Fluorescent quantitative PCR primer group for identifying high-virulence klebsiella pneumoniae, application and detection method

Abstract

The embodiment of the invention relates to a fluorescent quantitative PCR primer group for identifying high-virulence klebsiella pneumoniae, application and a detection method, the primer group comprises one or more primer pairs in SEQ ID NO: 1 to SEQ ID NO: 26, and the identification accuracy of the high-virulence klebsiella pneumoniae can be improved.

Description

Technical Field

[0001] The present application relates to the field of biological detection technology, and more specifically, to a primer set, application and detection method for fluorescent quantitative PCR for identifying highly virulent Klebsiella pneumoniae. Background Art

[0002] Klebsiella pneumoniae is an increasingly important bacterial pathogen that can cause severe organ damage and life-threatening diseases. One of its notable characteristics is the ability to acquire new genes. Currently, the pathogen is mainly divided into two pathological types: classic Klebsiella pneumoniae (cKP) and hypervirulent Klebsiella pneumoniae (hvKp). Hypervirulent Klebsiella pneumoniae is highly mucoid and highly toxic, and is prone to cause critical illnesses such as pneumonia, liver abscesses, and bloodstream infections. It is difficult to treat and has a high mortality rate. Existing virulence detection methods have shortcomings, and multiple methods are usually required in clinical practice to improve accuracy, which brings great confusion to both doctors and patients.

[0003] The characteristics of hvKp and its differences from cKp have not been fully investigated. Although hvKp has been described as a highly virulent pathogen, there are increasing reports of infections caused by hvKp in the community. Clinical epidemiological studies have shown that hvKp infection can occur at any age and that infected individuals often present with multiple sites of infection, which may subsequently undergo metastatic dissemination, making K. pneumoniae an unusual member of the Enterobacteriaceae family. hvKp can infect almost any site in the human body. These infectious syndromes include liver abscesses, pneumonia, necrotizing fasciitis, endophthalmitis, and meningitis. The hyperviscosity phenotype, which was initially thought to be susceptible and specific to hvKp strains, has now been shown to be a non-exclusive marker, as not all hvKp strains are hyperviscosity and some cKp strains also have this feature. Therefore, it is inaccurate to use the hyperviscosity phenotype alone to define hvKp strains. Similarly, the chromosomally encoded aerobic bacteriocin (iutA) receptor or iroE gene is also present in many cKp strains and cannot be used alone to define hvKp. The presence of K1 or K2 capsular type alone cannot be used as a criterion for identifying hvKp. However, hvKp has acquired virulence genes present on large virulence plasmids (such as pK2044 and pLVPK) and integrated chromosomal elements (ICEs) that confer a high virulence phenotype, which are called biomarkers on virulence plasmids.

[0004] Traditional methods for detecting virulence genes of Klebsiella pneumoniae mainly rely on PCR technology to detect a series of known virulence genes, such as rmpA, rmpA2, magA, etc. These genes are believed to be related to the virulence characteristics of bacteria, and in theory their presence or absence can be used as an indicator to assess the virulence of strains. However, recent studies have shown that these traditional virulence gene detection methods have several limitations in actual clinical applications: the ubiquity of virulence genes, which are often detected even in classical strains. For example, genes such as rmpA and magA can also be detected in some Klebsiella pneumoniae strains with lower virulence. This finding suggests that simply detecting the presence of these genes is not sufficient to accurately assess the virulence level of strains.

[0005] The virulence genes of Klebsiella pneumoniae include capsule genes, lipopolysaccharide, pili, outer membrane proteins, nitrogen source utilization system and iron carrier system. However, the experimental results found that whether it is through the detection of pili or virulence genes to identify hvKp, the accuracy is not high enough compared with the gold standard - the greater wax moth model. Summary of the invention

[0006] The present application is provided to solve the above defects in the prior art. A primer set, application and detection method for fluorescent quantitative PCR for identifying highly virulent Klebsiella pneumoniae are needed to improve the accuracy of identifying highly virulent Klebsiella pneumoniae.

[0007] In a first aspect of the present application, a primer set for fluorescent quantitative PCR for identifying highly virulent Klebsiella pneumoniae is provided, wherein the primer set comprises any one or more of the following primer pairs:

[0008] The primer pair shown in SEQ ID NO: 1 and SEQ ID NO: 2 for the gene expressed by ACO54002.1;

[0009] The primer pair shown in SEQ ID NO:3 and SEQ ID NO:4 for the AOP87411.1 expressed gene;

[0010] The primer pair shown in SEQ ID NO:5 and SEQ ID NO:6 for the AVX34350.1 expressed gene;

[0011] The primer pair shown in SEQ ID NO:7 and SEQ ID NO:8 for the gene expressed by AWF76568.1;

[0012] The primer pair shown in SEQ ID NO:9 and SEQ ID NO:10 for the CTQ24123.1 expressed gene;

[0013] The primer pair shown in SEQ ID NO:11 and SEQ ID NO:12 for the EPA91234.1 expressed gene;

[0014] The primer pair shown in SEQ ID NO: 13 and SEQ ID NO: 14 for the gene expressed by WP_000872613.1;

[0015] The primer pair shown in SEQ ID NO: 15 and SEQ ID NO: 16 for the gene expressed by WP_000888080.1;

[0016] The primer pair shown in SEQ ID NO: 17 and SEQ ID NO: 18 for the gene expressed by WP_008502228.1;

[0017] The primer pair shown in SEQ ID NO: 19 and SEQ ID NO: 20 for the gene expressed by WP_008502230.1;

[0018] The primer pair shown in SEQ ID NO:21 and SEQ ID NO:22 for the gene expressed by WP_009310051.1;

[0019] The primer pair shown in SEQ ID NO:23 and SEQ ID NO:24 for the gene expressed by WP_020324597.1;

[0020] The primer pair shown in SEQ ID NO: 25 and SEQ ID NO: 26 for the gene expressed by WP_001091224.1.

[0021] In a second aspect of the present application, a fluorescent quantitative PCR kit for identifying highly virulent Klebsiella pneumoniae is provided, wherein the kit comprises the primer set described in any embodiment of the present application.

[0022] The third aspect of the present application provides a use of the primer set described in any embodiment of the present application or the kit described in any embodiment of the present application in identifying highly virulent Klebsiella pneumoniae.

[0023] The fourth aspect of the present application provides a use of the primer set described in any embodiment of the present application or the kit described in any embodiment of the present application in the preparation of a product for identifying highly virulent Klebsiella pneumoniae.

[0024] The fifth aspect of the present application provides a method for identifying highly virulent Klebsiella pneumoniae, the method comprising the following steps: 1) extracting RNA of the bacteria to be tested; 2) reverse transcribing the extracted RNA; 3) using cDNA as a template and the primer set according to claim 1 to perform PCR amplification to obtain a PCR product; and determining the type of the bacteria to be tested based on the sequence of the PCR product.

