A primer set, application and detection method for identifying high virulence type klebsiella pneumoniae by fluorescent quantitative PCR

Abstract

The embodiment of the application relates to a primer group, application and detection method of a fluorescent quantitative PCR for identifying high-toxicity Klebsiella pneumoniae, wherein the primer group comprises a primer pair of one or more of SEQ ID NO: 1 to SEQ ID NO: 26, and the primer group can improve the identification accuracy of the high-toxicity Klebsiella pneumoniae.

Description

Technical Field

[0001] This application relates to the field of biodetection technology, and more specifically, to a primer set, application, and detection method for quantitative real-time PCR of highly virulent Klebsiella pneumoniae. Background Technology

[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 its ability to acquire new genes. Currently, this pathogen is mainly classified into two pathological types: classic Klebsiella pneumoniae (cKP) and hypervirulent Klebsiella pneumoniae (hvKP). Hypervirulent Klebsiella pneumoniae is highly mucoid and virulent, easily causing severe illnesses such as pneumonia, liver abscesses, and bloodstream infections, and is difficult to treat with a high mortality rate. Existing virulence detection methods are insufficient, and multiple methods are usually combined 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 are not fully understood. Although hvKp is described as a highly virulent pathogen, reports of hvKp infections in the community are increasing. Clinical epidemiological studies have shown that hvKp infection can occur at any age, and infected individuals often exhibit multiple sites of infection, followed by potential metastatic transmission, making Klebsiella pneumoniae an unusual member of the Enterobacteriaceae family. hvKp can infect almost any part of the human body. These infectious syndromes include liver abscess, pneumonia, necrotizing fasciitis, endophthalmitis, and meningitis. The high-viscosity phenotype, initially thought to be susceptible and specific to hvKp strains, has now been shown not to be the sole marker, as not all hvKp strains are high-viscosity, and some cKp strains also possess this characteristic. Therefore, using the high-viscosity phenotype alone to define hvKp strains is inaccurate. Similarly, the chromosome-encoded aerobicin (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 types cannot be used alone as a criterion for identifying hvKp. However, hvKp has acquired virulence genes on highly virulence plasmids (such as pK2044 and pLVPK) and integrated chromosomal elements (ICEs) that confer a high virulence phenotype; these elements are referred to as biomarkers on virulence plasmids.

[0004] Traditional methods for detecting Klebsiella pneumoniae virulence genes primarily rely on PCR technology to detect a series of known virulence genes, such as rmpA, rmpA2, and magA. These genes are believed to be related to bacterial virulence characteristics, and theoretically, their presence or absence can serve as an indicator of strain virulence. However, recent studies have shown that these traditional virulence gene detection methods have several limitations in practical clinical applications: the widespread presence of virulence genes, even in classic strains, frequently leads to their detection. For example, genes such as rmpA and magA can be detected in some low-virulence Klebsiella pneumoniae strains. This finding suggests that simply detecting the presence of these genes is insufficient to accurately assess the virulence level of a strain.

[0005] The virulence genes of Klebsiella pneumoniae include capsule genes, lipopolysaccharides, pili, outer membrane proteins, nitrogen utilization systems, and siderophore systems. However, experimental results show that the accuracy of identifying hvKp by detecting pili or virulence genes is not as high as the gold standard—the large wax moth model. Summary of the Invention

[0006] This application addresses the aforementioned deficiencies in the prior art. There is a need for a primer set, application, and detection method for quantitative real-time PCR to identify highly virulent Klebsiella pneumoniae, which can improve the accuracy of identifying highly virulent Klebsiella pneumoniae.

[0007] A first aspect of this application provides a primer set for quantitative real-time PCR identification of highly virulent Klebsiella pneumoniae, said primer set comprising any one or more primer pairs as follows:

[0008] Primer pairs shown in SEQ ID NO:1 and SEQ ID NO:2 targeting the ACO54002.1 gene expression gene;

[0009] Primer pairs shown in SEQ ID NO:3 and SEQ ID NO:4 targeting the AOP87411.1 expression gene;

[0010] Primer pairs shown in SEQ ID NO:5 and SEQ ID NO:6 targeting the AVX34350.1 expression gene;

[0011] Primer pairs shown in SEQ ID NO:7 and SEQ ID NO:8 targeting the AWF76568.1 expression gene;

[0012] Primer pairs shown in SEQ ID NO:9 and SEQ ID NO:10 targeting the CTQ24123.1 gene expression gene;

[0013] Primer pairs shown in SEQ ID NO:11 and SEQ ID NO:12 targeting the EPA91234.1 expression gene;

[0014] Primer pairs shown in SEQ ID NO:13 and SEQ ID NO:14 targeting the gene expressed by WP_000872613.1;

[0015] Primer pairs shown in SEQ ID NO:15 and SEQ ID NO:16 targeting the gene expressed by WP_000888080.1;

[0016] Primer pairs shown in SEQ ID NO:17 and SEQ ID NO:18 targeting the gene expressed by WP_008502228.1;

[0017] Primer pairs shown in SEQ ID NO:19 and SEQ ID NO:20 targeting the gene expressed by WP_008502230.1;

[0018] Primer pairs shown in SEQ ID NO:21 and SEQ ID NO:22 targeting the gene expressed by WP_009310051.1;

[0019] Primer pairs shown in SEQ ID NO:23 and SEQ ID NO:24 targeting the gene expressed by WP_020324597.1;

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

[0021] A second aspect of this application provides a kit for identifying highly virulent Klebsiella pneumoniae using real-time PCR, the kit comprising the primer set described in any embodiment of this application.

