent of traditional microorganism culture, and can rapidly detect suspected pathogens in clinical samples. The single molecular DNA or RNA in the sample is used for recording the composition of the base by the current change when passing through the nanopore, so that the sequencing of the whole genome is completed. The main advantage of nanopore sequencing is that on one hand, the nanopore sequencing has an ultra-long reading length, can generate sequences of 1kb to more than 100kb, and the longer sequences have greater advantages in the aspects of analyzing pathogenic bacteria, 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 performed in real time, so that the species information of microorganisms in the sample and the drug resistance/virulence gene information carried by the microorganisms can be obtained rapidly. That is, when nanopore sequencing is combined with an efficient genomic nucleic acid extraction and library building method, detection results can be obtained within 4-6 hours, thereby helping clinical diagnosis and treatment of critically ill patients.

Disclosure of Invention

In order to solve the technical problems, the application is based on multiplex PCR library construction and combines the high-flux nanopore sequencing technology to detect bacteria and drug resistance/virulence genes in blood samples.

Specifically, the application provides the following technical scheme:

the application firstly provides a targeting primer group for establishing a blood infection metagenome sample drug resistance/virulence gene library based on a nanopore sequencing platform, wherein the primer is aimed 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.

Further, the primer group specifically aims at genes: 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 primer concentration ratio is: SEQ ID NO.1/2:SEQ ID NO.3/4:SEQ ID NO.5/6:SEQ ID NO.7/8:SEQ ID NO.9/10:SEQ ID NO.11/12: SEQ ID NO.13/14: SEQ ID NO.15/16: SEQ ID NO.17/18: SEQ ID NO.19/20: SEQ ID NO.21/22: SEQ ID NO.23/24: SEQ ID No.25/26:SEQ ID No.27/28:SEQ ID No.29/30:SEQ ID No.31/32:SEQ ID No.33/34:SEQ ID No.35/36:SEQ ID No.37/38:SEQ ID No.39/40:SEQ ID No.41/42:SEQ ID No.43/44:SEQ ID No. 45/46=3:3:3:3:3:3:3 (1-2): 3:3:3:3:3 (1-2): 3:3:3:3:3:3:3:3:3:3:3:3).

Preferably, the primer concentration ratio is: SEQ ID NO.1/2:SEQ ID NO.3/4:SEQ ID NO.5/6:SEQ ID NO.7/8:SEQ ID NO.9/10:SEQ ID NO.11/12: SEQ ID NO.13/14: SEQ ID NO.15/16: SEQ ID NO.17/18: SEQ ID NO.19/20: SEQ ID NO.21/22: SEQ ID NO.23/24: SEQ ID NO. 25/26:27/28:29/30:31/32:33/34:35/36:37 SEQ ID NO.39/40 SEQ ID NO.41/42 SEQ ID NO.43/44 SEQ ID NO. 45/46=3:3:3:3:2:3:3:3:3:3:2:3:3:3:3:3:3:3:3:3:3:3).

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

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

The application also provides a kit for constructing a drug resistance/virulence gene library of a blood infection metagenome sample for a nanopore sequencing platform, which comprises any one of the primer groups.

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

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

The application also provides application of any one of the primer groups in preparing a detection kit for detecting drug resistance/virulence genes of blood infection metagenome samples.

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

1) Carrying out host DNA treatment on the infected sample;

2) Adding DNA/RNA lysate to perform sample pretreatment;

3) Extracting nucleic acid from the treated sample;

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

5) Purifying the amplified product;

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

The application also provides a blood infection sample drug resistance/virulence gene detection method, which comprises the library construction method and further comprises the steps of sequencing and biological analysis.

The application has the beneficial technical effects that:

the application establishes a group of effective targeted amplification system through exploring and establishing multi-targeted drug-resistant/virulence flora and gene combination and designing, screening and optimizing a targeted primer system, and the system can comprehensively amplify and effectively construct drug-resistant virulence genes in blood infection metagenome samples and ensure the detection comprehensiveness and accuracy in a third-generation nanopore sequencing platform.

The rapid detection method based on the targeted determination of drug resistance/toxicity genes, which is developed by the application, can finish the detection of drug resistance/toxicity in a blood sample within 6 hours at maximum, and has the advantages of high sensitivity, high flux, high detection speed, accurate result and the like.

Drawings

FIG. 1 shows agarose gel electrophoresis results, wherein M is Marker, and the Marker interval is 200bp-2000bp;1 represents group 1; and 2 represents group 2.