[0025] The present invention provides a primer set, application and detection method for fluorescent quantitative PCR for identifying highly virulent Klebsiella pneumoniae in various embodiments, and proposes a primer set for fluorescent quantitative PCR. The experimental results show that the primer set of the present invention can more accurately identify Klebsiella pneumoniae as highly virulent Klebsiella pneumoniae, and can more accurately distinguish it from classical Klebsiella pneumoniae. The problem that the traditional gene presence detection method may not be able to accurately evaluate the virulence level of bacteria is solved. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In the drawings, which are not necessarily drawn to scale, the same reference numerals may describe similar parts in different views. The same reference numerals with letter suffixes or different letter suffixes may represent different instances of similar parts. The accompanying drawings generally illustrate various embodiments by way of example and not limitation, and together with the specification and claims, are used to illustrate the embodiments applied for. When appropriate, the same reference numerals are used throughout the drawings to refer to the same or similar parts. Such embodiments are illustrative and are not intended to be exhaustive or exclusive embodiments of the present apparatus or method.

[0027] Figure 1 The genome structure of the highly virulent strain WX071 according to Experimental Example 1 of the present application is shown;

[0028] Figure 2 The genome structure of the classical strain WX045 according to Experimental Example 1 of the present application is shown;

[0029] Figure 3 A diagram showing the number of genes differentially expressed according to the transcriptome data of the validation example of the present application;

[0030] Figure 4 The GO enrichment analysis result diagram according to the verification example of this application is shown;

[0031] Figure 5 The KEGG enrichment analysis result diagram according to the verification example of this application is shown;

[0032] Figure 6 The electrophoresis diagram of the highly virulent Klebsiella pneumoniae WX090 and the electrophoresis diagram of the low-virulence strain WX077 according to the verification example of the present application are shown;

[0033] Figure 7The ratio of the number of strains of Klebsiella pneumoniae with different survival rates according to the verification example of the present application is shown;

[0034] Figure 8 The expression ratio diagram of 13 genes according to the verification example of the present application is shown;

[0035] Fig. 9 A graph showing the relationship between the survival rate of G. mellonella and the detection rates of 13 candidate genes according to the verification example of the present application. DETAILED DESCRIPTION

[0036] In order to enable those skilled in the art to better understand the technical solution of the present application, the present application is described in detail below in conjunction with the accompanying drawings and specific implementation examples. The embodiments of the present application are further described in detail below in conjunction with the accompanying drawings and specific implementation examples, but are not intended to limit the present application.

[0037] The words "first", "second" and similar words used in this application do not indicate any order, quantity or importance, but are only used to distinguish. The words "include" or "comprise" and similar words mean that the elements before the word include the elements listed after the word, and do not exclude the possibility of including other elements.

[0038] According to an embodiment of the present application, a primer set for fluorescent quantitative PCR for identifying highly virulent Klebsiella pneumoniae is provided, wherein the primer set includes any one or more of the following primer pairs:

[0039] The primer pair shown in SEQ ID NO: 1 and SEQ ID NO: 2 for the gene expressed by ACO54002.1;

[0040] The primer pair shown in SEQ ID NO:3 and SEQ ID NO:4 for the AOP87411.1 expressed gene;

[0041] The primer pair shown in SEQ ID NO:5 and SEQ ID NO:6 for the AVX34350.1 expressed gene;

[0042] The primer pair shown in SEQ ID NO:7 and SEQ ID NO:8 for the gene expressed by AWF76568.1;

[0043] The primer pair shown in SEQ ID NO:9 and SEQ ID NO:10 for the CTQ24123.1 expressed gene;

[0044] The primer pair shown in SEQ ID NO:11 and SEQ ID NO:12 for the EPA91234.1 expressed gene;

[0045] The primer pair shown in SEQ ID NO: 13 and SEQ ID NO: 14 for the gene expressed by WP_000872613.1;

[0046] The primer pair shown in SEQ ID NO: 15 and SEQ ID NO: 16 for the gene expressed by WP_000888080.1;

[0047] The primer pair shown in SEQ ID NO: 17 and SEQ ID NO: 18 for the gene expressed by WP_008502228.1;

[0048] The primer pair shown in SEQ ID NO: 19 and SEQ ID NO: 20 for the gene expressed by WP_008502230.1;

[0049] The primer pair shown in SEQ ID NO:21 and SEQ ID NO:22 for the gene expressed by WP_009310051.1;

[0050] The primer pair shown in SEQ ID NO:23 and SEQ ID NO:24 for the gene expressed by WP_020324597.1;

[0051] The primer pair shown in SEQ ID NO: 25 and SEQ ID NO: 26 for the gene expressed by WP_001091224.1.

[0052] Among the above 13 primer pairs, DMA of high-virulence strains and low-virulence strains was detected by electrophoresis, and it was found that they all existed in high-virulence strains and partially existed in low-virulence strains, but the detection rate of these 13 primer pairs in high-virulence strains was very high, while the detection rate in low-virulence strains was 0. Therefore, the 13 primer pairs are specific for the identification of high-virulence strains and can be used as effective markers to distinguish high-virulence and low-virulence Klebsiella pneumoniae.

[0053] The primer sequences of the primers in this application are shown in Table 1.

[0054] Table 1 Primer sequences for identifying highly virulent Klebsiella pneumoniae

[0055]

[0056]

[0057] According to the embodiments of the present application, a kit for fluorescent quantitative PCR for identifying highly virulent Klebsiella pneumoniae is also provided, wherein the kit comprises the primer set described in any embodiment of the present application.

[0058] According to the embodiments of the present application, there is also provided a use of the primer set described in any embodiment of the present application or the kit described in any embodiment of the present application in identifying highly virulent Klebsiella pneumoniae.

[0059] According to the embodiments of the present application, there is also provided a use of the primer set described in any embodiment of the present application or the kit described in any embodiment of the present application in the preparation of a product for identifying highly virulent Klebsiella pneumoniae.

[0060] According to an embodiment of the present application, a method for identifying highly virulent Klebsiella pneumoniae is also provided, and the identification method comprises the following steps: 1) extracting RNA of the bacteria to be tested; 2) reverse transcribing the extracted RNA; 3) using cDNA as a template and the primer set described in claim 1 to perform PCR amplification to obtain a PCR product; and determining the type of the bacteria to be tested based on the sequence of the PCR product.