[0022] A third aspect of this application provides the use of the primer set or kit described in any embodiment of this application in the identification of highly virulent Klebsiella pneumoniae.

[0023] A fourth aspect of this application provides the use of the primer set or kit described in any embodiment of this application in the preparation of products for identifying highly virulent Klebsiella pneumoniae.

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

[0025] The various embodiments of this application provide primer sets, applications, and detection methods for identifying highly virulent Klebsiella pneumoniae using quantitative real-time PCR. Primer sets for quantitative real-time PCR are proposed, and experimental results show that the primer sets of this application can more accurately identify Klebsiella pneumoniae as highly virulent Klebsiella pneumoniae and more precisely distinguish it from classical Klebsiella pneumoniae. This solves the problem that traditional gene presence detection methods may not be able to accurately assess the virulence level of bacteria. Attached Figure Description

[0026] In drawings that are not necessarily drawn to scale, the same reference numerals may describe similar parts in different views. The same reference numerals with or without letter suffixes may indicate different instances of similar parts. The drawings generally illustrate various embodiments by way of example rather than limitation and are used, together with the description and claims, to illustrate the claimed embodiments. Where appropriate, the same reference numerals are used in all drawings to refer to the same or similar parts. Such embodiments are illustrative and not intended to be exhaustive or exclusive embodiments of the apparatus or method.

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

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

[0029] Figure 3 A graph showing the number of differentially expressed genes in transcriptome data based on the validation examples of this application;

[0030] Figure 4 The graph shows the GO enrichment analysis results based on the verification example of this application;

[0031] Figure 5 The results of KEGG enrichment analysis based on the verification example of this application are shown in the figure.

[0032] Figure 6 The electrophoresis diagrams of highly virulent Klebsiella pneumoniae WX090 and low-virulence strain WX077 according to the verification examples of this application are shown.

[0033] Figure 7The proportion of Klebsiella pneumoniae strains with different survival rates according to the verification examples of this application is shown.

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

[0035] Figure 9 The graph shows the relationship between the survival rate of the large wax moth and the detection rate of 13 candidate genes in the validation example of this application. Detailed Implementation

[0036] To enable those skilled in the art to better understand the technical solutions of this application, the application will be described in detail below with reference to the accompanying drawings and specific embodiments. The embodiments of this application will be further described in detail below with reference to the accompanying drawings and specific examples, but these are not intended to limit the scope of this application.

[0037] The terms “first,” “second,” and similar terms used in this application do not indicate any order, quantity, or importance, but are merely used for distinction. Terms such as “including” or “comprising” mean that the element preceding the term covers the element listed after the term, and do not exclude the possibility of covering other elements as well.

[0038] According to embodiments of this application, a primer set for real-time PCR identification of highly virulent Klebsiella pneumoniae is provided, the primer set comprising any one or more primer pairs as follows:

[0039] Primer pairs shown in SEQ ID NO:1 and SEQ ID NO:2 targeting the ACO54002.1 gene expression gene;

[0040] Primer pairs shown in SEQ ID NO:3 and SEQ ID NO:4 targeting the AOP87411.1 expression gene;

[0041] Primer pairs shown in SEQ ID NO:5 and SEQ ID NO:6 targeting the AVX34350.1 expression gene;

[0042] Primer pairs shown in SEQ ID NO:7 and SEQ ID NO:8 targeting the AWF76568.1 expression gene;

[0043] Primer pairs shown in SEQ ID NO:9 and SEQ ID NO:10 targeting the CTQ24123.1 gene expression gene;

[0044] Primer pairs shown in SEQ ID NO:11 and SEQ ID NO:12 targeting the EPA91234.1 expression gene;

[0045] Primer pairs shown in SEQ ID NO:13 and SEQ ID NO:14 targeting the gene expressed by WP_000872613.1;

[0046] Primer pairs shown in SEQ ID NO:15 and SEQ ID NO:16 targeting the gene expressed by WP_000888080.1;

[0047] Primer pairs shown in SEQ ID NO:17 and SEQ ID NO:18 targeting the gene expressed by WP_008502228.1;

[0048] Primer pairs shown in SEQ ID NO:19 and SEQ ID NO:20 targeting the gene expressed by WP_008502230.1;

[0049] Primer pairs shown in SEQ ID NO:21 and SEQ ID NO:22 targeting the gene expressed by WP_009310051.1;

[0050] Primer pairs shown in SEQ ID NO:23 and SEQ ID NO:24 targeting the gene expressed by WP_020324597.1;

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

[0052] Of the 13 primer pairs mentioned above, DMA was detected by electrophoresis of highly virulent and low-virulence strains. The results showed that all DMA pairs were present in highly virulent strains, while some were present in low-virulence strains. However, the detection rate of all 13 primer pairs was very high in highly virulent strains, while the detection rate in low-virulence strains was 0%. Therefore, these 13 primer pairs are specific for the identification of highly virulent strains and can serve as effective markers for distinguishing between highly virulent and low-virulence Klebsiella pneumoniae.