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

FIG. 3 shows the results of detection performance test of drug-resistant primers.

Detailed Description

Embodiments of the present application will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present application and should not be construed as limiting the scope of the present application. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Partial term definition

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.

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

The term "about" in the present application means a range of accuracy that one skilled in the art can understand while still guaranteeing the technical effect of the features in question. The term generally means a deviation of + -10%, preferably + -5%, from the indicated value.

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.

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

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 method for detecting the drug resistance/toxicity of the blood infection metagenome sample is based on the targeting primer design to target and build a library for the DNA of the blood metagenome sample, and combines nanopore sequencing to realize the rapid detection of the drug resistance/toxicity of the blood infection sample, 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 produced. The preferred in vivo infection of the present application is bacterial resistance to antibiotics.

The "virulence" refers to virulence such as invasiveness and toxin generated by metabolism of bacteria, viruses, fungi and the like. Virulence factors can be encoded on mobile genetic elements (e.g., plasmids, gene islands, phages, etc.) and subjected to horizontal gene transfer, rendering harmless bacteria dangerous pathogens.

In some embodiments, the targeting primer is directed against 11 beta lactam drug resistance genes, 5 carbapenem drug resistance genes, 1 pair polymyxin drug resistance genes, 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.3/4:SEQ ID NO.5/6:SEQ ID NO.7/8:SEQ ID NO.9/10:SEQ ID NO.11/12: SEQ ID NO.13/14: SEQ ID NO.15/16: SEQ ID NO.17/18: SEQ ID NO.19/20: SEQ ID NO.21/22: SEQ ID NO.23/24: SEQ ID No.25/26:SEQ ID No.27/28:SEQ ID No.29/30:SEQ ID No.31/32:SEQ ID No.33/34:SEQ ID No.35/36:SEQ ID No.37/38:SEQ ID No.39/40:SEQ ID No.41/42:SEQ ID No.43/44:SEQ ID No. 45/46=3:3:3:3:3:3:3 (1-2): 3:3:3:3:3 (1-2): 3:3:3:3:3:3:3:3:3:3:3:3).

In some specific embodiments, the primer concentration ratio is: SEQ ID NO.1/2:SEQ ID NO.3/4:SEQ ID NO.5/6:SEQ ID NO.7/8:SEQ ID NO.9/10:SEQ ID NO.11/12: SEQ ID NO.13/14: SEQ ID NO.15/16: SEQ ID NO.17/18: SEQ ID NO.19/20: SEQ ID NO.21/22: SEQ ID NO.23/24: SEQ ID NO.25/26:SEQ ID NO.27/28:SEQ ID NO.29/30:SEQ ID NO.31/32:SEQ ID NO.33/34:SEQ ID NO.35/36:SEQ ID NO.37/38:SEQ ID NO.39/40:SEQ ID NO.41/42:SEQ ID NO.43/44:SEQ ID NO. 45/46=3:3:3:3:3:3:2:3:3:3:2:3:3:3:3:3:3:3:3:3:3:3:3:3:3:3:3:3:3:3:3:3:3:3.

The application is illustrated below in connection with specific embodiments.

Experimental example basic method flow establishment of the application

1. Process for removing host

The infected samples were subjected to host DNA removal treatment.

2. Sample pretreatment

Adding DNA/RNA cleavage buffer into the treated sample, fully mixing, and centrifuging for collection.

3. Nucleic acid extraction

The method adopts a commercial kit to extract nucleic acid, and the extraction mode is used for extracting according to the instruction of the kit.

4. Targeted amplification

Based on the designed drug resistance/toxicity primer system, the Novamat biotechnology Co., ltd 2X Phanta Flash M aster Mix (P510) kit is used for targeted amplification, and the specific system is as follows

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

Because the primer is a drug-resistant/virulence primer, 10uM stock solution is diluted by using Low TE (10mM Tris,0.1mM EDTA,pH8.0) of biological engineering (Shanghai) Co., ltd, diluted into 2uM working solution, and 2ul to 20ul of PCR reaction systems are taken from each working solution when the primers are mixed together, so that the final concentration of the primer is ensured to be 0.2uM per primer.

5. Amplification product purification

Commercial purification magnetic beads are selected for purification, and the purification mode is performed according to the requirements of a magnetic bead purification instruction.