[0061] In some embodiments, determining the type of the bacteria to be tested according to the sequence of the PCR product includes: when the PCR product contains one or more of ACO54002.1 expression gene, AOP87411.1 expression gene, AVX34350.1 expression gene, AWF76568.1 expression gene, CTQ24123.1 expression gene, EPA91234.1 expression gene, WP_000872613.1 expression gene, WP_000888080.1 expression gene, WP_008502228.1 expression gene, WP_008502230.1 expression gene, WP_009310051.1 expression gene, WP_020324597.1, and WP_001091224.1, determining that the bacteria to be tested is a highly virulent Klebsiella pneumoniae.

[0062] In some embodiments, determining the type of the bacteria to be tested according to the sequence of the PCR product includes: when the PCR product contains any one or more of the WP_008502230.1 expression gene, the EPA91234.1 expression gene, the WP_009310051.1 expression gene, the CTQ24123.1 expression gene and the AWF76568.1 expression gene, determining that the bacteria to be tested is a highly virulent Klebsiella pneumoniae.

[0063] In some embodiments, the extraction of RNA of the bacteria to be tested includes: centrifuging the bacterial solution to remove the supernatant to obtain cell solids; mixing the cell solids with a buffer solution, lysing the mixture, adsorbing the mixture on an adsorption column, deproteinizing the adsorption column, rinsing and centrifuging the mixture to obtain the RNA of the bacteria to be tested.

[0064] In some embodiments, the method for synthesizing cDNA includes: RNA or mRNA is mixed with oligonucleotide, reverse transcription kit, reverse transcription kit and water and incubated to obtain cDNA.

[0065] In this application, highly virulent Klebsiella pneumoniae is referred to as highly virulent strain or highly virulent Klebsiella pneumoniae; and classical Klebsiella pneumoniae is referred to as low-virulence strain or low-virulence Klebsiella pneumoniae.

[0066] Experimental Example 1-Sequencing of Klebsiella pneumoniae genome

[0067] The genome sequencing of Klebsiella pneumoniae was completed by Biomarker Biotechnology Co., Ltd. The experimental subjects were 10 strains of Klebsiella pneumoniae (coded as WX060, WX071, WX090, WX117, WX120, WX045, WX075, WX077, WX079, and WX132, respectively). Among these 10 strains of Klebsiella pneumoniae, there were 5 strains of highly virulent Klebsiella pneumoniae and 5 strains of classical Klebsiella pneumoniae, all of which were purchased from Thermo Fisher Scientific, USA.

[0068] 1. Sequencing principle:

[0069] PacBio sequencing technology uses SMRT chips as sequencing carriers. In the nanopores inside the SMRT chip, DNA polymerase binds to the template, and four bases (dNTPs) are labeled with four-color fluorescence. During the base pairing stage, the addition of different bases will emit different colors of light, and the type of base added can be determined based on the wavelength and peak value of the light.

[0070] 2. Experimental process:

[0071] The experimental process was performed according to the standard protocol provided by PacBio (PacBio Technology Co., Ltd.), including sample quality detection, library construction, library quality detection and library sequencing. The library construction includes the following steps:

[0072] 1) Use g-TUBE to shear DNA samples;

[0073] 2) Repair the damaged DNA samples;

[0074] 3) Perform end repair on DNA;

[0075] 4) Connect the dumbbell type connector;

[0076] 5) performing exonuclease digestion;

[0077] 6) Use the BluePippin automated nucleic acid recovery system to screen target fragments and obtain a sequencing library.

[0078] 3. Information analysis mainly includes the following steps:

[0079] 1) Quality control of raw data, filtering CCS reads that are too short;

[0080] 2) Genome assembly: assemble the filtered CCS reads de novo and correct the assembled genome;

[0081] 3) Analysis of genome components, mainly including: analysis of repetitive sequences, coding genes, non-coding RNA, prophages, gene islands, CRISPR, etc.;

[0082] 4) Functional annotation, mainly including general databases such as Nr, Uniprot, COG, KEGG and other databases, as well as proprietary database annotations such as CAZyme, PHI, and CARD;

[0083] 5) Genome map analysis, including: genome circle map, genome map display tools, and genome map.

[0084] 4. Klebsiella pneumoniae genome sequencing results

[0085] 1) Klebsiella pneumoniae genome size and plasmids

[0086] The sequencing results showed that the genome size of all bacteria was about 5-6Mb. All bacteria were composed of chromosomes and several plasmids. As shown in Table 2, all bacteria carried at least one plasmid. The genome structure of the highly virulent strain WX071 is as follows: Figure 1 The genome structure of the classical strain WX045 is shown in Figure 2 As shown, the highly virulent strain WX071 carries 4 plasmids, while the classical strain WX045 carries 5 plasmids.

[0087] Table 2 Genome assembly of 10 clinically isolated Klebsiella pneumoniae strains

[0088]

[0089]

[0090] 2) Structural analysis of the genomes of 10 clinically isolated Klebsiella pneumoniae strains

[0091] According to the data of the genome, gene prediction was performed using the software Prodigal v2.6.3, which uses a dynamic programming algorithm to predict genes in newly sequenced genomes with high accuracy. The statistical information of the specific gene prediction results is shown in Table 3. The results show that the number of genes of these 10 strains of Klebsiella pneumoniae is about 5,000 genes. RepeatMaskerv4.0.5 software is used to compare the bacterial genome with a known repetitive sequence database (such as Repbase) to search for repetitive sequences in the genome. The repetitive sequence ratio of highly toxic strains is 0.49-0.55%, while the repetitive sequence ratio of low toxic strains has a larger span of 0.32-0.94%. The software Infernal v1.1.3 can accurately predict the three types of rRNA in the genome based on the covariance model. The number of non-coding RNAs in most strains is about 115, among which the number of non-coding RNAs in the highly toxic strain WX117 reaches 240, while the number of non-coding RNAs in the low toxic strain WX075 reaches 286. CRISPR (Clustered Regularly Interspaced Palindromic Repeats) is a continuous DNA region in the genome. The CRISPR region, together with some Cas-related proteins, forms an immune system that is widely present in bacteria and archaea, which prevents external nucleic acids from entering bacterial cells. The CRT v1.2 software was used to predict the CRISPR sequence of the genome, and the statistical results are shown in Table 3. The number of CRISPR sequences in highly virulent strains is relatively distributed between 15 and 18, while the number of CRISPR sequences in low-virulence strains is between 14 and 62, among which the number of CRISPR sequences in strain WX079 is as high as 62. Genomic islands often have specific functions. The software IslandPath-DIMOB v0.2 is a commonly used software for predicting genomic islands in bacterial genomes. The number of genomic islands is shown in Table 3. Only strain WX075 contains 3 genomic islands, and the others have 4 genomic islands. The software antiSMASH v5.0 is the most widely used tool for identifying and analyzing biosynthetic gene clusters (BGCs) in bacterial and fungal genome sequences. Gene clusters encoding all known broad chemical classes of secondary metabolites were accurately identified. The results showed that most strains contained five gene clusters, and strains WX17, WX075, and WX079 contained four gene clusters. The promoter region is a key regulatory region that allows genes to be transcribed or repressed, and can also be determined experimentally. Using the tool PromPredictv1, genomic DNA with different GC contents was analyzed and used as a universal standard for predicting promoter regions in microbial genomes.Promoters within 500 bp upstream of the gene and with a prediction confidence higher than level 2 were selected as prediction results. The results are shown in Table 3: It shows that the number of promoters in all highly virulent strains is 141, while in the low-virulence strains, the number of promoters in most strains is more than 50, and only the strain WX075 has a promoter number as high as 187.