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

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

[0055]

[0056]

[0057] According to embodiments of this application, a kit for identifying highly virulent Klebsiella pneumoniae using real-time PCR is also provided, the kit comprising the primer set described in any embodiment of this application.

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

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

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

[0061] In some embodiments, determining the type of the test bacterium based on the sequence of the PCR product includes: if the PCR product contains one or more of the following genes: ACO54002.1, AOP87411.1, AVX34350.1, AWF76568.1, CTQ24123.1, EPA91234.1, WP_000872613.1, WP_000888080.1, WP_008502228.1, WP_008502230.1, WP_009310051.1, WP_020324597.1, and WP_001091224.1, then the test bacterium is determined to be a highly virulent type of Klebsiella pneumoniae.

[0062] In some embodiments, determining the type of the test bacterium based on the sequence of the PCR product includes: if the PCR product contains any one or more of the following gene expression genes: WP_008502230.1, EPA91234.1, WP_009310051.1, CTQ24123.1, and AWF76568.1, then the test bacterium is identified as a highly virulent type of Klebsiella pneumoniae.

[0063] In some embodiments, the extraction of RNA from the target bacteria includes: centrifuging the bacterial culture to remove the supernatant and obtaining cell solids; mixing the cell solids with a buffer solution and then subjecting the mixture to lysis and adsorption on an adsorption column; and then subjecting the adsorbed adsorption column to protein removal, rinsing, and centrifugation to obtain the RNA from the target bacteria.

[0064] In some embodiments, the method for synthesizing cDNA includes: incubating RNA or mRNA with oligonucleotides, a reverse transcription kit, a reverse transcription kit, and water to obtain cDNA.

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

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

[0067] The genome sequencing of Klebsiella pneumoniae was performed 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). Of these 10 strains, 5 were highly virulent and 5 were classic strains, all purchased from Thermo Fisher Scientific, Inc., USA.

[0068] 1. Sequencing principle:

[0069] PacBio sequencing technology uses SMRT chips as the sequencing vector. Within the nanopores of the SMRT chip, DNA polymerase binds to the template, and four bases (dNTPs) are labeled with four different colors of fluorescence. During the base pairing phase, the addition of different bases emits different colors of light; the type of base added can be determined based on the wavelength and peak value of the light.

[0070] 2. Experimental Procedure:

[0071] The experimental procedure was performed according to the standard protocol provided by PacBio, including sample quality testing, library construction, library quality testing, and library sequencing. Library construction included the following steps:

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

[0073] 2) Repair damage to broken DNA samples;

[0074] 3) Perform end repair on DNA;

[0075] 4) Connect the dumbbell-shaped connector;

[0076] 5) Perform exonuclease digestion;

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

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

[0079] 1) Raw data quality control, filtering out CCS reads that are too short;

[0080] 2) Genome assembly: The filtered CCS reads are assembled de novo, and the assembled genome is corrected.

[0081] 3) Genomic component analysis, mainly including: repetitive sequence, coding gene, non-coding RNA, prophage, gene island, CRISPR, etc.

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

[0083] 5) Genome mapping analysis, including: genome loop maps, genome mapping tools, and genome maps.

[0084] 4. Klebsiella pneumoniae genome sequencing results

[0085] 1) Klebsiella pneumoniae genome size and plasmids

[0086] Sequencing results showed that the genome size of all bacteria was approximately 5-6 Mb. All bacteria consisted of chromosomes and several carried plasmids. According to Table 2, all bacteria carried at least one plasmid. The genome structure of the highly virulent strain WX071 is shown below. Figure 1 As shown, the genome structure of the classic strain WX045 is as follows: Figure 2 As shown, the highly virulent strain WX071 carries 4 plasmids, while the classic 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] Based on genomic data, gene prediction was performed using Prodigal v2.6.3 software. This software uses a dynamic programming algorithm to predict genes in the newly sequenced genome, demonstrating high accuracy. Statistical information on the specific gene prediction results is shown in Table 3. The results showed that the number of genes in these 10 Klebsiella pneumoniae strains was approximately 5000. RepeatMasker v4.0.5 software was used to compare the bacterial genome with known repetitive sequence databases (such as Repbase) to search for repetitive sequences in the genome. The proportion of repetitive sequences in highly virulent strains ranged from 0.49% to 0.55%, while the proportion of repetitive sequences in low-virulence strains varied more widely, ranging from 0.32% to 0.94%. Infernal v1.1.3 software, based on a covariance model, accurately predicted the three types of rRNA in the genome. Most strains had approximately 115 non-coding RNAs, with the highly virulent strain WX117 having 240 non-coding RNAs, while the low-virulence strain WX075 had 286 non-coding RNAs. CRISPR (Clustered Regularly Interspaced Palindromic Repeats) is a continuous DNA region in the genome. CRISPR regions, along with some Cas-related proteins, constitute the immune system widely present in bacteria and archaea, which prevents external nucleic acids from entering bacterial cells. CRISPR prediction of the genome was performed using CRT v1.2 software, and the results are shown in Table 3. The number of CRISPR sequences in highly virulent strains ranged from 15 to 18, while the number of CRISPR sequences in low-virulence strains ranged from 14 to 62, with strain WX079 having as many as 62 CRISPR sequences. Genomic islands often have specific functions. IslandPath-DIMOB v0.2 software is commonly used to predict genomic islands in bacterial genomes; the number of genomic islands is shown in Table 3. Only strain WX075 contained 3 genomic islands, while the others had 4. antiSMASH v5.0 software is the most widely used tool for identifying and analyzing biosynthetic gene clusters (BGCs) in bacterial and fungal genome sequences. Gene clusters encoding secondary metabolites across a wide range of known chemical classes were accurately identified. Results showed that most strains contained five gene clusters, while strains WX17, WX075, and WX079 contained four. Promoter regions are crucial regulatory areas that enable gene transcription or repression and can be determined experimentally. The tool PromPredictv1 was used to analyze genomic DNA with varying GC contents, serving as a general 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: the number of promoters in all highly virulent strains is 141, while the number of promoters in most low-virulence strains is around 50, and only strain WX075 has as many as 187 promoters.

