CN117025801A

CN117025801A - HAP/VAP pathogenic bacteria and related drug-resistant gene rapid detection product

Info

Publication number: CN117025801A
Application number: CN202310714764.6A
Authority: CN
Inventors: 李丹; 竭晶; 宋磊; 管青天; 雒晶晶; 刘兵
Original assignee: First Hospital Jinlin University
Current assignee: First Hospital Jinlin University
Priority date: 2023-06-16
Filing date: 2023-06-16
Publication date: 2023-11-10
Anticipated expiration: 2043-06-16
Also published as: CN117025801B

Abstract

The application relates to the technical field of gene detection, in particular to a product and a method for rapidly detecting HAP/VAP pathogenic microorganisms and drug-resistant genes. The method provided by the application has the advantages of rapid detection, high sensitivity, good specificity, high accuracy and the like aiming at clinical samples.

Description

HAP/VAP pathogenic bacteria and related drug-resistant gene rapid detection product

Technical Field

The application relates to the technical field of gene detection, in particular to a product, a method and application for rapidly detecting HAP and VAP pathogenic microorganisms and related drug-resistant genes thereof.

Background

Hospital-acquired pneumonia (HAP) and Ventilator Associated Pneumonia (VAP) are the most common hospital-acquired infections in our country. HAP refers to pneumonia that occurs newly in hospitals during hospitalization of patients without receiving invasive mechanical ventilation, without being in the latent stage of pathogenic infection, and at a time of not less than 48 hours after hospitalization. VAP refers to pneumonia occurring after a patient is subjected to a tracheal cannula or tracheotomy and mechanical ventilation for 48 hours, and pneumonia occurring within 48 hours after a patient is mechanically ventilated and removed and tube drawn.

Both pneumonia is affected by severe disease and host basic disease, and early diagnosis and treatment after diagnosis are difficult. Once the etiology and its susceptibility results are determined, the antibacterial agent should be selected targeted and the mode of administration determined in combination with the drug metabolism and pharmacokinetics (PK/PD) and host conditions.

The detection of HAP and VAP is based on traditional culture technology, but the traditional culture technology is slow and insensitive, and especially the effect is more general after the detection is contacted with antibiotics. Molecular diagnostics can solve this problem by providing rapid results that are not affected by current antibiotics, and currently more common molecular diagnostics techniques mostly employ PCR or sequencing techniques. However, the HAP/VAP infectious bacteria have low detection sensitivity in partial tissue fluid and peripheral blood due to low released content, and have low nucleic acid extraction efficiency on pathogenic microorganisms with thicker cell walls, such as mould and candida infection, so that the molecular clinical detection rate and sensitivity are generally low in practice, and the existing method cannot solve the problem well.

In view of this, the present application has been proposed.

Disclosure of Invention

In order to solve the technical problems, the application develops a detection method and a detection system aiming at HAP and VAP, which are rapid in detection, high in sensitivity, good in specificity and high in accuracy based on the whole technical route of the attached figures 1-2.

Specifically, the application develops a rapid joint inspection method for nosocomial infection, which is based on targeted amplification and high-throughput sequencing technology and aims at 5 common nosocomial infection pathogenic bacteria: coli, acinetobacter baumannii, pseudomonas aeruginosa, klebsiella pneumoniae and staphylococcus aureus, and 2 pathogenic fungi: aspergillus (Aspergillus niger, aspergillus flavus, aspergillus fumigatus) and Candida (Candida albicans, candida glabrata) were subjected to rapid, high-sensitivity nucleic acid detection against 10 pathogenic microorganisms and the corresponding 17 drug resistance genes.

Accordingly, a first object of the present application is to provide a product for rapid and high sensitivity detection of HAP and VAP pathogenic microorganisms and drug resistance genes;

a second object of the present application is to provide a method for rapid detection of HAP and VAP pathogenic microorganisms and drug resistance genes.

Specifically, the technical scheme adopted by the application is as follows:

the application firstly provides a rapid detection primer composition for HAP/VAP pathogenic microorganisms and drug-resistant genes, wherein the primer is aimed at clinical pathogenic bacteria, pathogenic aspergillus and pathogenic candida.

Further, the primers are directed against Acinetobacter baumannii, pseudomonas aeruginosa, klebsiella pneumoniae, staphylococcus aureus, escherichia coli, aspergillus fumigatus, aspergillus flavus, aspergillus niger, candida albicans, and Candida glabrata.