[0092] Table 3 Genome structures of 10 clinically isolated Klebsiella pneumoniae strains

[0093]

[0094] 3) Subcellular localization of proteins encoded by genes in the genomes of 10 clinically isolated Klebsiella pneumoniae strains

[0095] The signal peptide is a short peptide segment at the N-terminus of the protein initially synthesized by the ribosome. After being synthesized by the ribosome, the peptide segment is recognized by the signal peptide recognition particle, and is guided to the biomembrane (cell membrane or organelle membrane surface, bacteria only have cell membrane) under the action of related proteins and signal peptide recognition particles, and then the signal peptide sequence is removed to form a mature protein. The software SignalPv4.0 is used to predict whether the protein contains a signal peptide and predict the specific signal peptide removal site, as shown in Table 4. The results show that the number of signal peptides in all strains is between 477-524. There is no obvious difference between high-virulence and low-virulence strains.

[0096] Transmembrane proteins are proteins that exist in large quantities on the cell membrane. The transmembrane regions of these proteins contain one or more transmembrane helices, and the other regions are located on the surface of the cell membrane. According to the characteristics of the transmembrane region and the non-transmembrane region, the software TMHMM v2.0 designed based on the hidden Markov model can accurately predict whether the protein is a transmembrane helix, and find out the specific predicted transmembrane region and non-transmembrane region, as shown in Table 4. The results show that the number of transmembrane proteins in all strains is between 1221-1333, and there is no obvious difference in number between high virulence and low virulence.

[0097] Secretory proteins are proteins secreted outside the cell during the life of microorganisms. Secretory proteins can often improve the adaptability of bacteria to the environment. For example, some secretory proteins are proteases or polysaccharide degrading enzymes, which degrade macromolecular nutrients (proteins or polysaccharides) that cannot be directly absorbed and utilized by bacteria in the environment into small molecules that can be absorbed and utilized. Some secretory proteins are toxic to other bacteria (antibiotics, etc.), inhibiting the growth of other types of bacteria around them. Proteins with signal peptides can be guided to the cell membrane surface or outside the cell by signal peptides, and proteins located on the cell membrane generally have transmembrane helices. In the above-predicted proteins containing signal peptides, the proteins containing transmembrane helices are removed, and the remaining proteins are secretory proteins. As shown in Table 4, the number of secretory proteins is between 395-524, and there is no obvious difference in number between high toxicity and low toxicity.

[0098] Table 4 Subcellular localization of gene-encoded proteins from 10 clinically isolated Klebsiella pneumoniae strains

[0099]

[0100]

[0101] Through Experimental Example 1, it can be obtained that among the 10 clinically isolated Klebsiella pneumoniae strains, there is no obvious difference between the highly toxic Klebsiella pneumoniae and the classical Klebsiella pneumoniae in terms of plasmid genome size, number of genes, ratio of repeat sequences, number of non-coding RNAs and CRISPR sequences in the highly toxic and classical strains. In addition, there are no significant differences in gene islands and gene clusters (including metabolic functions and antibiotic resistance, etc.) between the highly toxic and low toxic strains. It can be obtained that there are certain limitations in evaluating the toxicity of strains based solely on genome sequencing data. Genome sequencing can only reveal the existence of genes, but cannot provide the expression of genes in a specific environment. There may be significant differences in the expression levels of toxic genes in different strains, but these differences cannot be directly observed through genome sequencing. For example, some toxic genes may not be expressed in classical strains due to transcriptional regulation or other mechanisms, but are expressed at high levels in highly toxic strains. Genome sequencing data provides a comprehensive perspective on the structure of bacterial genomes, but there are still limitations in its analysis and interpretation.

[0102] Compared with the existing belief that highly toxic Klebsiella pneumoniae often contains large plasmids, which carry multiple virulence genes and significantly enhance the pathogenicity of bacteria, in this study, through genome sequencing technology, we found that there was no significant difference in the number of plasmids between highly toxic and low toxic strains, and even some low toxic strains had more plasmids. Plasmids are small DNA molecules independent of chromosomes in bacteria that can carry antibiotic resistance genes, virulence genes, and metabolic genes, giving host bacteria a variety of survival advantages. The traditional view is that the number and size of plasmids are directly related to the virulence of bacteria, especially in highly toxic Klebsiella pneumoniae. However, the results of genome sequencing in this application show that the number of plasmids is not the only factor that determines the virulence of bacteria. On the contrary, some low-toxic strains may contain a larger number of plasmids, indicating that other factors may play a role in the expression of virulence.

[0103] Therefore, it can be concluded from Experimental Example 1 that there is no significant difference between highly virulent Klebsiella pneumoniae and low-virulence Klebsiella pneumoniae in terms of the number of signal peptides, transmembrane proteins, secreted proteins, number of plasmids, genome size, number of genes, repeat ratio, number of non-coding RNAs, and CRISPR sequences, and the accuracy of identifying highly virulent Klebsiella pneumoniae through these aspects is poor.

[0104] Validation Example - Transcriptome Sequencing of Klebsiella Pneumoniae

[0105] The experimental subjects were 10 strains of Klebsiella pneumoniae (coded as high virulence: WX060, WX071, WX090, WX117, WX120; low virulence: WX045, WX075, WX077, WX079, WX132). Among these 10 strains of Klebsiella pneumoniae, 5 strains of high virulence Klebsiella pneumoniae and 5 strains of classic Klebsiella pneumoniae were purchased from Thermo Fisher Scientific, USA.