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

[0093]

[0094] 3) Subcellular localization of gene-encoded proteins from 10 clinically isolated Klebsiella pneumoniae strains

[0095] A signal peptide is a short peptide segment at the N-terminus of a protein initially synthesized by the ribosome. After synthesis by the ribosome, this peptide is recognized by signal peptide recognition granules and guided to the biological membrane (cell membrane or organelle membrane surface; bacteria only have a cell membrane) under the action of related proteins and signal peptide recognition granules. The signal peptide sequence is then cleaved, forming the mature protein. The software SignalPv4.0 was used to predict whether a protein contains a signal peptide and to predict the specific signal peptide cleavage site, as shown in Table 4. The results showed that the number of signal peptides ranged from 477 to 524 in all strains. There was no significant difference between highly virulent and low-virulence strains.

[0096] Transmembrane proteins are abundant on the cell membrane. These proteins have transmembrane regions containing one or more transmembrane helices, while the remaining regions are located on the cell membrane surface. Based on the characteristics of transmembrane and non-transmembrane regions, the software TMHMM v2.0, designed based on a Hidden Markov Model, can accurately predict whether a protein is a transmembrane helix and identify the specific transmembrane and non-transmembrane regions, as shown in Table 4. The results show that the number of transmembrane proteins in all strains ranges from 1221 to 1333, with no significant difference in number between high and low virulence.

[0097] Secretory proteins are proteins secreted extracellularly during the life cycle of microorganisms. Secretory proteins often enhance the adaptability of bacteria to their environment. For example, some secretory proteins are proteases or polysaccharide-degrading enzymes, degrading large molecular nutrients (proteins or polysaccharides) that cannot be directly absorbed and utilized by the bacteria into smaller, absorbable molecules. Other secretory proteins have toxic effects on other bacteria (antibiotics, etc.), inhibiting the growth of surrounding bacteria. Proteins carrying signal peptides can be guided to the cell membrane surface or extracellular space by these peptides. Proteins localized to the cell membrane generally possess transmembrane helices. By removing transmembrane helices from the predicted signal peptide-containing proteins, the remaining proteins are the secretory proteins. As shown in Table 4, the number of secretory proteins ranges from 395 to 524, with no significant difference in number between high and low toxicity levels.

[0098] Table 4. Subcellular localization of gene-encoded proteins from 10 clinical isolates of Klebsiella pneumoniae

[0099]

[0100]

[0101] Experiment 1 showed that among 10 clinically isolated Klebsiella pneumoniae strains, there were no significant differences between highly virulent and classic Klebsiella pneumoniae strains in terms of plasmid genome size, gene number, repetitive sequence ratio, non-coding RNA quantity, and CRISPR sequence. Furthermore, there were no significant differences in gene islands and gene clusters (including metabolic function and antibiotic resistance) between highly virulent and low-virulence strains. This demonstrates that relying solely on genome sequencing data to assess strain virulence has limitations. Genome sequencing can only reveal the presence of genes, not their expression under specific environmental conditions. The expression levels of virulence genes may differ significantly among different strains, but these differences cannot be directly observed through genome sequencing. For example, some virulence genes may not be expressed in classic strains due to transcriptional regulation or other mechanisms, but may be highly expressed in highly virulent strains. While genome sequencing data provides a comprehensive view of bacterial genome structure, its analysis and interpretation still have limitations.

[0102] Compared to the existing view that highly virulent Klebsiella pneumoniae often contains large plasmids carrying multiple virulence genes, significantly enhancing bacterial pathogenicity, this study, through genome sequencing, found no significant difference in plasmid numbers between highly virulent and low-virulence strains; in fact, some low-virulence strains even had more plasmids. Plasmids are small, extrachromosomal DNA molecules in bacteria that can carry antibiotic resistance genes, virulence genes, and metabolic genes, conferring various survival advantages on the host bacteria. Traditionally, it has been believed that plasmid number and size are directly related to bacterial virulence, especially in highly virulent Klebsiella pneumoniae. However, the results of genome sequencing in this application indicate that plasmid number is not the sole determinant of bacterial virulence. Conversely, some low-virulence strains may contain a greater number of plasmids, suggesting that other factors may play a role in virulence expression.