6. Library construction

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

7. Purifying magnetic beads: and (5) purifying by using a commercial kit.

8. Library loading: pre-machine preparation was performed using the ONT (Oxford Nanopore) SQK-PBK004 kit.

9. Sequencing runs: and sequencing by adopting a three-generation nanopore sequencing platform Gridion.

10. And (5) letter generation analysis: and carrying out belief analysis on sequencing off-machine data.

Example 1 drug resistance/virulence Gene selection

In view of the differences in microbial flora during human blood infection, the drug resistance and virulence response 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 critical for clinical evaluation. The detection process not only needs to take the comprehensiveness of detection into consideration, avoids the problem of missed detection, but also needs to ensure that the selected target flora or genes can be effectively detected, and avoids the problems of false negative and the like.

In order to realize comprehensive and effective detection of drug resistance and virulence of blood infection samples, the application combines the prior theory, and finally takes beta-lactamase resistant gram-negative bacillus, carbapenem resistant enterobacter/acinetobacter baumannii, vancomycin resistant enterococci and the like as research objects after the prior mass belief prediction and wet experiment demonstration. The corresponding exploration logic is as follows:

pathogenic bacteria containing VIM gene are generally selected from genus Pseudomonas aeruginosa, klebsiella pneumoniae, escherichia coli, enterobacter cloacae, etc., and can tolerate carbapenem antibiotics;

pathogenic bacteria containing MecA gene are generally selected from the genus Staphylococcus aureus, and are tolerant to beta-lactam antibiotics;

the pathogenic bacteria containing CTX-M_group8/25 gene are usually selected from the genus Escherichia coli, klebsiella pneumoniae, salmonella enterica, etc., and can tolerate cefotaxime antibiotics;

pathogenic bacteria containing OXA-24 gene are generally selected from genus Acinetobacter baumannii, acinetobacter pittii, etc., and can tolerate beta lactam antibiotics;

pathogenic bacteria containing the oxa_23 gene are generally selected from the genus Acinetobacter baumannii, klebsiella pneumoniae, etc., and are resistant to beta-lactam antibiotics;

pathogenic bacteria containing the blaTEM_bs gene are generally selected from the genus Klebsiella pneumoniae, which are tolerant to beta-lactam antibiotics;

the pathogenic bacteria containing the ctx_m_group1 gene are generally selected from the genus Escherichia coli, which is resistant to antibiotics of the cefotaxime class;

the pathogenic bacteria containing the ctx_m_group9 gene are generally selected from the genus Escherichia, which is resistant to antibiotics of the cefotaxime class;

pathogenic bacteria containing KPC gene are generally selected from genus Klebsiella pneumoniae, escherichia coli, acinetobacter baumannii, etc., and can tolerate beta lactam antibiotics;

the pathogenic bacteria containing the ctx_m_grou2 gene are generally selected from the genus Escherichia coli, which is resistant to antibiotics of the cefotaxime type;

the pathogenic bacteria containing the oxa_48 gene are typically selected from the genus Klebsiella pneumoniae, which are tolerant to beta-lactam antibiotics;

the pathogen containing the blatem_esbl gene is typically selected from the genus Klebsiella pneumoniae, which is tolerant to beta lactam antibiotics;

pathogenic bacteria containing SHV gene are generally selected from genus Klebsiella pneumoniae, klebsiella oxytoca, escherichia coli, etc., and are tolerant to beta-lactam antibiotics;

the pathogenic bacteria containing the orfX-SCCmec gene are typically selected from the genus coagulase negative staphylococci and are tolerant to beta-lactam antibiotics;

pathogenic bacteria containing mcr genes are generally selected from the genera Escherichia coli, klebsiella pneumoniae, salmonella sp, etc., and are tolerant to polymyxin antibiotics;

pathogenic bacteria containing IMP gene are generally selected from genus Acinetobacter baumannii, pseudomonas putida, klebsiella oxytoca, etc., and can tolerate carbapenem antibiotics;

pathogenic bacteria containing NDM gene are generally selected from the genus Escherichia coli, klebsiella pneumoniae, providencia stuartii, etc., and can tolerate carbapenem antibiotics;

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

the pathogen containing the vanB gene is typically selected from the genus Enterococcus faecalis and is resistant to vancomycin antibiotics.