[0106] The transcriptome sequencing of 10 strains of Klebsiella pneumoniae was completed by Beijing Biomarker Biotechnology Co., Ltd. The transcriptome sequencing of each Klebsiella pneumoniae strain was carried out according to the following steps: First, the total RNA of the sample was extracted using the Trizol method, and the sample quality was checked using the ThermoNanoDropOne (ultra-micro UV-visible photometer) and Agilent4200TapeStation (UV gel imager). After the test was qualified, the ribosomal RNA was removed using the EpicentreRibo-Zero rRNARemovalKit. Then, the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB Next UltraII RNA strand-specific library preparation kit) was used for library construction. The specific steps included: 1. mRNA was sheared into short fragments; 2. Using mRNA as a template, a six-base random primer (random hexamers) was used to synthesize a strand of cDNA; 3. Buffer, dNTPs (dTTP in deoxynucleotide dNTP was replaced by dUTP), DNA Polymerase I (DNA polymerase I) and RNase H (ribonucleotidase H) were added to synthesize a second strand of cDNA; 4. Double-stranded cDNA was purified using AMPure XP beads (DNA fragment sorting magnetic bead kit); 5. The second strand of cDNA containing U was degraded using USER enzyme (uracil-specific excision reagent enzyme); 6. The purified double-stranded cDNA was end-repaired, A-tailed and connected to the sequencing adapter; 7. Fragment size selection was performed using AMPure XP beads; 8. Finally, PCR amplification was performed, and the PCR product was purified using AMPure XP beads to obtain the final transcriptome library.

[0107] NEBNextUltra IIDirectional RNA Library Prep Kit for Illumina, cDNA first-strand synthesis kit and AMPure XP beads were purchased from Beijing Adlai Biotechnology Co., Ltd.

[0108] The constructed transcriptome library needs to be quality checked. For qualified transcriptome libraries, PE150 sequencing will be performed using the Illumina high-throughput sequencing platform. The raw image data files obtained by sequencing are converted into raw sequencing sequences (Raw Reads) through base calling analysis. The results are stored in the FASTQ (abbreviated as fq) file format, which contains the sequencing sequences (Reads) and their corresponding sequencing quality information. After obtaining the raw sequencing sequences (Raw Reads), if there is species reference information, bioinformatics analysis will be performed.

[0109] A total of 5101 transcribed gene products were detected in the transcriptome library sequencing results of 5 highly virulent Klebsiella pneumoniae strains and 5 classical Klebsiella pneumoniae strains. After comparing these two types of strains, a total of 475 genes were found to be differentially expressed, such as Figure 3 As shown, 287 genes were up-regulated and 188 genes were down-regulated.

[0110] After GO and KEGG enrichment analysis of differentially expressed genes, the GO enrichment analysis results are shown in the figure Figure 4 As shown in Figure 2, many metabolic processes were significantly enriched, including nucleic acid metabolism, intracellular nitrogen metabolism, and intracellular aromatic compound metabolism. Figure 5 As shown in the figure, the KEGG enrichment results showed that pathways such as pyruvate metabolism, porphyrin metabolism and ABC transport were significantly enriched.

[0111] It was also found in the transcriptome data that some genes showed significantly high expression in highly toxic strains, of which 13 genes were expressed in 5 highly toxic strains and not expressed at all in 5 low toxic strains, as shown in Table 5. The functions of these genes involve hemolysin expression regulation, OsmC-like proteins, and transposon-related elements. In addition, two genes encoding unknown proteins among these 13 genes were highly expressed in highly toxic strains. According to the transcriptome data, no independently expressed genes were found in low toxic strains, and almost all genes in low toxic strains were expressed in high toxic strains.

[0112] Table 5 13 genes specifically expressed in highly virulent strains

[0113]

[0114]

[0115] The presence of these 13 specific genes was detected using the genomic DNA of 5 highly virulent Klebsiella pneumoniae strains and 5 low-virulence Klebsiella pneumoniae strains as templates. The results showed that all 13 genes were present in the 5 highly virulent strains, and some of the genes were also present in the low-virulence strains. The electrophoresis diagram of the highly virulent Klebsiella pneumoniae WX090 and the electrophoresis diagram of the low-virulence strain WX077 are shown in Figure 2. Figure 6 As shown, it can be obtained that 10 genes were detected in the low toxicity strain WX077. The electrophoresis data of all gene detections are shown in Table 6.

[0116] Table 6 Detection of 13 highly toxic genes

[0117]

[0118] Verification of the transcription of 13 genes in clinically isolated Klebsiella pneumoniae

[0119] Virulence verification of the greater wax moth model

[0120] A total of 308 Klebsiella pneumoniae strains were collected, and their specimen sources, infection sites, basic information of patients, and clinical outcomes were analyzed. Specimen sources included sputum (245 strains, accounting for 79.5%), midstream urine (26 strains, accounting for 8.4%), bronchoalveolar lavage fluid (12 strains, accounting for 3.9%), pleural effusion bile (11 strains, accounting for 3.6%), blood drainage fluid (8 strains, accounting for 2.6%), and secretions (6 strains, accounting for 1.9%). It can be inferred from the source of the specimens that lung infection is the most common, followed by urinary system and bloodstream infection.

[0121] The basic information of the patients showed that there were 220 male patients, accounting for 71.4%; there were 88 female patients, accounting for 28.6%. The average age of the patients was 63.2±16.4 years old, mainly the elderly. Most patients had underlying diseases such as hypertension or diabetes, including 155 patients with hypertension (50.3%), 50 patients with coronary heart disease (16.2%), 99 patients with diabetes (32.1%), 71 patients with a history of tumors (23.1%), and 73 patients with cerebral infarction (23.7%). In terms of lifestyle, there were 114 patients (37.0%) who smoked and 62 patients (20.1%) who drank.

[0122] In the clinical data analysis, 266 patients (86.4%) used antibiotics for infection control; 42 patients (13.6%) did not use antibiotics or the use of antibiotics was ineffective. The median duration of antibiotic use was 12 days (8.0 to 19.0 days). The median length of hospital stay was 14 days (10.0 to 21.0 days). In terms of clinical outcomes, 35 patients (11.4%) had rapid disease progression and ineffective treatment; 273 patients (88.6%) improved after treatment (Table 1.2).

[0123] 12 hours after the injection of 308 clinically isolated Klebsiella pneumoniae strains, 13 strains infected wax moths all died, and 120 hours after the injection, 54 strains infected wax moths all died, accounting for 4.3% and 17.6% respectively. 12 hours after the injection of 308 clinically isolated Klebsiella pneumoniae strains, 165 strains infected wax moths had a mortality rate of more than 50%, and 241 strains infected wax moths had a mortality rate of more than 50%, accounting for 53.7% and 78.1% respectively; 12 hours after the injection of 308 clinically isolated Klebsiella pneumoniae strains, 218 strains infected wax moths had a mortality rate of more than 20%, and 289 strains infected wax moths had a mortality rate of more than 20%, accounting for 53.7% and 78.1% respectively (see Figure 7). In order to further analyze the correlation between its virulence and clinical virulence genes and other indicators, this study defined the clinically isolated Klebsiella pneumoniae with a mortality rate of more than 90% after 12 hours of injection as a high-virulence strain. By this standard, 42 high-virulence strains were screened out. The strains with a mortality rate of less than 10% in the greater wax moth at 120 hours were defined as low-virulence strains. By this standard, 29 low-virulence strains were screened out.