[0103] Therefore, Experiment 1 shows that there are no significant differences between highly virulent Klebsiella pneumoniae and low-virulence Klebsiella pneumoniae in terms of signal peptide number, transmembrane protein, secretory protein, plasmid number, genome size, gene number, repetition ratio, non-coding RNA number, and CRISPR sequence. Therefore, 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). Of these 10 strains, 5 were highly virulent and 5 were classic Klebsiella pneumoniae, all purchased from Thermo Fisher Scientific, Inc., USA.

[0106] Transcriptome sequencing of 10 Klebsiella pneumoniae strains was performed by Beijing Biomarker Biotechnology Co., Ltd. The transcriptome sequencing of each Klebsiella pneumoniae strain followed these steps: First, total RNA was extracted from the samples using the Trizol method, and sample quality control was performed using a Thermo NanoDropOne (ultra-micro UV-Vis spectrophotometer) and an Agilent 4200 TapeStation (UV gel imaging system). After passing the quality control, ribosomal RNA was removed using the Epicentre Ribo-Zero rRNA Removal Kit. The library was then constructed using the NEB Next Ultra II Directional RNA Library Prep Kit for Illumina. The specific steps included: 1. Fragmenting mRNA into short fragments; 2. Using mRNA as a template, synthesizing one-stranded cDNA with six-base random primers; 3. Adding buffer, dNTPs (replacing dTTPs with dUTPs in deoxyribonucleotide dNTPs), DNA Polymerase I, and RNase H to synthesize two-stranded cDNA; 4. Purifying the double-stranded cDNA using AMPure XPbeads; 5. Degrading the second strand of cDNA containing U using USER enzyme; 6. End repairing, adding A tails, and ligating sequencing adapters to the purified double-stranded cDNA; 7. Selecting fragment sizes using AMPure XPbeads; 8. Finally, performing PCR amplification and purifying the PCR product with AMPure XP beads to obtain the final transcriptome library.

[0107] The NEBNext Ultra II Directional RNA Library Prep Kit for Illumina, cDNA one-strand synthesis kit, and AMPure XPbeads were purchased from Beijing Adley Biotechnology Co., Ltd.

[0108] The constructed transcriptome libraries undergo quality control. Qualified transcriptome libraries will be sequenced using the Illumina high-throughput sequencing platform with PE150 sequencing. The raw image data files obtained from sequencing will be converted into raw sequencing reads through base calling analysis. The results are stored in FASTQ (fq) file format, which includes the sequencing reads and their corresponding sequencing quality information. After obtaining the raw sequencing reads, if species reference information is available, 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. Comparison between the two strains revealed 475 differentially expressed genes, such as... Figure 3 As shown, 287 genes were upregulated and 188 genes were downregulated.

[0110] After performing GO and KEGG enrichment analyses on differentially expressed genes, the GO enrichment analysis results are shown in Figure 1. Figure 4 As shown, many metabolic processes were significantly enriched, including nucleic acid metabolism, intracellular nitrogen metabolism, and intracellular aromatic compound metabolism. KEGG enrichment was observed in... Figure 5 As shown, the KEGG enrichment results indicate that pathways such as pyruvate metabolism, porphyrin metabolism, and ABC transport are significantly enriched.

[0111] Transcriptome data also revealed that some genes showed significant overexpression in highly virulent strains. Thirteen genes were expressed in five highly virulent strains but not at all in five low-virulence strains, as shown in Table 5. These genes are involved in the regulation of hemolysin expression, OsmC-like proteins, and transposon-related elements. Furthermore, two of these 13 genes encoding unknown proteins were highly expressed in the highly virulent strains. Based on the transcriptome data, no independently expressed genes were found in the low-virulence strains; almost all genes present in the low-virulence strains were also expressed in the highly virulent strains.

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

[0113]

[0114]

[0115] Using genomic DNA from five highly virulent and five low-virulence Klebsiella pneumoniae strains as templates, the presence of 13 specific genes was detected. The results showed that all 13 genes were present in the five highly virulent strains, while some genes were also present in the low-virulence strains. Electrophoresis images of highly virulent Klebsiella pneumoniae strain WX090 and low-virulence strain WX077 are shown below. Figure 6 As shown, the presence of 10 genes was detected in the low-virulence strain WX077. Electrophoresis data for all gene detections are shown in Table 6.

[0116] Table 6. Detection of 13 genes with high virulence.

[0117]

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

[0119] Verification of toxicity of the giant wax moth model

[0120] A total of 308 Klebsiella pneumoniae strains were collected, and their specimen sources, infection sites, patient characteristics, and clinical outcomes were analyzed. Specimen sources included sputum (245 strains, 79.5%), midstream urine (26 strains, 8.4%), bronchoalveolar lavage fluid (12 strains, 3.9%), pleural and peritoneal effusions (11 strains, 3.6%), blood drainage fluid (8 strains, 2.6%), and secretions (6 strains, 1.9%). Based on the specimen sources, pulmonary infection was the most common, followed by urinary tract infection and bloodstream infection.

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

[0122] In the clinical data analysis, 266 patients (86.4%) received antibiotics for infection control, while 42 patients (13.6%) did not receive antibiotics or received ineffective antibiotics. The median duration of antibiotic use was 12 days (range 8.0 to 19.0 days). The median length of hospital stay was 14 days (range 10.0 to 21.0 days). Regarding clinical outcomes, 35 patients (11.4%) experienced rapid disease progression and were unresponsive to treatment, while 273 patients (88.6%) showed improvement after treatment (Table 1.2).