Pathogenic bacteria containing CTX-M gene are usually selected from the genus Escherichia coli, klebsiella pneumoniae, enterobacteriaceae, etc., and are tolerant to beta-lactam antibiotics;

pathogenic bacteria containing the orfx-SCC gene are generally selected from the genus Staphylococcus, and are tolerant to beta-lactam antibiotics;

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

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

the pathogenic bacteria comprising 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 established drug resistance/virulence genes of the present application 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, and finally determining that the specific genes 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 resistance/toxicity amplification system of the system is used as a high-weight amplification system, and in the primer design process, not only the amplification efficiency of a single primer is considered, but also the compatibility of each primer in the system is considered, and meanwhile, the sequencing platform is matched. In this example, for the genes determined in example 1, the optimal target amplification region is screened according to the gene sequence, and meanwhile, the target primer is designed, screened and filtered based on the characteristic requirement of the nanopore sequencing platform, so as to comprehensively optimize the primer sequence.

1. Primer sequence design optimization

For reasons of space, this example exemplifies primer design optimization of MecA gene (optimization of the raw end and wet end, respectively).

1. Collection of DNA sequences of target genes

The MecA resistance gene sequence was downloaded from the NDARO database.

2. Target gene multiple sequence alignment and target sequence selection

The MAFFT algorithm is used for carrying out multiple sequence comparison on the sequence of the MecA gene, the self-research algorithm of the comprehensive company is adopted in a relatively conservative window of the MecA gene, more than 50% of species with more than 20bp continuous regions are set to have base identity at the same position, and the region with highest identity is selected as a target.

3. Designing primers

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

4. Examination of melting temperature of MecA primer

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

5. Prediction of nonspecific amplification of host DNA by MecA primers

Primer-blast was used to predict the non-specific amplification of MecA on itself and between different primer pairs to host DNA where the host DNA was referenced to the human hg38 genome and then the number of complementary bases for non-specific amplification of the primer pairs was counted.

6. Predicting the tendency of MecA to form dimers

Primer-pooler software was used to examine the complementarity between the Primer pairs themselves and the different Primer pairs and predict dimer formation propensity. The lowest Δg value of dimers that MecA primers can form was counted.

7. Primer selection and pooling

The primer pairs are comprehensively evaluated and selected according to the identification degree, coverage, melting temperature deviation degree, nonspecific amplification complementary base number and dimer delta G value of the primers.

Preliminarily designing a plurality of groups of primer sequences aiming at MecA through the letter generation optimization, wherein partial results are as follows;

as can be seen from the amplification verification of the wet experiment, the amplification effect of the group2 is superior to that of the group1, and is shown in the 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 the group2 is about 500bp, which accords with the theoretical amplification length of 574bp, while the amplification length of the pair of primers in the group1 is less than 1400bp, which does not accord with the theoretical amplification length, and the primers in the group2 are selected.

2. Primer combination optimization

For the whole multiplex primer system, the application adopts a plurality of alternatives for designing primers for each different type of genes in an initial system. And then, carrying out a single primer amplification experiment on all the candidate primers, and aiming at checking the amplification capability and the amplification efficiency of the designed primers and eliminating part of the primers. In the embodiment, the drug resistance/toxicity primers of 44 pairs are tested together, and 10 pairs of primers which cannot be amplified are eliminated in the first single primer amplification experiment; and then carrying out multiplex system construction on the primers passing the test, and testing 30 pairs of primers in total, wherein 7 pairs of primers are eliminated due to incapacity of amplification, and 4 pairs of primers are eliminated due to incapacity of achieving the optimal amplification effect. The primer sequences are further regulated, and the remaining 23 pairs of primers are amplified normally and construct a multiple system successfully.

Through the optimization, the application finally determines the target primer sequence as follows:

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wherein R and Y are degenerate primers, r=aor G; y=c or T.

Example 3 primer concentration optimization

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

Through analysis, 2 pairs of amplification primers aiming at TEM in 23 pairs of drug-resistant/virulence primers are obviously higher than other drug-resistant/virulence primers, so that in order to ensure balanced amplification among the drug-resistant/virulence primers and reduce deviation values, proper adjustment of the input concentration and proportion of the primers is tried, and specific adjustment thinking comprises: 1) adjusting the concentration of 21 pairs of drug resistant/virulence primers alone, 2) adjusting the concentration of 2 pairs of blaTEM primers alone, 3) adjusting the concentration of 23 pairs of drug resistant/virulence primers as a whole.