[0124] 138 strains of Klebsiella pneumoniae were selected from 308 clinically isolated strains of Klebsiella pneumoniae. Total RNA of these 138 clinically isolated Klebsiella pneumoniae was extracted and reverse transcribed. 13 candidate genes were identified using cDNA as a template. The results showed that the ratios of the genes in different strains were different. Figure 8 As shown in Figure 2, as the virulence weakened, the detection ratio of virulence genes decreased significantly. Figure 8 As shown, Figure 8 The horizontal axis in the figure represents the strain number of 308 strains of Klebsiella pneumoniae. These data indicate that virulence genes may exist in the genome, and some virulence genes may not be transcribed. In order to further analyze the relationship between the 13 genes and the virulence of Klebsiella pneumoniae, the detection data of the 13 candidate genes and the data of the Greater Wax Moth model were used for Spearman correlation analysis. The results showed that the gene detection data at 12, 24, 48 and 72 hours and the virulence detected by the Greater Wax Moth model showed a significant correlation, and the survival rate of the Greater Wax Moth and the detection rate of the 13 candidate genes at different time points showed a significant negative correlation. Fig. 9 These data indicate that these 13 virulence genes are significantly correlated with the virulence of Klebsiella pneumoniae.

[0125] According to the detection of 13 candidate genes in high-virulence and low-virulence strains of Klebsiella pneumoniae, 18 high-virulence strains and 20 low-virulence strains were included in the 138 strains. The results showed that the detection rate of these 13 candidate genes in high-virulence strains was very high. Among them, the detection rates of the five genes WP_008502230.1, EPA91234.1, WP_009310051.1, CTQ24123.1 and AWF76568.1 in high-virulence strains were 100%, but not in low-virulence strains. Even the ACO54002.1 gene, which had the lowest detection rate in high-virulence strains, had a detection rate of 72.2%, while the detection rates of these genes in low-virulence strains were all 0 (see Table 7). In order to further analyze the feasibility of these 13 candidate genes as virulence identification markers for Klebsiella pneumoniae, their sensitivity and specificity were evaluated. The results showed that the identification sensitivity of genes such as WP_008502230.1, EPA91234.1, WP_009310051.1, and CTQ24123.1 was 100%, and even the lowest sensitivity of ACO54002.1 gene reached 72.22%. In terms of specificity, these 13 genes all showed 100% specificity. These data indicate that these 13 candidate genes can be used as effective markers to distinguish high-virulence and low-virulence Klebsiella pneumoniae.

[0126] Table 7 Correlation analysis between 13 candidate genes and virulence

[0127]

[0128]

[0129] Example 1

[0130] In this study, Klebsiella pneumoniae strains were isolated from 103 patients with slow clinical progression and 35 patients with rapid clinical progression. RNA was extracted according to the method of the validation example, and the extracted RNA was reverse transcribed. Then, PCR amplification was performed using cDNA as a template and the primers shown in Table 1 to obtain PCR products; and genetic testing was performed.

[0131] The method for extracting RNA from Klebsiella pneumoniae includes:

[0132] Use the bacterial RNA rapid extraction kit of Adelaide Biotechnology. The specific steps are as follows: collect 1 mL of bacterial solution (10 8 -10 9 Cells) into a 1.5mL centrifuge tube, remove as much supernatant as possible, and make sure that the amount of residual supernatant does not exceed 20μL / 100μLTE. Resuspend the cells fully in 100μL (5×10 8cells) or 200 μL (5 × 10 8 -7.5×10 8 Cells were resuspended in TE (10mM Tris-HCl, 1mM EDTA) with 1mg / mL lysozyme or lysostaphin added to TE, or lysozyme was added after resuspension. Incubate at room temperature (15-25°C) for 5min / lysozyme, or at 37°C for 15min / lysostaphin to lyse the cell wall. Vortex for 10s every 2min to help break the cell wall. Add 350μL (if 100μLTE / enzyme) or 700μL (if 200μLTE / enzyme) of Lysis Buffer RLT, pipette to mix, and then manually shake vigorously for 20s to fully lyse. Next, add 250μL 96-100% ethanol (if 100μLTE / 350μL RLT) or 500μL 96-100% ethanol (if 200μLTE / 700μL RLT) and pipette to mix immediately. Then, transfer the mixture (no more than 700 μL each time, can be added twice) to the adsorption column RA, centrifuge at 13,000 rpm for 60 seconds, and discard the waste liquid. Next, add 700 μL of deproteinization solution RW1, let it stand at room temperature for 30 seconds, then centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid. Add 500 μL of rinse solution RW, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid. Repeat the step of adding 500 μL of rinse solution RW, put the adsorption column RA back into the empty collection tube, and centrifuge at 13,000 rpm for 2 minutes to remove as much ethanol as possible from the rinse solution. Finally, put the adsorption column RA into an RNase-free centrifuge tube, add 30-50 μL of RNase-free water to the middle part of the adsorption membrane according to the expected RNA yield, let it stand at room temperature for 1 minute, and then centrifuge at 12,000 rpm for 1 minute.

[0133] The steps for cDNA synthesis are as follows: add the following components: RNA / mRNA 50-500 ng, Oligo (dT) 1 μL, 5×TRUE Reaction Mix 4 μL, gDNA Remover 1 μL, RNase free H2O to 20 μL. After gentle mixing, incubate at 42°C for 30 min and heat at 80°C for 5 min.

[0134] PCR reaction: qPCR reaction solution (2×TaqPCRMix (KT201) purchased from Tiangen Biochemical Technology Co., Ltd.) was prepared according to the reagent instructions, and PCR amplification reaction was performed using cDNA as a template and the primer set described in Table 1. During the PCR process, fluorescent dye was added to monitor the increase of PCR products in real time, thereby quantitatively analyzing the expression of the target gene and determining the type of expressed gene.

[0135] The 13 specific genes include: ACO54002.1 expression gene, AOP87411.1 expression gene, AVX34350.1 expression gene, AWF76568.1 expression gene, CTQ24123.1 expression gene, EPA91234.1 expression gene, WP_000872613.1 expression gene, WP_000888080.1 expression gene, WP_008502228.1 expression gene, WP_008502230.1 expression gene, WP_009310051.1 expression gene, WP_020324597.1 expression gene, and WP_001091224.1 expression gene. If any one or more of the following are detected, the Klebsiella pneumoniae is determined to be a highly virulent type of Klebsiella pneumoniae.