[0123] Of the 308 clinically isolated Klebsiella pneumoniae strains, 13 strains resulted in the complete death of all large wax moths infected 12 hours after injection, and 54 strains resulted in the complete death of all large wax moths infected 120 hours after injection, representing 4.3% and 17.6% respectively. Of the 308 clinically isolated Klebsiella pneumoniae strains, 165 strains resulted in a mortality rate exceeding 50% in large wax moths infected 12 hours after injection, and 241 strains resulted in a mortality rate exceeding 50% in large wax moths infected 120 hours after injection, representing 53.7% and 78.1% respectively. Of the 308 clinically isolated Klebsiella pneumoniae strains, 218 strains resulted in a mortality rate exceeding 20% ​​in large wax moths infected 12 hours after injection, and 289 strains resulted in a mortality rate exceeding 20% ​​in large wax moths infected 120 hours after injection, representing 53.7% and 78.1% respectively (see [link to article]). Figure 7To further analyze the correlation between virulence and clinical virulence genes and other indicators, this study defined clinically isolated Klebsiella pneumoniae strains with a mortality rate exceeding 90% within 12 hours after injection as highly virulent strains. Using this criterion, 42 highly virulent strains were screened. Strains with a mortality rate of less than 10% against the wax moth within 120 hours were defined as low-virulence strains. Using this criterion, 29 low-virulence strains were screened.

[0124] Of the 308 clinically isolated Klebsiella pneumoniae strains, 138 were selected. Total RNA was extracted from these 138 strains and reverse transcribed. Using cDNA as a template, 13 candidate genes were identified. The results showed the proportions of different strains in the *Klebsiella pneumoniae* strains, for example... Figure 8 As shown, the detection rate of virulence genes decreases significantly with decreasing virulence. Figure 8 As shown, Figure 8 The horizontal axis represents the strain number of the 308 Klebsiella pneumoniae strains. These data suggest that virulence genes may exist in the genome, and some virulence genes may not be transcribed. To further analyze the relationship between these 13 genes and the virulence of Klebsiella pneumoniae, Spearman correlation analysis was performed using the detection data of the 13 candidate genes and data from the large wax moth model. The results showed a significant correlation between the gene detection data at 12, 24, 48, and 72 hours and the virulence detected by the large wax moth model, and a significant negative correlation between the survival rate of the large wax moth and the detection rate of the 13 candidate genes at different time points. Figure 9 As shown in the figure, these data demonstrate a significant correlation between these 13 virulence genes and the virulence of Klebsiella pneumoniae.

[0125] A comparison was made of the detection rates of 13 candidate genes in highly virulent and low-virulence Klebsiella pneumoniae strains. Of the 138 strains, 18 were highly virulent and 20 were low-virulence. The results showed that the detection rate of these 13 candidate genes was very high in highly virulent strains. Specifically, the detection rates of five genes—WP_008502230.1, EPA91234.1, WP_009310051.1, CTQ24123.1, and AWF76568.1—were 100% in highly virulent strains but not detected in low-virulence strains. Even the gene ACO54002.1, which had the lowest detection rate in highly virulent strains, still reached 72.2%, while the detection rates of these genes in low-virulence strains were all 0% (see Table 7). To further analyze the feasibility of these 13 candidate genes as biomarkers for Klebsiella pneumoniae virulence identification, 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 gene with the lowest sensitivity, ACO54002.1, reached 72.22%. In terms of specificity, all 13 genes exhibited 100% specificity. These data indicate that these 13 candidate genes can serve as effective markers for distinguishing between highly virulent 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. Following the method of the validation case, RNA was extracted, reverse transcribed, and then PCR amplified using cDNA as a template and primers shown in Table 1 to obtain PCR products; gene detection was then performed.

[0131] Methods for extracting Klebsiella pneumoniae RNA include:

[0132] Using Adley Biotechnology's rapid bacterial RNA extraction kit, the specific steps are as follows: Centrifuge to collect 1 mL of bacterial culture (10... 8 -10 9 Transfer the cells to a 1.5 mL centrifuge tube, removing as much supernatant as possible, ensuring that the residual supernatant does not exceed 20 μL / 100 μL. Resuspend the cells thoroughly in 100 μL (5 × 10⁻⁶) centrifuge tube. 8200 μL (cells) or 200 μL (5 × 10⁻⁶ cells) 8 -7.5×10 8 Cells were placed in TE (10 mM Tris-HCl, 1 mM EDTA) with 1 mg / mL lysozyme or lysostaphin added, or lysozyme was added after resuspension. The cells were incubated at room temperature (15-25°C) for 5 min with lysozyme, or at 37°C for 15 min with lysostaphin, to lyse the cell wall. The cells were vortexed for 10 s every 2 min to aid in cell lysis. 350 μL (if using 100 μL LTE / enzyme) or 700 μL (if using 200 μL LTE / enzyme) of lysis buffer RLT was added, and the mixture was pipetted and then vigorously shaken manually for 20 s to ensure complete lysis. Next, 250 μL of 96-100% ethanol (if using 100 μL LTE / 350 μL RLT) or 500 μL of 96-100% ethanol (if using 200 μL LTE / 700 μL RLT) was added, and the mixture was immediately pipetted and vortexed. Next, transfer the mixture (no more than 700 μL at a time, added in two batches) to the adsorption column RA, centrifuge at 13,000 rpm for 60 s, and discard the waste liquid. Then, add 700 μL of protein removal buffer RW1, incubate at room temperature for 30 s, centrifuge at 12,000 rpm for 30 s, and discard the waste liquid. Add 500 μL of wash buffer RW, centrifuge at 12,000 rpm for 30 s, and discard the waste liquid. Repeat the step of adding 500 μL of wash buffer RW, place the adsorption column RA back into the empty collection tube, and centrifuge at 13,000 rpm for 2 min to remove as much residual ethanol as possible from the wash buffer. Finally, place the adsorption column RA into an RNase-free centrifuge tube, add 30-50 μL of RNase-free water to the middle of the adsorption membrane according to the expected RNA yield, incubate at room temperature for 1 min, and centrifuge at 12,000 rpm for 1 min.

