Kit for amplifying capsular serotype, virulence gene and drug-resistant gene of high-virulence klebsiella pneumoniae and application of kit

Abstract

The invention discloses a kit for amplifying a capsular serotype, a virulence gene and a drug-resistant gene of high-virulence klebsiella pneumoniae and application of the kit, and belongs to the technical field of microbiological detection. The primer combination comprises primers for amplifying the following target fragments: five virulence genes, two capsular serotype marker genes and seven drug-resistant genes; the five virulence genes are an iucA gene, an iroB gene, a peg344 gene, an rmpA gene and an rmpA2 gene; the two capsular serotypes are a K1 capsular serotype and a K2 capsular serotype; the seven drug-resistant genes are a CTXM gene, an SHV gene, an OXA-23 gene, an MCR gene, an NDM gene, a KPC gene and an OXA-48 gene. According to the invention, high-throughput synchronous detection of common serotypes, virulence and drug resistance genes of hvKp is realized, an advanced comprehensive diagnosis tool is provided for clinic, and the kit has remarkable innovation advantages and application value.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial detection, and particularly to a kit for amplifying capsular serotypes, virulence genes and drug resistance genes of hypervirulent Klebsiella pneumoniae and its application. Background Art

[0002] Klebsiella pneumoniae, as an important pathogenic bacterium for hospital- and community-acquired infections, has attracted much attention due to its wide range of infections and severe clinical consequences. In recent years, a variant of Klebsiella pneumoniae with high virulence, hypervirulent Klebsiella pneumoniae (hvKp), has gradually emerged as a new challenge in the clinical and public health fields. Compared with classical Klebsiella pneumoniae (cKp), hvKp has higher virulence characteristics, can infect healthy individuals of any age group, and tends to cause multi-site infections and subsequent extensive metastatic spread, leading to a more complex and severe disease course clinically. hvKp has extensive drug resistance to a variety of antibiotics and has evolved into multidrug-resistant hypervirulent Klebsiella pneumoniae (MDR-hvKp), which greatly limits the effectiveness of traditional antibiotic treatment regimens and may lead to treatment failure, thereby increasing the risk of patient death, and this phenomenon is particularly prominent in drug-resistant bacterial infections. In addition, the infection rate of hvKp has been continuously increasing globally in the past few decades, and this trend has exacerbated the severity of the problem and posed a serious threat to public health.

[0003] hvKp has a high mucoid phenotype. Traditionally, the string test is a commonly used method for the preliminary identification of hvKp. This method evaluates the high mucoid characteristics of strains by measuring the length of the viscous string formed by stretching the bacterial colonies on an agar plate. However, this method has certain limitations. Especially in the population of immunocompromised or patients with comorbidities, its identification ability is greatly limited. In addition, studies have confirmed that not all hvKp strains have high mucoid characteristics, which further reveals the limitations of traditional detection methods. With the continuous progress of molecular biology, more and more virulence genes have been identified as biological markers of hvKp, including rmpA, rmpA2, iroB, iucA, and peg344, etc. The distribution of these virulence genes varies among different capsular serotypes, and the K1 serotype has the highest positive rate. In addition, siderophores have also been confirmed to have strong predictive value for hvKp, which provides a new means for the detection of hvKp.

[0004] Based on these findings, researchers have developed various molecular biology methods, such as detection of hvKp-related virulence genes, high-throughput sequencing, and mass spectrometry identification, etc., for differentiating hvKp from cKp. However, despite certain progress made by these methods in laboratory research, they still face numerous challenges in clinical applications, such as some methods not being comprehensively validated, lacking means for comprehensive detection of virulence and drug resistance genes, etc. At the same time, the current laboratory detection methods for hvKp involve multiple steps, including isolation and culture, identification, virulence detection, and drug susceptibility testing, etc. Clinically, multiple methods often need to be used in combination, resulting in long time consumption, high cost, and difficulty in meeting the comprehensive requirements of high throughput, high accuracy, high sensitivity, and low cost. Therefore, further optimizing the detection method, improving the detection efficiency and accuracy, remains the key to meeting the urgent need for rapid clinical diagnosis. Summary of the Invention

[0005] An object of the present invention is to provide a kit for amplifying capsular serotypes, virulence genes, and drug resistance genes of hypervirulent Klebsiella pneumoniae and its application, so as to solve the problems existing in the above-mentioned prior art. The present invention realizes high-throughput synchronous detection of common serotypes, virulence, and drug resistance genes of hvKp. After sample extraction, transcription and amplification can be completed in a single tube, reducing the probability of sample contamination. It is faster, more accurate than traditional methods, and has a wider detection range, providing an advanced comprehensive diagnostic tool for clinical practice, with significant innovative advantages and application value.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a primer combination for simultaneously amplifying capsular serotypes, virulence genes, and drug resistance genes of hypervirulent 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 5 virulence genes are iucA gene, iroB gene, peg344 gene, rmpA gene, and rmpA2 gene;

[0009] The 2 capsular serotypes are K1 capsular serotype and K2 capsular serotype;

[0010] The 7 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; the primer sequences for amplifying the MCR gene are shown in SEQ ID NO.25-26; the primer sequences for amplifying the NDM gene are shown in SEQ ID NO.27-28; the primer sequences for amplifying the KPC gene are shown in SEQ ID NO.31-32; the 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 in the preparation of a kit for detecting highly virulent Klebsiella pneumoniae.

[0013] Optionally, the primer combination detects 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 primer combination.

[0015] Optionally, it further comprises amplification primers for the internal reference gene rcsA of Klebsiella pneumoniae and the internal reference gene IC for system quality control.

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

[0017] Optionally, it further comprises a multiplex PCR reaction premix.