[0136] As shown in Table 8, there are significant differences in the distribution of different genes in the two groups of patients. The detection rate of most genes in the rapid clinical progression group was significantly higher than that in the slow clinical progression group. For example, the detection rates of kp.q.WP_008502230.1 in the slow clinical progression group and the rapid clinical progression group were 19.4% and 51.4%, respectively, with a P value of <0.001. Similarly, the detection rates of kp.q.EPA91234.1 in the two groups were 32% and 57.1%, respectively, with a P value of =0.008. These results indicate that these 13 specific genes may play an important role in the rapid progression of the disease, providing important clues for further research.

[0137] Table 8 Correlation between the 13 candidate genes and clinical progression

[0138]

[0139]

[0140] In summary, the genome sequencing of high-virulence and low-virulence Klebsiella pneumoniae was performed, and it was found that there were no significant differences between these strains in terms of plasmids, genome size, number of genes, ratio of repeat sequences, number of non-coding RNAs, and CRISPR sequences. In addition, there were no significant differences in gene islands and gene clusters (including metabolic functions and antibiotic resistance, etc.) between high-virulence and low-virulence strains.

[0141] Plasmid number is not the only factor determining bacterial virulence. On the contrary, some strains with low virulence may contain more plasmids, indicating that other factors may play a role in the expression of virulence.

[0142] Thirteen genes specifically expressed in highly virulent strains, including genes related to the regulation of hemolysin expression, mobile genetic elements, antioxidant defense, and transcriptional regulation. These genes may enhance bacterial virulence through multiple mechanisms, such as disrupting host cells, resisting oxidative stress, and regulating the expression of other virulence genes. Thirteen genes specifically expressed in highly virulent Klebsiella pneumoniae were discovered through transcriptome sequencing. These genes were also present in low-virulent strains but were not transcriptionally expressed. Through further expanding the sample size and validating 138 clinical strains and statistically analyzing the results, it was found that among 18 highly virulent strains and 20 low-virulent strains, the detection rates of these 13 candidate genes were relatively high in highly virulent strains. The detection rates of five genes, WP_008502230.1, EPA91234.1, WP_009310051.1, CTQ24123.1, and AWF76568.1, in highly virulent strains were 100%. The detection rate of the gene ACO54002.1, which had the lowest detection rate, was also as high as 72.2% in highly virulent strains. These 13 genes were not detected in low-virulent strains, and the detection rate was 0%. The statistical differences of all 13 indicators were very significant, P < 0.001. Through the sensitivity and specificity analysis of these 13 indicators, it was found that the specificity was as high as 100%. Except for the sensitivity of four indicators, ACO54002.1 (72.22%), AVX34350.1 (77.78%), WP_020324597.1 (88.89%), and WP_001091224.1 (88.89%), which were lower than 90%, the rest were higher than 94%, and the sensitivity of five indicators was as high as 100%. At the same time, the spearman correlation analysis was used to statistically analyze the survival rate of Galleria mellonella at different times and the detection ratios of 13 candidate genes. The correlation coefficients at 12h, 24h, 48h, and 72h were -0.77, -0.77, -0.74, and -0.75, respectively. The absolute values of the correlation coefficients were between 0.7< / r / <0.9, all of which were strongly correlated. This indicates that the more of these 13 newly discovered indicators are detected in clinical strains, the lower the survival rate of the strains infecting Galleria mellonella and the higher the virulence of the strains.

[0143] The clinical information of 138 strain infections was collected and summarized, including 103 patients with slow clinical progression and 35 patients with rapid clinical progression. The detection rates of the transcription levels of 13 new indicators were analyzed, showing that the distribution of different genes in the two groups of patients also had significant differences. The detection rate of most genes in the rapid clinical progression group was significantly higher than that in the slow clinical progression group. Except for AWF76568.1, which had no statistically significant difference between the two cohorts, the detection rates of the remaining 12 indicators in the rapid clinical progression cohort were significantly higher than those in the slow clinical progression cohort, and there were statistical differences. Among them, the differences in the six genes WP_008502230.1, WP_008502228.1, WP_000888080.1, WP_000872613.1, WP_001091224.1, and WP_020324597.1 were P<0.001. For example, the detection rates of WP_008502230.1 in the slow and rapid clinical progression groups were 19.4% and 51.4%, respectively, with a P value of <0.001. Similarly, the detection rates of EPA91234.1 in the two groups were 32% and 57.1%, respectively, with a P value of =0.008. Based on these data, it is shown that the expression of the transcription level of the 13 candidate genes can be used as a new indicator to distinguish clinically highly virulent and low-virulent Klebsiella pneumoniae. Compared with the previous method of evaluating bacterial virulence only by the presence or absence of virulence genes, this finding emphasizes the importance of detecting gene expression levels and regulatory mechanisms in virulence evaluation, breaking the traditional detection thinking and barriers.

[0144] The 13 candidate genes specifically expressed included genes related to hemolysin expression regulation, mobile genetic elements, antioxidant defense, and transcriptional regulation. Hemolysin expression regulatory proteins significantly increase the pathogenicity of bacteria by destroying the host cell membrane; mobile genetic element proteins promote the horizontal transfer of genes, increasing the diversity and adaptability of bacterial genomes; antioxidant defense-related proteins enhance the ability of bacteria to resist host immune responses; and transcriptional regulators regulate the expression of multiple virulence genes, allowing bacteria to survive under antibiotic pressure. The functions of these genes indicate that highly virulent strains not only rely on virulence genes on plasmids, but also integrate multiple metabolic pathways and defense mechanisms through complex gene regulatory networks to enhance their adaptability and pathogenicity. For example, DsbA family oxidoreductases and SDR family oxidoreductases are involved in protein folding and multiple metabolic pathways. The high expression of these genes may improve the survival of bacteria in the host. In addition, the active transposase indicates that genome recombination and evolution play an important role in highly virulent strains, which may help bacteria quickly adapt to the host environment and immune pressure.

[0145] The 13 specific genes found in this application are not very related to the virulence genes of Klebsiella pneumoniae reported previously. The functions of the proteins encoded by these genes mainly involve processes such as regulation and resistance, mobile transposition, anti-oxidation, metabolism-related, and protein folding and modification. In addition, the functions of the proteins encoded by two genes are still unclear. This shows that the virulence mechanism of Klebsiella pneumoniae may be more complex and diverse than previously understood. First of all, the role of mobile transposition and resistance-related genes in bacterial virulence cannot be ignored. Transposases are a class of enzymes that can promote the movement of DNA fragments in the genome. This mobility can lead to genome recombination and increase gene diversity and adaptability. Active transposases in highly toxic strains may help these bacteria adapt to environmental changes more quickly, especially to survive under antibiotic pressure. In addition, transposons may carry virulence genes or antibiotic resistance genes and spread to other strains through horizontal gene transfer, further enhancing the pathogenicity of bacteria.