[0133] The steps for cDNA synthesis are as follows: Add the following components: 50-500 ng RNA / mRNA, 1 μL Oligo(dT), 4 μL 5×TRUE ReactionMix, 1 μL gDNARemover, and 20 μL RNase-free H2O. Gently mix and incubate at 42°C for 30 min, then heat at 80°C for 5 min.

[0134] PCR reaction: qPCR reaction solution (2×TaqPCRMix (KT201) purchased from Tiangen Biotech Co., Ltd.) was prepared according to the reagent instructions. Using cDNA as a template, PCR amplification was performed using 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 level of the target gene and determining the type of expressed gene.

[0135] The 13 specific genes, including ACO54002.1, AOP87411.1, AVX34350.1, AWF76568.1, CTQ24123.1, EPA91234.1, WP_000872613.1, WP_000888080.1, WP_008502228.1, WP_008502230.1, WP_009310051.1, WP_020324597.1, and WP_001091224.1, determine whether Klebsiella pneumoniae is a highly virulent strain.

[0136] As shown in Table 8, the distribution of different genes differed significantly between the two patient groups. The detection rate of most genes was significantly higher in the rapidly progressing group than in the slowly progressing group. For example, the detection rate of kp.q.WP_008502230.1 was 19.4% in the slowly progressing group and 51.4% in the rapidly progressing group (P < 0.001). Similarly, the detection rate of kp.q.EPA91234.1 was 32% in the two groups and 57.1% in the rapidly progressing group (P = 0.008). These results suggest 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 shows the correlation between the 13 candidate genes and clinical progress.

[0138]

[0139]

[0140] In summary, genome sequencing of highly virulent and low-virulence Klebsiella pneumoniae strains revealed no significant differences between them in terms of plasmids, genome size, gene number, repetitive sequence ratio, non-coding RNA quantity, and CRISPR sequences. Furthermore, there were no significant differences in gene islands and gene clusters (including metabolic function and antibiotic resistance) between the highly virulent and low-virulence strains.

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

[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 various 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 enlargement of the sample size to validate 138 clinical strains and statistical analysis of 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, with a detection rate of 0%. The statistical differences in all 13 indicators were highly significant, with P < 0.001. Through sensitivity and specificity analysis of these 13 indicators, it was found that the specificity was as high as 100%. Except for the four indicators ACO54002.1 (72.22%), AVX34350.1 (77.78%), WP_020324597.1 (88.89%), and WP_001091224.1 (88.89%) whose sensitivities were lower than 90%, the rest were higher than 94%, and the sensitivities of five indicators were as high as 100%. At the same time, using spearman correlation analysis to statistically analyze the survival rates of Galleria mellonella at different times and the detection ratios of 13 candidate genes, the correlation coefficients at 12 h, 24 h, 48 h, and 72 h 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, indicating that the more of these 13 newly discovered indicators were detected in clinical strains, the lower the survival rate of the strains infecting Galleria mellonella and the higher the virulence of the strains.

[0143] Clinical information from 138 cases of bacterial strain infection was collected and summarized, including 103 patients with slow clinical progression and 35 patients with rapid clinical progression. Analysis of the detection rate of 13 new indicators (transcriptional levels) revealed significant differences in the distribution of different genes between the two groups. The detection rate of most genes was significantly higher in the rapidly progressing group than in the slowly progressing group. Except for AWF76568.1, which showed no statistically significant difference between the two cohorts, the detection rates of the other 12 indicators were significantly higher in the rapidly progressing cohort than in the slowly progressing cohort, with statistically significant differences. Among these, the differences for 6 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-progression group and the rapid-progression group were 19.4% and 51.4%, respectively (P < 0.001). Similarly, the detection rates of EPA91234.1 in the two groups were 32% and 57.1%, respectively (P = 0.008). These data demonstrate that the transcriptional expression levels of 13 candidate genes can serve as a novel indicator to differentiate between clinically highly virulent and low-virulence Klebsiella pneumoniae. This finding, compared to previous methods that assessed bacterial virulence solely based on the presence or absence of virulence genes, emphasizes the importance of detecting gene expression levels and regulatory mechanisms in virulence evaluation, breaking down traditional detection paradigms and barriers.