a) Preparation of Mixed pure bacterial nucleic acid

Mixed pure bacterial nucleic acid: commercial ATCC strains were used. 4 ATCC strains with drug resistance genes are selected for activation culture, and pure bacterial nucleic acid is obtained by extracting with a micro sample genome DNA extraction kit (DP 316) of Tiangen biochemical technology (Beijing) limited company after the culture. The presence of drug resistance genes is known for all 4 important pathogenic microorganisms selected, 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) Independent adjustment of 21 pairs of drug resistance/virulence primer concentrations

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

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

2) Separate adjustment of 2 pairs of blaTEM primer concentrations

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

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

3) Overall adjustment of 23 pair drug resistance/virulence primer concentration

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

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

test protocol 7 the other drug resistance/virulence primers were 0.15uM, whereas the TEM primer concentration was 0.1uM;

test scheme 8 the other drug resistant/virulence primers were 0.15uM, whereas the TEM primer concentration was 0.05uM. '548

c) Agarose gel electrophoresis detection

Electrophoresis was performed using 1% agarose gel at 120V for 30min at a loading of 5ul, and the results were as shown in FIG. 2 for a total of 8 sets of test results.

As to the electrophoresis result, the bands were single and concentrated in 500-800bp because the amplified length of the primer was substantially the same for the drug resistance of the above four bacteria, KPC amplified length should be 798bp, vanA amplified length should be 805bp, vanB amplified length should be 805bp, mecA amplified length should be 574bp, so the bands would be indistinguishable together and would fall on the range of 500-800 bp.

1) Results of individual adjustment of 21 pairs of drug resistance/virulence primer concentrations: test protocol 1 amplified bands were brighter than test protocol 2, so 21 pairs of drug resistant/virulence primer concentrations of 0.15uM could be initially used as the primer concentration to be determined. Test protocol 1 amplified the band somewhat more upward than test protocol 2, i.e., more toward KPC, vanA, vanB, while test protocol 2 more toward mecA, both alternatives.

2) Results of 2 pairs of blaTEM primer concentrations were adjusted individually: test scheme 3 and test scheme 4 were independent adjustments of TEM drug-resistant primer concentrations, and it was evident that the amplification efficiency was stronger than the sum of the other drug-resistant primers, and also stronger than test scheme 1 and test scheme 2, but the difference between test scheme 3 and test scheme 4 was not significant, both from the amplified fragment size and from the amplified brightness, and thus could be used as alternatives.

3) Overall 23 results for drug resistance/virulence primer concentration: test protocol 5 did not differ much in strip size and strip brightness compared to test protocol 6, while test protocol 7 was stronger in strip brightness than test protocol 8, with more significant amplification. If the 4 protocols were compared simultaneously, it was found that the bands for tests 7 and 8 were somewhat higher than for tests 5 and 6, while the upper half of the band was brighter, so tests 7 and 8 were biased more toward amplification of the TEM gene.

In summary, test scheme 7 (other drug resistant/virulence primers were 0.15uM and TEM primer concentration was 0.1 uM) amplified with significantly higher efficiency than test scheme 5 and test scheme 6, while amplifying TEM genes than test scheme 8.

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

SEQ ID NO.1/2:SEQ ID NO.3/4:SEQ ID NO.5/6:SEQ ID NO.7/8:SEQ ID NO.9/10:SEQ ID NO.11/12: SEQ ID NO.13/14: SEQ ID NO.15/16: SEQ ID NO.17/18: SEQ ID NO.19/20: SEQ ID NO.21/22: SEQ ID NO.23/24: SEQ ID NO. 25/26:27/28:29/30:31/32:33/34:35/36:37/38=3:3:3:3:3: (1-2) 3:3:3:3:3 (1-2) 3:3:3:3:3:3:3:3:3:3:3; preferably 3:3:3:3:3:2:3:3:3:3:3:2:3:3:3:3:3:3:3:3:3:3:3:3:3, more preferably, the other drug resistant/virulence primers in the system are 0.15uM and TEM primer concentration is 0.1uM.

Example 4 sequencing detection Performance evaluation

The system was evaluated by sequencing the whole flow.

a) Preparation of Mixed pure bacterial nucleic acid

Mixed pure bacterial nucleic acid: commercial ATCC strains were used. Several ATCC strains with drug resistance genes are selected for activation culture, and pure bacterial nucleic acid is obtained by extracting with a micro sample genome DNA extraction kit (DP 316) of Tiangen biochemical technology (Beijing) limited company after the culture. Several pathogenic microorganisms were selected for the known presence of 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) Sample pretreatment and warehouse establishment machine

The mixed pure bacterial nucleic acid is treated according to the steps 3) to 9) of the experimental scheme of the application, and all samples are constructed into qualified sequencing libraries

c) On-machine detection

And (5) performing on-machine detection on the library constructed by the mixed pure bacterial nucleic acid, and adopting the same sequencing depth.