[0146] In addition, although exemplary embodiments have been described herein, the scope includes any and all embodiments based on the present application with equivalent elements, modifications, omissions, combinations (e.g., various embodiments intersecting schemes), adaptations or changes. The elements in the claims will be interpreted broadly based on the language adopted in the claims, and are not limited to the examples described in this specification or during the implementation of the application, and the examples will be interpreted as non-exclusive. Therefore, this specification and examples are intended to be considered as examples only, and the true scope and spirit are indicated by the following claims and the full scope of their equivalents.

[0147] The above description is intended to be illustrative rather than restrictive. For example, the above examples (or one or more of them) can be used in combination with each other. For example, those of ordinary skill in the art can use other embodiments when reading the above description. In addition, in the above-mentioned specific embodiments, various features can be grouped together to simplify the application. This should not be interpreted as a feature of an application that does not require protection being necessary for any claim. On the contrary, the subject matter of the present application may be less than all the features of the embodiments of a specific application. Thus, the following claims are incorporated into the specific embodiments as examples or embodiments, wherein each claim is independently used as a separate embodiment, and it is considered that these embodiments can be combined with each other in various combinations or arrangements. The scope of the present invention should be determined with reference to the full scope of the equivalent forms of the attached claims and these claims.

[0148] The above embodiments are only exemplary embodiments of the present application and are not intended to limit the present invention. The protection scope of the present invention is defined by the claims. Those skilled in the art may make various modifications or equivalent substitutions to the present invention within the essence and protection scope of the present application, and such modifications or equivalent substitutions shall also be deemed to fall within the protection scope of the present invention.

Claims

1. A primer set for fluorescent quantitative PCR for identifying Klebsiella pneumoniae, characterized in that: The primer set includes any one or more of the following primer pairs: The primer pair shown in SEQ ID NO: 1 and SEQ ID NO: 2 for the gene expressed by ACO54002.1; The primer pair shown in SEQ ID NO:3 and SEQ ID NO:4 for the AOP87411.1 expressed gene; The primer pair shown in SEQ ID NO:5 and SEQ ID NO:6 for the AVX34350.1 expressed gene; The primer pair shown in SEQ ID NO:7 and SEQ ID NO:8 for the gene expressed by AWF76568.1; The primer pair shown in SEQ ID NO:9 and SEQ ID NO:10 for the CTQ24123.1 expressed gene; The primer pair shown in SEQ ID NO:11 and SEQ ID NO:12 for the EPA91234.1 expressed gene; The primer pair shown in SEQ ID NO: 13 and SEQ ID NO: 14 for the gene expressed by WP_000872613.1; The primer pair shown in SEQ ID NO: 15 and SEQ ID NO: 16 for the gene expressed by WP_000888080.1; The primer pair shown in SEQ ID NO: 17 and SEQ ID NO: 18 for the gene expressed by WP_008502228.1; The primer pair shown in SEQ ID NO: 19 and SEQ ID NO: 20 for the gene expressed by WP_008502230.1; The primer pair shown in SEQ ID NO:21 and SEQ ID NO:22 for the gene expressed by WP_009310051.1; The primer pair shown in SEQ ID NO:23 and SEQ ID NO:24 for the gene expressed by WP_020324597.1; The primer pair shown in SEQ ID NO: 25 and SEQ ID NO: 26 for the gene expressed by WP_001091224.

1.

2. The primer set according to claim 1, characterized in that The primer set includes any one or more of the following primer pairs: The primer pair shown in SEQ ID NO:7 and SEQ ID NO:8 for the gene expressed by AWF76568.1; The primer pair shown in SEQ ID NO:9 and SEQ ID NO:10 for the CTQ24123.1 expressed gene; The primer pair shown in SEQ ID NO:11 and SEQ ID NO:12 for the EPA91234.1 expressed gene; The primer pair shown in SEQ ID NO: 19 and SEQ ID NO: 20 for the gene expressed by WP_008502230.1; The primer pair shown in SEQ ID NO: 21 and SEQ ID NO: 22 for the gene expressed by WP_009310051.

1.

3. A kit for fluorescent quantitative PCR for identifying highly virulent Klebsiella pneumoniae, characterized in that: The kit comprises the primer set according to claim 1 or 2.

4. Use of the primer set according to any one of claims 1 to 2 or the kit according to claim 3 in identifying highly virulent Klebsiella pneumoniae.

5. Use of the primer set according to any one of claims 1 to 2 or the kit according to claim 3 in preparing a product for identifying highly virulent Klebsiella pneumoniae.

6. A method for identifying highly virulent Klebsiella pneumoniae, characterized in that: The identification method comprises the following steps: 1) Extracting RNA from the bacteria to be tested; 2) Reverse transcription of the extracted RNA; 3) using the cDNA as a template and the primer set of claim 1 to perform PCR amplification to obtain a PCR product; The type of bacteria to be tested is determined based on the sequence of the PCR product.

7. The identification method according to claim 6, characterized in that: Determining the type of the bacteria to be tested according to the sequence of the PCR product includes: when the PCR product contains one or more of ACO54002.1 expression gene, AOP87411.1 expression gene, AVX34350.1 expression gene, AWF76568.1 expression gene, CTQ24123.1 expression gene, EPA91234.1 expression gene, WP_000872613.1 expression gene, WP_000888080.1 expression gene, WP_008502228.1 expression gene, WP_008502230.1 expression gene, WP_009310051.1 expression gene, WP_020324597.1, and WP_001091224.1, determining that the bacteria to be tested is a highly virulent Klebsiella pneumoniae.

8. The identification method according to claim 6, characterized in that: Determining the type of the bacteria to be tested according to the sequence of the PCR product includes: when the PCR product contains any one or more of the WP_008502230.1 expression gene, the EPA91234.1 expression gene, the WP_009310051.1 expression gene, the CTQ24123.1 expression gene and the AWF76568.1 expression gene, determining that the bacteria to be tested is a highly virulent Klebsiella pneumoniae.

9. The identification method according to claim 6, characterized in that: The method for extracting RNA of the bacteria to be tested comprises: centrifuging the bacterial solution to remove the supernatant to obtain cell solids; mixing the cell solids with a buffer solution, lysing the mixture, adsorbing the mixture on an adsorption column, deproteinizing the adsorption column, rinsing the adsorbed mixture, and centrifuging the mixture to obtain RNA of the bacteria to be tested.

10. The identification method according to claim 6, characterized in that: The synthesis method of cDNA includes: RNA or mRNA is mixed with oligonucleotide, reverse transcription kit, reverse transcription kit and water to obtain cDNA.

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