A kit for amplifying high virulence klebsiella pneumoniae capsule serotypes, virulence genes and drug resistance genes and application thereof

Abstract

The application discloses a kit for amplifying high-toxicity Klebsiella pneumoniae capsule serotypes, virulence genes and drug resistance genes and application thereof and belongs to the technical field of microorganism detection. The primer combination comprises primers for amplifying the following target fragments: 5 virulence genes, 2 capsule serotype marker genes and 7 drug resistance genes; the 5 virulence genes are iucA gene, iroB gene, peg344 gene, rmpA gene and rmpA2 gene; the 2 capsule serotypes are K1 capsule serotype and K2 capsule serotype; and the 7 drug resistance genes are CTXM gene, SHV gene, OXA-23 gene, MCR gene, NDM gene, KPC gene and OXA-48 gene. The application realizes high-throughput synchronous detection of common serotypes, virulence and drug resistance genes of hvKp, provides an advanced comprehensive diagnostic tool for the clinic and has significant innovation advantages and application value.

Description

Technical Field

[0001] This invention relates to the field of microbial detection technology, and in particular to a kit for amplifying the capsular serotype, virulence gene, and drug resistance gene of highly virulent Klebsiella pneumoniae, and its application. Background Technology

[0002] Klebsiella pneumoniae, a significant pathogen causing hospital- and community-acquired infections, has garnered considerable attention due to its widespread infection range and severe clinical consequences. In recent years, a highly virulent variant of Klebsiella pneumoniae—hypervirulent Klebsiella pneumoniae (hvKp)—has emerged, posing a new challenge in clinical and public health fields. Compared to classic Klebsiella pneumoniae (cKp), hvKp exhibits higher virulence, capable of infecting healthy individuals of any age, and tends to cause multi-site infections and subsequent widespread metastasis, leading to more complex and severe disease progression. hvKp exhibits extensive resistance to multiple antibiotics, evolving into multidrug-resistant hypervirulent Klebsiella pneumoniae (MDR-hvKp), significantly limiting the effectiveness of traditional antibiotic treatments and potentially leading to treatment failure, thereby increasing the risk of patient death—a phenomenon particularly pronounced in drug-resistant infections. Furthermore, the global infection rate of HVKp has been rising continuously over the past few decades, a trend that exacerbates the severity of the problem and poses a serious threat to public health.

[0003] hvKp exhibits a high-slim phenotype, and traditionally, the string test is a common method for preliminary identification of hvKp. This method assesses the high-slim characteristic of the strain by measuring the length of the sticky string formed when bacterial colonies are stretched on an agar plate. However, this method has certain limitations, especially in immunocompromised or comorbid patients, where its identification ability is significantly limited. Furthermore, studies have confirmed that not all hvKp strains possess high-slim characteristics, further revealing the limitations of traditional detection methods. With the continuous advancement of molecular biology, an increasing number of virulence genes have been identified as biomarkers for hvKp, including rmpA, rmpA2, iroB, iucA, and peg344. The distribution of these virulence genes varies among different capsular serotypes, with the K1 serotype showing the highest positive rate. In addition, siderophores have also been shown to have strong predictive value for hvKp, providing a new means for hvKp detection.

[0004] Based on these findings, researchers have developed various molecular biology methods, such as hvKp-related virulence gene detection, high-throughput sequencing, and mass spectrometry identification, to distinguish hvKp from cKp. However, despite these advancements in laboratory research, their clinical application still faces numerous challenges, including the lack of comprehensive validation of some methods and the absence of integrated methods for detecting virulence and resistance genes. Furthermore, current laboratory detection methods for hvKp involve multiple steps, including isolation and culture, identification, virulence detection, and drug susceptibility testing. Clinically, multiple methods often need to be used in combination, leading to long processing times, high costs, and difficulty in meeting the comprehensive requirements of high throughput, high accuracy, high sensitivity, and low cost. Therefore, further optimizing detection methods and improving detection efficiency and accuracy remain crucial to meeting the urgent clinical need for rapid diagnosis. Summary of the Invention