Furthermore, the primer is further aimed at the following drug resistance genes related to the pathogenic bacteria: CTX-M, OXA-23, TEM, KPC, GES, NDM, IMP, VIM, OXA-48, mcr-1, mecA, vanA, vanB, kayG, inhA, embB, pncA.

Further, the primer is specifically designed by the following method:

targeting region selection design:

1) Downloading target genome sequences in Genebank, wherein the number of genome downloads is not less than 2000 strains;

2) Screening in genome sequences of all strains of the same species, and selecting specific sequences between species which are conserved in the species and have the sequence length of 250-550bp; the method comprises the steps of carrying out a first treatment on the surface of the Specific: taking 20-25bp as a node, screening in a genome one by one, and requiring that the comparison rate of the intraspecific conserved sequences in all strains exceeds more than 85%; the specific comparison rate between species is not compared with more than 96%, and the complete sequence of human genome is not compared with more than 99% in a region of more than 5 kb;

3) The number of the target areas found by each species is not less than 50, and the target areas are ranked from high to low in comparison rate;

4) The coordinates of each species need to fall on the same genome coordinate position, can not fall on the target area of the same genome, a second genome is used, and the number of the genome used in the target area of each species is not more than 5;

5) The target region selects a region with GC content of about 50% and single base/multiple base repetition less than 5;

6) The target region is not involved in plasmid, mitochondrial and 16S conserved regions;

7) The number of target areas per pathogenic species is not less than 5.

v. primer preliminary design step:

1) The primer length is 45-49bp, the GC content of the primer is 35% -65%, the Tm value is 65-66 ℃, the 3 'end of the primer avoids the 3 rd and 4 th positions of codons, no complementary sequence larger than 7bp exists between the primer and the primer, and the primer is 5'

The terminal and intermediate Δg values are at least 1.4 times the 3' terminal Δg value;

2) The 5' -end of the primer is provided with an illuminea sequencing tail sequence;

3) The sequence interval of primer amplification is between 250 and 550bp;

primer sequence optimization and adjustment.

Further, the sequence of the primer is specifically shown as SEQ ID NO.1-290, or at least more than 90% homology with SEQ ID NO. 1-290.

The application also provides a product for HAP/VAP pathogenic microorganisms and drug resistance genes, which comprises any one of the primer combinations.

Further, the product is a kit.

Further, the product also comprises enzyme for amplification, buffer solution, internal reference, quality control product and the like.

The application also provides application of any of the primer combinations in preparation of a rapid detection product of HAP/VAP pathogenic microorganisms and drug resistance genes.

The application also provides application of any of the primer combinations in the rapid detection of HAP/VAP pathogenic microorganisms and drug-resistant genes.

The application also provides a method for rapidly detecting HAP/VAP pathogenic microorganisms and drug-resistant genes, which comprises the step of amplifying a sample by utilizing any one of the primer compositions.

Further, the method also comprises the steps of sequencing, letter analysis and the like.

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

1) The correlation degree between the currently reported drug-resistant genotype and drug-resistant phenotype still has a gap, and the existing detection method has low drug-resistant related gene coverage, so that the related drug-resistant genes are difficult to detect with high sensitivity.

2) The application realizes high sensitivity, high specificity and high accuracy of the whole detection system by deeply designing and optimizing primer sequences and combinations (including targeted genes/loci, primer sequences, GC matching degree, generic and generic common primer design and the like) of the whole system, can be used for detecting pathogenic bacteria of a low-to-2 copy/reaction system, effectively solves the problem that the pathogenic bacteria of the HAP/VAP in tissue fluid and peripheral blood are difficult to detect due to low released content, and simultaneously has the advantages of complete flow, short time and the like by deeply designing and optimizing the primer sequences and combinations of the whole system.

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, a flow chart of amplification, library-building and sequencing of a target site according to the application;

FIG. 2, a roadmap of the overall technology of the application;

FIG. 3, results of detection before and after GC/coverage optimization of Pseudomonas aeruginosa;

FIG. 4, detection results before and after the optimization of the GC/coverage of Acinetobacter baumannii;

FIG. 5, aspergillus fumigatus GC/coverage optimization before and after detection results;

FIG. 6, results of detection before and after GC/coverage optimization of Aspergillus flavus;

FIG. 7, GC content comparison results before and after overall optimization of all primers;

FIG. 8, optimized primer sequences;

FIG. 9, optimized primer sequences;

FIG. 10, optimized primer sequences;

fig. 11, clinical specimen test results.