As a result, the detected gene was shown on the left side in FIG. 3, and VanA and VanB were included in Vancomycin resistance gene. Therefore, the method can effectively detect the beta-lactam antibiotic drug resistance genes such as KPC, SHV and the like and the glycopeptide antibiotic drug resistance genes such as vanA, vanB and the like carried by bacteria in clinical samples, and can provide basis for drug sensitive phenotype prediction. Wherein the detection sensitivity of the glycopeptide antibiotic resistance gene is slightly better than that of the beta-lactam antibiotic resistance gene (related to pathogen load).

Example 5 clinical sample validation and detection Limit evaluation

Since part of the drug resistance and virulence genes are not clinically common like KPC/SHV, verification of part of the drug resistance virulence genes is performed using plasmids. The present example verifies the validity and accuracy of the method of the present application by performing detection verification on a real clinical sample, a laboratory plasmid sample, or a laboratory microbial culture sample, by comparing with the conventional mNSS detection result and culture result.

And 3) completing all library building sequencing through the experimental example steps 1-9), wherein the sequencing time on the machine is 2 hours. And the samples are normally built into libraries and sequencing data are obtained, and the depth of the sequencing data is consistent.

Specific comparative test results are shown in the following table:

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in terms of detection accuracy, the detection results obtained by the method are all consistent with the identification results obtained by the traditional method. Wherein, for 6 clinical samples (2 sputum/1 alveolar lavage liquid/3 blood samples), the consistency of the detection result and the mNSS detection result is 100% (3/3), the consistency of the multiple targeting drug resistance/toxicity detection result and the blood culture result of the blood samples is 100% (4/4), and the consistency of the multiple targeting drug resistance/toxicity detection result and the mNSS detection result is 100% (4/4). Likewise, the method of the application can detect 100% of other plasmids and pure bacteria.

In terms of detection sensitivity, the detection sensitivity of the method of the application is at least one order of magnitude higher than that of mNGS by comparing the lowest detection concentrations of mNGS and the method of the application in the above table.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present application, and not for limiting the same; although the application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the application.

Claims (7)

1. The targeting primer group for establishing a blood infection metagenome sample drug resistance/virulence gene library based on a nanopore sequencing platform is characterized by aiming 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; the targeting primer group aims at genes: 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; the primer sequences in the targeting primer group are shown in SEQ ID NO. 1-46.

2. The set of targeting primers according to claim 1, wherein the concentration ratio of the primers is: SEQ ID NO.1/2 SEQ ID NO.3/4 SEQ ID NO.5/6 SEQ ID NO.7/8 SEQ ID NO.9/10 SEQ ID NO.11/12: SEQ ID NO.13/14 SEQ ID NO.15/16 SEQ ID NO.17/18 SEQ ID NO.19/20 SEQ ID NO.21/22 SEQ ID NO.23/24: SEQ ID No.25/26 SEQ ID No.27/28 SEQ ID No.29/30 SEQ ID No.31/32 SEQ ID No.33/34 SEQ ID No.35/36 SEQ ID No.37/38 SEQ ID No.39/40 SEQ ID No.41/42 SEQ ID No.43/44 SEQ ID No. 45/46=3:3:3:3:3:3:3 (1-2): 3:3:3:3 (1-2): 3:3:3:3:3:3:3:3:3:3:3).

3. A composition comprising the targeting primer set of any one of claims 1-2.

4. A kit for nanopore sequencing platform for drug resistance/virulence gene banking against blood infected metagenomic samples, characterized in that it comprises the targeting primer set of any one of claims 1-2.

5. Use of a set of targeting primers according to any of the claims 1-2 in the construction of a sequencing library of drug resistance/virulence genes of a blood-infected metagenomic sample.

6. Use of the targeting primer set according to any one of claims 1-2 for the preparation of a detection kit for a drug resistance/virulence gene of a blood infection metagenomic sample.

7. A method for constructing a library of blood-infected samples, comprising the steps of:

1) Carrying out host DNA treatment on the infected sample;

2) Adding DNA/RNA lysate to perform sample pretreatment;

3) Extracting nucleic acid from the treated sample;

4) Carrying out targeted amplification enrichment by using the targeted primer group of claim 2;

5) Purifying the amplified product;

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