[0005] The purpose of this invention is to provide a kit for amplifying serotypes, virulence genes, and drug resistance genes of highly virulent Klebsiella pneumoniae capsular serotypes and their applications, thereby addressing the problems existing in the prior art. This invention achieves high-throughput simultaneous detection of common hvKp serotypes, virulence genes, and drug resistance genes. After sample extraction, transcription and amplification can be completed in a single tube, reducing the probability of sample contamination. Compared to traditional methods, it is faster, more accurate, and covers a wider detection range, providing an advanced comprehensive diagnostic tool for clinical use, demonstrating significant innovative advantages and application value.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a primer combination for simultaneously amplifying the capsular serotype, virulence gene, and drug resistance gene of highly virulent Klebsiella pneumoniae, including primers for amplifying the following target fragments: 5 virulence genes, 2 capsular serotype marker genes, and 7 drug resistance genes;

[0008] The five virulence genes are iucA, iroB, peg344, rmpA, and rmpA2.

[0009] The two capsular serotypes are K1 capsular serotype and K2 capsular serotype;

[0010] The seven drug resistance genes are CTXM gene, SHV gene, OXA-23 gene, MCR gene, NDM gene, KPC gene and OXA-48 gene;

[0011] The primer sequences for amplifying the iucA gene are shown in SEQ ID NO. 9–10; the primer sequences for amplifying the iroB gene are shown in SEQ ID NO. 5–6; the primer sequences for amplifying the peg344 gene are shown in SEQ ID NO. 7–8; the primer sequences for amplifying the rmpA gene are shown in SEQ ID NO. 11–12; the primer sequences for amplifying the rmpA2 gene are shown in SEQ ID NO. 13–14; the primer sequences for amplifying the marker gene of the K1 capsular serotype are shown in SEQ ID NO. 1–2; the primer sequences for amplifying the marker gene of the K2 capsular serotype are shown in SEQ ID NO. 3–4; the primer sequences for amplifying the CTXM gene are shown in SEQ ID NO. 19–20; the primer sequences for amplifying the SHV gene are shown in SEQ ID NO. 21–22; the primer sequences for amplifying the OXA-23 gene are shown in SEQ ID NO. 23–24; and the primer sequences for amplifying the MCR gene are shown in SEQ ID NO. 19–20. Primer sequences for amplifying the NDM gene are shown in SEQ ID NO. 25-26; primer sequences for amplifying the KPC gene are shown in SEQ ID NO. 31-32; and primer sequences for amplifying the OXA-48 gene are shown in SEQ ID NO. 29-30.

[0012] The present invention also provides the application of the primer combination described herein in the preparation of a kit for detecting highly virulent Klebsiella pneumoniae.

[0013] Optionally, the primer combination can be used to detect the capsular serotype, virulence, and drug resistance of the highly virulent Klebsiella pneumoniae.

[0014] The present invention also provides a kit for detecting highly virulent Klebsiella pneumoniae, comprising the aforementioned primer combination.

[0015] Optionally, primers for amplifying the internal reference gene rcsA of Klebsiella pneumoniae and the internal reference gene IC for system quality control are also included.

[0016] Optionally, the amplification primer sequences for the internal reference gene rcsA are shown in SEQ ID NO.15-16; and the amplification primer sequences for the internal reference gene IC are shown in SEQ ID NO.17-18.

[0017] Optionally, a multiplex PCR reaction premix may also be included.

[0018] The present invention also provides the application of the primer combination or the kit described herein in the detection of highly virulent Klebsiella pneumoniae for non-disease diagnostic purposes.

[0019] Optionally, the primer combination or the kit can be used to detect the capsular serotype, virulence, and drug resistance of the highly virulent Klebsiella pneumoniae.

[0020] Optionally, the detection is based on multiplex fluorescent PCR amplification and capillary electrophoresis.

[0021] The present invention discloses the following technical effects:

[0022] This invention designs 14 primer pairs covering serotypes, virulence genes, and drug resistance genes of highly virulent Klebsiella pneumoniae capsular serotypes. Using multiplex fluorescent labeling and capillary electrophoresis, it achieves high-throughput simultaneous detection of common hvKp serotypes, virulence genes, and drug resistance genes. After sample extraction, transcription and amplification can be completed in a single tube, reducing the probability of sample contamination. Compared to traditional methods, this is faster, more accurate, and covers a wider detection range, providing an advanced comprehensive diagnostic tool for clinical use, demonstrating significant innovative advantages and application value. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 Capillary electrophoresis images of 16 target genes (including 14 pathogenic genes related to highly virulent Klebsiella pneumoniae and 2 internal reference genes) identified using the method of this invention;

[0025] Figure 2 The target DNA concentration in Example 3 was 1×10⁻⁶. 6 At copies / μL, capillary electrophoresis images of all 16 target genes (including 14 pathogenic genes and 2 internal reference genes) showed obvious amplification peaks.