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.

Experimental example 1, screening and determination of HAP/VAP-infected bacteria, drug resistance Gene and drug resistance site

1) Selecting pathogenic bacteria; hospital-acquired pneumonia and ventilator-associated pneumonia are the most common hospital-acquired infections, and are difficult to diagnose and treat. The pathogenic bacteria which are common in HAP/VAP of China and are analyzed comprise Acinetobacter baumannii, pseudomonas aeruginosa, klebsiella pneumoniae and the like; meanwhile, the main differential diagnosis of nosocomial infections is fungal infections caused by dysbacteriosis, such as aspergillus and candida. Therefore, in order to define the etiology diagnosis of the nosocomial infection as soon as possible, the application is the basis for timely treatment of patients, and is helpful for improving the success rate of the rescue of the nosocomial acquired pneumonia, shortening the ICU hospitalization time, reducing the huge social and economic burden brought by the nosocomial acquired infection, and finally selecting Acinetobacter baumannii, pseudomonas aeruginosa, klebsiella pneumoniae, staphylococcus aureus, escherichia coli, aspergillus fumigatus, aspergillus flavus, aspergillus niger, candida albicans and candida glabrata for detection according to the comprehensive clinical requirements, the actual detection compatibility matching degree and the like.

2) Selection logic or experimental data of the corresponding drug resistance genes/drug resistance sites;

bacterial resistance presents a further challenge to treatment of HAP/VAP, and pathogens infected in the hospital are susceptible to drug resistance, and the drug resistance mechanisms of the pathogens are analyzed to include b-lactamases produced by enterobacteriaceae, including ESBL, ampC enzymes, and polypeptides, hydronase enases, penicillins, fluoroquinolones, sulfonamides, tetracyclines, and the like, which have an increasing trend in recent years.

Therefore, the application combines the series drug resistance mechanism of the pathogenic bacteria with the determined types, and early pre-experiment evaluation detection accessibility problems and the like, and finally screens the following 17 drug resistance genes: CTX-M, OXA-23, TEM, KPC, GES, NDM, IMP, VIM, OXA-48, mcr-1, mecA, vanA, vanB, kayG, inhA, embB, pncA.

Experimental example 2, corresponding primer design, optimization and determination

Experimental example one, targeting region selection and primer Primary design

1. Selective design of targeting regions

1) Downloading target genome sequences in Genebank, wherein the number of the downloaded genomes is not less than 2000 strains per species;

2) Screening in the homologous 2000 strain genome sequences, and selecting intraspecies conservation and interspecific specific sequences with the sequence length of 250-550bp;

3) Taking 25bp as a node, screening in genome one by one, and requiring that the comparison rate of the intraspecific conserved sequences in 2000 strains is more than 85%. The specific comparison rate between species is not compared with 96% or more, and the complete sequence of human genome is not compared with 99% or more in a region of more than 5 kb.

4) The number of the target areas found by each species cannot be less than 50, and the target areas are ranked from high to low according to the comparison rate;

5) The coordinates of each species should fall on the same genome coordinate position, and cannot fall on the target area of the same genome, a second genome is used, and the number of the genome used in the target area of each species cannot exceed 5;

6) The GC content of the target region is about 50%, and the single base/multiple base repetition is less than 5;

7) The target region cannot be related to plasmid region, mitochondrial region, 16S conserved region.

2. Primer primary design:

1) The length of the primer is 45-49bp, the GC content of the primer is 35-65%, the more the primer is close to 50%, the more the Tm value is close to 65-66 ℃, and the 3' end of the primer avoids the 3 rd and 4 th positions of codons. The primer itself and the complementary sequence larger than 7bp do not exist between the primers, and the 5 'end and the middle delta G value of the primer are 1.4 times of the delta G value of the 3' end; wherein, the TM calculation formula: tm=0.41 (% of GC) -674.5/l+81.36;

note that: l: primer base number; % of GC: primer GC content; % of gc=gc number/total number of primer bases;

3) The number of the primers in each target area is finally not less than 5;

4) The amplicon sequence interval is between 250-550 bp.