[0144] The 13 candidate specifically expressed genes included those related to hemolysin expression regulation, mobile genetic elements, antioxidant defense, and transcriptional regulation. Hemolysin expression regulatory proteins significantly enhance bacterial pathogenicity by disrupting the host cell membrane; mobile genetic element proteins promote horizontal gene transfer, increasing bacterial genomic diversity and adaptability; antioxidant defense-related proteins enhance the bacteria's ability to resist the host immune response; and transcriptional regulators regulate the expression of multiple virulence genes, enabling bacteria to survive under antibiotic stress. The functions of these genes suggest that highly virulent strains not only rely on plasmid-based virulence genes but also enhance their adaptability and pathogenicity through a complex gene regulatory network that integrates multiple metabolic pathways and defense mechanisms. For example, DsbA family oxidoreductases and SDR family oxidoreductases are involved in protein folding and multiple metabolic pathways; high expression of these genes may enhance bacterial survival within the host. Furthermore, transposase activity indicates that genomic recombination and evolution play important roles in highly virulent strains, potentially helping bacteria rapidly adapt to the host environment and immune stress.

[0145] The 13 specific genes identified in this application are not closely related to previously reported Klebsiella pneumoniae virulence genes. The proteins encoded by these genes primarily involve regulatory processes related to resistance, transposition, antioxidation, metabolism, and protein folding and modification. Furthermore, the functions of proteins encoded by two genes remain unclear. This suggests that the virulence mechanisms of Klebsiella pneumoniae may be more complex and diverse than previously understood. First, the roles of transposition and resistance-related genes in bacterial virulence cannot be ignored. Transposases are enzymes that facilitate the movement of DNA fragments within the genome; this mobility can lead to genomic recombination, increasing genetic diversity and adaptability. Active transposases in highly virulent strains may help these bacteria adapt more quickly to environmental changes, particularly surviving under antibiotic pressure. Furthermore, transposons may carry virulence genes or antibiotic resistance genes, which can be disseminated to other strains through horizontal gene transfer, further enhancing bacterial pathogenicity.

[0146] Furthermore, although exemplary embodiments have been described herein, their scope includes any and all embodiments based on this application that have equivalent elements, modifications, omissions, combinations (e.g., schemes involving intersections of various embodiments), adaptations, or alterations. Elements in the claims will be interpreted broadly based on the language used in the claims and are not limited to the examples described in this specification or during the implementation of this application, which will be interpreted as non-exclusive. Therefore, this specification and examples are intended to be considered illustrative only, and the true scope and spirit are indicated by the full scope of the following claims and their equivalents.

[0147] The above description is intended to be illustrative and not restrictive. For example, the above examples (or one or more of them) can be used in combination with each other. Other embodiments may be used by those skilled in the art upon reading the above description. Furthermore, in the above detailed description, various features may be grouped together to simplify the application. This should not be construed as an intention that a feature of an unclaimed application is necessary for any claim. Rather, the subject matter of this application may be less than all the features of an embodiment of a particular application. Thus, the following claims are incorporated herein by reference as examples or embodiments, wherein each claim is independently considered as a separate embodiment, and these embodiments are contemplated as being possible in various combinations or arrangements with each other. The scope of the invention should be determined by reference to the appended claims and the full scope of their equivalents.

[0148] The above embodiments are merely exemplary embodiments of this application and are not intended to limit the present invention. The scope of protection of the present invention is defined by the claims. Those skilled in the art can make various modifications or equivalent substitutions to the present invention within the spirit and scope of this application, and such modifications or equivalent substitutions should also be considered to fall within the scope of protection of the present invention.

Claims

1. A primer set for quantitative real-time PCR identification of Klebsiella pneumoniae, characterized in that, The primer set includes any one or more primer pairs as follows: Primer pairs shown in SEQ ID NO:7 and SEQ ID NO:8 targeting the AWF76568.1 expression gene; Primer pairs shown in SEQ ID NO:9 and SEQ ID NO:10 targeting the CTQ24123.1 gene expression gene; Primer pairs shown in SEQ ID NO:11 and SEQ ID NO:12 targeting the EPA91234.1 expression gene; Primer pairs shown in SEQ ID NO:19 and SEQ ID NO:20 targeting the gene expressed by WP_008502230.1; Primer pairs shown in SEQ ID NO:21 and SEQ ID NO:22 for the gene expressed by WP_009310051.

1.

2. A kit for identifying highly virulent Klebsiella pneumoniae using real-time quantitative PCR, characterized in that, The kit contains the primer set as described in claim 1.

3. The application of the primer set of claim 1 or the kit of claim 2 in the identification of highly virulent Klebsiella pneumoniae.

4. The use of the primer set of claim 1 or the kit of claim 2 in the preparation of products for identifying highly virulent Klebsiella pneumoniae.

5. A method for identifying highly virulent Klebsiella pneumoniae, characterized in that, The identification method described is not for diagnostic purposes; The identification method includes the following steps: 1) Extract RNA from Klebsiella pneumoniae; 2) Reverse transcribe the extracted RNA; 3) Using cDNA as a template, PCR amplification is performed using the primer set described in claim 1 to obtain PCR products; the type of bacteria to be tested is determined according to the sequence of the PCR products; if the PCR products contain any one or more of the following gene expression genes: WP_008502230.1, EPA91234.1, WP_009310051.1, CTQ24123.1, and AWF76568.1, the Klebsiella pneumoniae is determined to be a highly virulent type of Klebsiella pneumoniae.

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