[0026] Figure 3 The target DNA concentration in Example 3 was 1×10⁻⁶. 2 At copies / μL, capillary electrophoresis images of all 16 target genes (including 14 pathogenic genes and 2 internal reference genes) showed obvious amplification peaks. Detailed Implementation

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0030] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0031] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0032] This invention targets 14 key pathogenic genes associated with hvKp, including five core virulence genes (iucA, iroB, peg344, rmpA, and rmpA2), two important serotype markers (K1 and K2), and seven common drug resistance genes in highly virulent Klebsiella pneumoniae (hvKp): CTXM, SHV, OXA-23, MCR, NDM, KPC, and OXA-48, and designs specific primers accordingly. By combining multiplex fluorescent labeling with capillary electrophoresis, transcription and amplification of these 14 targets can be completed within a single tube, significantly reducing the risk of sample contamination. Combined with a first-generation sequencer, this enables rapid, high-throughput, and highly sensitive detection.

[0033] In some specific embodiments of the present invention, the above detection is achieved by a kit containing amplification primers for the above 14 detection targets. The kit also includes amplification primers for the internal reference gene rcsA of Klebsiella pneumoniae and the internal reference gene IC for system quality control, as well as a multiplex PCR reaction premix.

[0034] In some specific embodiments of the present invention, the multiplex PCR reaction premix is: C199P1 Multiplex PCR Master Mix, purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., specification 50T.

[0035] Optionally, the test kit can detect a variety of clinical samples, including sputum, throat swabs, bronchoalveolar lavage fluid, pleural and peritoneal effusions, and puncture fluid.

[0036] Optionally, the detection includes the following steps:

[0037] After extracting the genome from the sample, multiplex fluorescent PCR amplification was performed using the primers in Table 1. The amplification products were then detected by capillary electrophoresis. The electrophoresis results were analyzed using GeneMapper software, and the PCR product fragments of each gene were distinguished by comparison, thereby achieving high-throughput simultaneous detection.

[0038] The present invention will now be described in detail with reference to specific embodiments.

[0039] Example 1 Primer Design and Synthesis

[0040] This invention screened five core virulence genes (iucA, iroB, peg344, rmpA, and rmpA2), two key capsular serotype markers (K1 and K2), and seven common hvKp resistance genes (CTXM, SHV, OXA-23, MCR, NDM, KPC, and OXA-48), totaling 14 genes as detection targets. Two internal control genes were also included (rcsA is the internal reference gene for Klebsiella pneumoniae, and IC is the systemic quality control internal reference gene). Primers were designed using Oligo 7.0 software, and the 5′ ends of the primers were labeled with FAM fluorescent dye. BLAST alignment confirmed a high degree of match between the primer sequences and the target genes; the specific primer sequences are shown in Table 1. All primers were synthesized by Suzhou Genewise Biotechnology Co., Ltd.

[0041] Table 1 Primer sequence information

[0042]

[0043] Example 2: Detection of hvKp

[0044] 1. Strains screening

[0045] Klebsiella pneumoniae strains, including highly virulent Klebsiella pneumoniae (hvKp) and classic Klebsiella pneumoniae (cKp), were collected from the Department of Laboratory Medicine of Taian Central Hospital. To ensure the accuracy and reliability of the test results, the Antu fully automated microbial mass spectrometry detection system Auto ms1000 was used for preliminary identification, the biological characteristics were verified by the filamentation experiment, and molecular-level confirmation was carried out by sequencing technology.

[0046] 2. Extract the strain's genome

[0047] Genomic DNA of the strain was extracted. After the concentration was determined by NanoDrop micro-spectrophotometer, it was stored at 20°C for later use.