Based on the above steps, a targeting primer system for the above 10 pathogenic bacteria and 17 drug-resistant genes was preliminarily designed.

Experimental example two, adjustment and optimization of primer targeting sequence

For reasons of space, this section of content shows only examples of partial adjustment optimizations.

1. Primer sequence adjustment based on GC/match

Only considering the matching degree, the GC content span of the primer is large; when the PCR annealing temperature is set to be uniform, primers with higher TM values can be unbound, while primers with lower TM values than the annealing temperature can be subjected to nonspecific amplification, so that non-target signals can be generated.

In order to solve the above problems, the application adjusts and improves the sequence of the initial primer, taking pseudomonas aeruginosa, acinetobacter baumannii, aspergillus fumigatus and aspergillus flavus as examples, the part describes the comparison results before and after the optimization adjustment, and specifically, see the following table and fig. 3-6, and fig. 7 shows the GC content comparison results before and after the overall optimization of all the primers. From the optimized result, the adjusted sequence has better GC/matching degree, so that the amplification effect is better.

2. Further adjustment of primer sequences

For amplification of pathogenic microorganisms such as candida genes or loci with thicker cell walls, the present application adjusts candida albicans to "genus + species" common primers with "species" specific primer sequences.

Specific: because the candida cell wall is thicker, the yield in the extraction process is lower, the candida load in the sample is lower, only 100 copy/reaction sensitivity can be achieved by simply designing the species-specific primers, the design thought is adjusted, the genus-specific primers and the species-specific primers are combined and used, the numbers of the genus-specific primers and the species-specific primers are respectively searched, the number of the genus-specific primers is gradually increased from 2 genus-specific primers, and finally 3 genus-specific primers plus 7 species-specific primers are selected to judge together, the LOD can be as low as 10 copy/reaction, and the specificity reaches 100%. Continued addition of genus-specific primers results in reduced recognition specificity and non-targeted candida species are also recognized.

Through the adjustment, the primer sequence of the application for candida finally comprises the following steps:

similarly, for 17 drug resistance genes, there are also problems of primer targeting region screening, primer specificity and sensitivity optimization, and the reasons are not repeated.

In summary, the above series of primer sequence adjustments or optimizations, the final established optimal primer set of the present application is shown in FIGS. 8-10.

Experimental example 3 establishment of the method System of the application

1. The detection method comprises the following steps:

multiplex PCR reaction the system (10 ul) for each pool primer is as follows:

the PCR reaction was as follows:

after the end, 2ul of stop buffer was added and the mixture was allowed to stand for 30 minutes with a final volume of 12ul

1B. First round amplification reaction product purification

10ul of TE buffer is added in each reaction, 1.3X volume of magnetic beads are added, the mixture is fully and uniformly mixed, the mixture is placed on a magnetic rack for standing for 1 minute, the supernatant is discarded, the mixture is washed twice with freshly prepared 80% alcohol, and 10ul of TE buffer is added after the mixture is dried in the air and fully and uniformly mixed.

2A digestion reaction

The above 1B product was added:

6ul of nuclease-free water

2ul CP Reagent buffer

2ul CP Digestion Reagent

And taking down the centrifuge tube from the magnetic rack, fully and uniformly mixing, and standing at 37 ℃ for 10 minutes. After finishing, add 2ul of termination buffer

2B digest purification

Adding 1.3X volume of magnetic beads into the 2A product pipe, fully and uniformly mixing, placing on a magnetic rack, standing for 1 minute, removing the supernatant, washing twice with freshly prepared 80% alcohol, airing, and adding 10ul of TE buffer for fully and uniformly mixing.

3A second amplification

Sequentially adding into the 2B pipe

The PCR reaction was as follows:

3B second round amplification product purification

Adding 1X volume of magnetic beads into the 2A product pipe, fully and uniformly mixing, placing on a magnetic rack, standing for 1 minute, removing the supernatant, washing twice with freshly prepared 80% alcohol, airing, and adding 11ul of TE buffer for fully and uniformly mixing. Placed on a magnetic rack and after final clarification 10ul of library was aspirated and refrigerated at 4 ℃.