[0048] 3. Preparation of multiplex PCR reaction system

[0049] The PCR reaction system is shown in Table 2.

[0050] Table 2 PCR reaction system

[0051] reaction system Final concentration volume 4×C199P1 multiplex PCR premix 1× 6.25μL primer mix 300nM 6μL Template DNA / 2μL Nuclease-free water / 10.75μL

[0052] The PCR reaction procedure is shown in Table 3.

[0053] Table 3 PCR reaction procedure

[0054]

[0055] Using extracted genomic DNA from clinical samples as templates, multiplex fluorescent PCR was performed using primers in Table 1 and following the reaction systems and procedures in Tables 2 and 3. After the reaction, the products were collected for later use.

[0056] 4. Target detection and genotype analysis

[0057] Sample preparation: Add 9 μL of highly deionized formamide (HiDi) and 1 μL of multiplex PCR product to each well of the 96-well sample plate.

[0058] Capillary electrophoresis: Capillary electrophoresis was performed using a 3500Dx gene analyzer to detect FAM fluorescence signals.

[0059] Data analysis: The capillary electrophoresis results were analyzed using GeneMapper software, and the differences were made by comparing the size of the PCR product fragments of each gene.

[0060] Peak diagram display: Peak diagram of capillary electrophoresis is as follows Figure 1 As shown, the horizontal axis represents the segment length, and the vertical axis represents the peak height.

[0061] Depend on Figure 1It can be seen that the capillary electrophoresis peak diagram contains all the characteristic amplification peaks of a total of 16 target genes, including 14 core pathogenic genes and 2 internal reference genes, proving that the primers designed using Table 1 can comprehensively identify and characterize the target analytes.

[0062] Significance of detection peaks: Each detection peak uniquely corresponds to a pathogen. Only when the pathogen is actually present in the sample will the corresponding amplification peak be generated in capillary electrophoresis.

[0063] Performance indicators of the detection method in Example 3

[0064] 1. Specificity test

[0065] The multiplex PCR detection method of this invention was used to detect 10 clinically highly virulent Klebsiella pneumoniae strains, 5 cases of common Klebsiella pneumoniae, and 5 other Enterobacteriaceae bacteria screened in Example 2. The detection results were verified using next-generation sequencing. The concordance rate between the detection results of this invention and the gold standard (next-generation sequencing) was compared. The strain information and specific detection results are shown in Tables 4 and 5, respectively.

[0066] Table 4. Strain Information

[0067]

[0068] Table 5 Specific detection results

[0069] bacterial strain DNA Target gene detection rate Nonspecific amplification rate Highly virulent Klebsiella pneumoniae 100％(10 / 10) 0％ Klebsiella pneumoniae 0％(0 / 5) 0％ Other Enterobacteriaceae 0％(0 / 5) 0％

[0070] As shown in Table 5, the multiplex PCR detection method of the present invention can detect all highly virulent Klebsiella pneumoniae, while ordinary Klebsiella pneumoniae and other Enterobacteriaceae cannot be detected.

[0071] 2. Sensitivity

[0072] The genomic DNA of the strain extracted in Example 2 was diluted (10). 6 -10 1 (Copies / μL), each concentration was repeated 3 times. The limit of detection (LoD) was determined, and a detection rate ≥95% was considered acceptable. Results are shown in Table 6. DNA concentration was 1×10⁻⁶ copies / μL. 6 copies / μL and 1×10 2 The capillary electrophoresis images of the amplification products at copies / μL are as follows: Figure 2 and Figure 3 As shown.

[0073] Table 6 Sensitivity Detection Results

[0074]

[0075] Conclusion: LoD is 1×10 2 copies / μL.

[0076] 3. Repeatability verification

[0077] Two highly virulent Klebsiella pneumoniae positive samples and two other Enterobacter strains from Table 4 were selected. Nucleic acid extraction and PCR amplification were performed according to the conditions in Example 2, and the tests were repeated 10 times. The results are shown in Table 7. The test results of each sample were consistent.

[0078] Table 7 Repeatability verification results

[0079]

[0080] 4. Clinical sample validation

[0081] Twenty clinical samples (sputum, throat swabs, bronchoalveolar lavage fluid, pleural effusion, and puncture fluid, etc.) containing Klebsiella pneumoniae, collected from the Department of Laboratory Medicine of Taian Central Hospital, were subjected to nucleic acid extraction and PCR amplification under the conditions of Example 2. Simultaneously, next-generation sequencing analysis was performed, and the concordance rate between this method and the "gold standard" (next-generation sequencing) was calculated (≥90% was considered acceptable). The results are shown in Table 8.

[0082] Table 8. Clinical Sample Validation Results

[0083]

[0084] Positive concordance rate = 11 / 14 × 100% = 91.6%; Negative concordance rate = 8 / 8 × 100% = 100%.

[0085] In summary, the high-throughput simultaneous multiplex PCR detection method of the present invention achieves high-throughput simultaneous detection of common hvKp serotypes, virulence and drug resistance genes through multiplex fluorescent labeling and capillary electrophoresis technology. It also has good specificity and high sensitivity, and is faster and more accurate than traditional methods.

[0086] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of a primer combination that simultaneously amplifies the capsular serotype, virulence gene, and drug resistance gene of highly virulent Klebsiella pneumoniae in the preparation of a kit for detecting highly virulent Klebsiella pneumoniae, characterized in that, Primers for amplifying the following target fragments include: 5 virulence genes, 2 capsular serotype marker genes, and 7 drug resistance genes; The five virulence genes are iucA, iroB, peg344, rmpA, and rmpA2. The two capsular serotypes are K1 capsular serotype and K2 capsular serotype; The seven drug resistance genes are CTXM gene, SHV gene, OXA-23 gene, MCR gene, NDM gene, KPC gene and OXA-48 gene; The primer sequences for amplifying the iucA gene are shown in SEQ ID NO. 9-10; the primer sequences for amplifying the iroB gene are shown in SEQ ID NO. 5-6; the primer sequences for amplifying the peg344 gene are shown in SEQ ID NO. 7-8; the primer sequences for amplifying the rmpA gene are shown in SEQ ID NO. 11-12; the primer sequences for amplifying the rmpA2 gene are shown in SEQ ID NO. 13-14; the primer sequences for amplifying the marker gene of the K1 capsular serotype are shown in SEQ ID NO. 1-2; the primer sequences for amplifying the marker gene of the K2 capsular serotype are shown in SEQ ID NO. 3-4; the primer sequences for amplifying the CTXM gene are shown in SEQ ID NO. 19-20; the primer sequences for amplifying the SHV gene are shown in SEQ ID NO. 21-22; the primer sequences for amplifying the OXA-23 gene are shown in SEQ ID NO. 23-24; and the primer sequences for amplifying the MCR gene are shown in SEQ ID NO. 19-20. Primer sequences for amplifying the NDM gene are shown in SEQ ID NO. 25-26; primer sequences for amplifying the KPC gene are shown in SEQ ID NO. 31-32; and primer sequences for amplifying the OXA-48 gene are shown in SEQ ID NO. 29-30.

2. The application according to claim 1, characterized in that, The primer combination was used to detect the capsular serotype, virulence, and drug resistance of the highly virulent Klebsiella pneumoniae.

3. A kit for detecting highly virulent Klebsiella pneumoniae, characterized in that, Includes the primer combination described in claim 1.

4. The reagent kit according to claim 3, characterized in that, It also includes amplification primers for the internal reference gene rcsA of Klebsiella pneumoniae and the internal reference gene IC for system quality control.

5. The reagent kit according to claim 4, characterized in that, The amplification primer sequences for the internal reference gene rcsA are shown in SEQ ID NO.15~16; the amplification primer sequences for the internal reference gene IC are shown in SEQ ID NO.17~18.

6. The reagent kit according to claim 3, characterized in that, It also includes multiplex PCR reaction premix.

7. The use of the primer combination as described in claim 1 or the kit as described in any one of claims 3-6 in the detection of highly virulent Klebsiella pneumoniae for non-disease diagnostic purposes.

8. The application according to claim 7, characterized in that, The primer combination or the kit is used to detect the capsular serotype, virulence, and drug resistance of the highly virulent Klebsiella pneumoniae.

9. The application according to claim 7, characterized in that, The detection is based on multiplex fluorescent PCR amplification and capillary electrophoresis.

Images