4A library quality inspection and mixing

The prepared library is firstly taken to 5ul, agarose gel electrophoresis is used for quality inspection, the normal library fragment size is about 400bp, and no detail dimer and nonspecific band exist. And then accurately quantifying the library concentration by using a Qubit4.0 fluorometer, and mixing libraries according to the quantitative result of the libraries, wherein the data amount of each pathogen library is required to be not less than 0.5M reads.

5A. High throughput sequencing

The concentration of the pooled library was accurately quantified using a qubit4.0 fluorometer and then diluted to a final concentration of 4nM. 5ul of freshly prepared 0.2N NaOH was added to a 5ul of 4nM mix, and after vortexing, 280g was centrifuged for 1min and left at room temperature for 5min to denature the library. The denatured library can be subjected to high throughput sequencing using Illumina MiSeq Reagent Nano Kit, sequencing platform: illumina Miseq, illumina NextSeq500/550, average data size per library was 0.3-0.5M reads, sequencing read length was PE100.

6A data filtration and analysis

The original data of the machine is firstly subjected to joint identification, the joints and the subsequent sequences of the joints are cut off by the ready, the ready with the double-end length larger than 60bp is reserved, then the low-quality filtration is performed, and the ready with Q30 more than 50% is reserved as high-quality data. And (3) carrying out primer identification on the high-quality data, reserving reads with the correct comparison of the two ends, and comparing with a pathogen database to finally determine the pathogen type and the pathogen content in the sample.

Example 4 Performance verification of the inventive method System

1. Single target strain sensitivity gradient repeatability test-staphylococcus aureus

Purchase of staphylococcus aureus was serially diluted to 1 copy/reaction, 2 copy/reaction, 3 copy/reaction 4 copy/reaction, 5 copy/reaction, 6 copy/reaction, 10 copy/reaction, and 100 copy/reaction; and 4 replicates were made in trace patterns of 1copy and 2 copy. The bacterial solutions with different gradients are uniformly mixed with 10ng of human gDNA, and library construction is carried out according to the method of example 3, and the specific evaluation results are as follows:

from the results, it can be seen that trace amounts of staphylococcus aureus as low as 1copy per reaction can be detected in single target experiments using this panel. On the premise of low copy/reaction, a plurality of pairs of primers still work stably, and the differences among reads produced by different primers are not more than 6 times, which means that the coverage of different primers is average, the working efficiency is uniform, and the result is true and reliable.

2. Single target strain sensitivity gradient repeatability test-escherichia coli

E.coli was purchased and diluted sequentially to 1 copy/reaction, 2 copy/reaction, 3 copy/reaction, 4 copy/reaction, 5 copy/reaction, 6 copy/reaction, 10 copy/reaction, and 100 copy/reaction; and 4-8 replicates were made in trace patterns of 1copy and 2 copy. The above bacterial solutions of different gradients were mixed with 10ng of human gDNA and library construction was performed as in example 3, with the following results:

from the results, it can be seen that a single target experiment with this panel can detect trace amounts of E.coli as low as 1 copy/reaction. On the premise of as low as 1 copy/reaction, a plurality of pairs of primers still work stably, and the differences among reads produced by different primers are not more than 6 times, which means that the coverage of different primers is average and the working efficiency is uniform.

3. Single target strain sensitivity gradient repeatability test-klebsiella pneumoniae

The purchase of klebsite, diluted sequentially to 1 copy/reaction, 2 copy/reaction, 3 copy/reaction 4 copy/reaction, 5 copy/reaction, 6 copy/reaction, 10 copy/reaction, and 100 copy/reaction; and 4-8 replicates were made in trace patterns of 1copy and 2 copy. The above bacterial solutions of different gradients were mixed with 10ng of human gDNA and library construction was performed as in example 3, with the following results:

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from the results, it can be seen that single target experiments with this panel can detect trace amounts of klebsiella pneumoniae as low as 3 copy/reaction. On the premise of 3 copy/reaction, a plurality of pairs of primers still work stably, and the differences among reads produced by different primers are not more than 6 times, which means that the coverage of different primers is average, the working efficiency is uniform, and the result is true and reliable.

4. Multi-target sensitivity gradient reproducibility test

Commercial E.coli, P.aeruginosa, staphylococcus aureus, candida albicans, acinetobacter baumannii, aspergillus fumigatus, aspergillus niger, candida parapsilosis were diluted sequentially to 1 copy/reaction, 10 copy/reaction, and 100 copy/reaction, and mixed in equal amounts; 3 replicates. The library construction was performed as in example 3 by mixing the above-mentioned mixed bacterial solutions with different gradients with 10ng of human gDNA, and the results were as follows

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It should be noted that trace amounts are calculated when the copy number is lower than 10, and the mixed target can be detected specifically and multiple targets in practice, so that the detection is very difficult; and the difference of the numbers of reads in different primer pairs of the same sample is not more than 6 times, which indicates that the working efficiency uniformity of different primers is good, the result is true and reliable, and the coverage is uniform.

Example 4 clinical sample testing

In this example, a total of 15 samples of clinically collected alveolar lavage fluid and blood were collected, and 40ul of samples were drawn for use. The procedure of example 3 was used for detection, the rest was shake-cultured in liquid medium, and drug-resistant culture was performed according to clinical manifestations to obtain clinical verification.

The detailed experimental parameters and experimental results are shown in fig. 11. The result shows that the detection result of the method is completely consistent with the clinical verification result for 15 samples with different types and different infections, so that the method still has extremely accurate detection advantage under the condition of real clinical low copy concentration.

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 primer composition for rapid detection of HAP/VAP pathogenic microorganisms and drug resistance genes, characterized in that the primer is directed against clinically pathogenic bacteria, aspergillus and candida;

preferably, the primers are directed against Acinetobacter baumannii, pseudomonas aeruginosa, klebsiella pneumoniae, staphylococcus aureus, escherichia coli, aspergillus fumigatus, aspergillus flavus, aspergillus niger, candida albicans, and Candida glabrata.

2. The primer composition of claim 1, wherein the primer is further directed to the following drug resistance genes: CTX-M, OXA-23, TEM, KPC, GES, NDM, IMP, VIM, OXA-48, mcr-1, mecA, vanA, vanB, kayG, inhA, embB and pncA.

3. The primer composition of claim 1, wherein the primer design comprises the steps of:

i. and (3) selecting and designing a target area:

1) Downloading genome sequences of target pathogenic species in Genebank, wherein the number of genome downloads is not less than 2000 strains per species;

2) Screening in genome sequences of all strains of the same species, and selecting specific sequences between species which are conserved in the species and have the sequence length of 250-550bp; the selecting comprises the following steps: taking 20-25bp as a node, screening in genome one by one,

the intraspecific conserved sequences are required to have the alignment rate of more than 85% in all strains; the specific comparison rate between species is below 96%, and the human genome complete sequence is more than 99% in a region of more than 5kb and cannot be compared;

4) Each seed coordinate needs to fall on the same genome coordinate position, and can not fall on the target area of the same genome, a second genome is used, and the number of genome used in the target area of each seed is not more than 5;

7) A number of target regions for each pathogenic species of no less than 5;

step ii, primer preliminary design:

1) The primer length is 45-49bp, the GC content of the primer is 35% -65%, the Tm value is 65-66 ℃, the 3' end of the primer avoids the 3 rd and 4 th positions of codons, no complementary sequence larger than 7bp exists between the primer and the primer, and the 5' end and the middle delta G value of the primer are at least 1.4 times of the delta G value of the 3' end;

3) The sequence interval of primer amplification is between 250 and 550bp;

primer sequence optimization and adjustment.

4. The primer composition of claim 3, wherein the primer sequence is as shown in SEQ ID NO.1-290 or has at least 90% homology with SEQ ID NO. 1-290.

5. A product for HAP/VAP pathogenic microorganisms and drug resistance genes, characterized in that the product comprises the primer composition according to any one of claims 1-4.

6. The product of claim 5, wherein the product is a kit.

7. The product of claim 6, further comprising an enzyme for amplification, a buffer, an internal reference, and a quality control.

8. The use of the primer composition according to any one of claims 1 to 5 in the preparation of a rapid detection product of HAP/VAP pathogenic microorganisms and drug-resistant genes, or in the rapid detection of HAP/VAP pathogenic microorganisms and drug-resistant genes.

9. A method for rapid detection of HAP/VAP pathogenic microorganisms and drug-resistant genes, comprising the step of amplifying a sample using the primer composition according to any one of claims 1 to 4.

10. The rapid test method of claim 9, further comprising the steps of sequencing and belief analysis.