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

[0019] Optionally, the primer combination or the kit detects the capsular serotype, virulence and drug resistance of the highly virulent Klebsiella pneumoniae.

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

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

[0022] The present invention designs 14 pairs of primer combinations covering the capsular serotypes, virulence genes and drug resistance genes of highly virulent Klebsiella pneumoniae. Through multiplex fluorescence labeling and capillary electrophoresis technology, high-throughput synchronous detection of common serotypes, virulence and drug resistance genes of hvKp is realized. After sample extraction, transcription and amplification can be completed in a single tube, reducing the probability of sample contamination. It is faster, more accurate than traditional methods, and has a wider detection range, providing an advanced comprehensive diagnostic tool for clinical use, with significant innovation advantages and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0024] Figure 1 Capillary electrophoresis diagram for identifying 16 target genes (including 14 highly virulent Klebsiella pneumoniae-related pathogenic genes and 2 internal reference genes) by the method of the present invention;

[0025] Figure 2 For Example 3, when the target DNA concentration is 1×10 6 copies / μL, the capillary electrophoresis diagrams of all 16 target genes (including 14 pathogenic genes and 2 internal reference genes) are shown, and obvious amplification peaks are presented in the figures;

[0026] Figure 3 For Example 3, when the target DNA concentration is 1×10 2 copies / μL, the capillary electrophoresis diagrams of all 16 target genes (including 14 pathogenic genes and 2 internal reference genes) are shown, and obvious amplification peaks are presented in the figures. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be construed as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention.

[0028] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0030] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0031] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0032] The present invention uses five core virulence genes, namely iucA, iroB, peg344, rmpA, and rmpA2, two important serotype markers, K1 and K2, and seven common drug-resistant genes of hypervirulent Klebsiella pneumoniae (hvKp), namely CTXM, SHV, OXA-23, MCR, NDM, KPC, and OXA-48, a total of 14 key pathogenic genes related to hvKp as detection targets and designs specific primers. Through the multiplex fluorescence labeling technology combined with capillary electrophoresis technology, the transcription and amplification of the above 14 detection targets can be completed in a single tube, significantly reducing the risk of sample contamination, and combined with a first-generation sequencer to achieve rapid, high-throughput, and high-sensitivity 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 the internal reference gene rcsA of Klebsiella pneumoniae, amplification primers for the internal reference gene IC for system quality control, and 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 Yeasen Biotechnology (Shanghai) Co., Ltd., with a specification of 50T.

[0035] Optionally, the detection targets of the kit include various clinical samples such as sputum, throat swabs, bronchoalveolar lavage fluid, pleural and peritoneal effusions, and puncture fluids.

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

[0037] After extracting the sample genome, perform multiplex fluorescence PCR amplification with the primers in Table 1, then perform capillary electrophoresis detection on the amplification products, analyze the electrophoresis results using GeneMapper software, and distinguish by comparing the fragment sizes of each gene PCR product, so as to achieve high-throughput synchronous detection.

[0038] The following will show the present invention in detail with specific embodiments.

[0039] Example 1 Primer Design and Synthesis

[0040] The present invention screened 14 genes in total, including five core virulence genes of iucA, iroB, peg344, rmpA, and rmpA2, two key capsular serotype markers of K1 and K2, and seven common drug-resistant genes of hvKp including CTXM, SHV, OXA-23, MCR, NDM, KPC, and OXA-48, as detection targets, and incorporated two internal standard genes (rcsA is an internal reference gene of Klebsiella pneumoniae, and IC is a system quality control internal reference gene). The primers were designed using Oligo7.0 software and labeled with FAM fluorescent dye at the 5′ end of the primers. After BLAST alignment, it was confirmed that the primer sequences highly matched the target genes. The specific primer sequences are shown in Table 1. All primers were synthesized by Genewiz Biotechnology Co., Ltd., Suzhou.

[0041] Table 1 Primer Sequence Information

[0042]

[0043] Example 2 Detection of hvKp

[0044] 1. Strain Screening

[0045] Klebsiella pneumoniae strains were collected from the clinical laboratory of Taian Central Hospital, including hypervirulent Klebsiella pneumoniae strains (hvKp) and classical Klebsiella strains (cKp). To ensure the accuracy and reliability of the test results, the Antu automatic microbial mass spectrometry detection system Auto ms1000 was used for preliminary identification, the string test was used to verify the biological characteristics, and sequencing technology was combined for molecular confirmation.

[0046] 2. Extract the genomic DNA of the strains

[0047] The genomic DNA of the strains was extracted. After measuring the concentration with a NanoDrop micro-spectrophotometer, it was stored at 20 °C for later use.

[0048] 3. Prepare the 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× 6.25 μL primer mix 300 nM 6 μL Template DNA / 2 μL Nuclease-free water / 10.75 μL

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

[0053] Table 3 PCR reaction program

[0054]

[0055] Using the genomic DNA of the clinical samples extracted as a template, the primers in Table 1 were used to perform multiplex fluorescence PCR reactions according to the reaction systems and reaction programs 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: Use a 3500Dx genetic analyzer for capillary electrophoresis to detect the FAM fluorescence signal.

[0059] Data analysis: Use GeneMapper software to analyze the capillary electrophoresis results and distinguish them by comparing the fragment sizes of the PCR products of each gene.

[0060] Peak map display: The peak map of capillary electrophoresis is as Figure 1 shown, where the horizontal axis represents the fragment length and the vertical axis represents the peak height.

[0061] By Figure 1It can be seen that the capillary electrophoresis peak pattern 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 in Table 1 can comprehensively identify and authenticate the target analytes.

[0062] Significance of the detection peak: Each detection peak uniquely corresponds to a pathogen, and only when the pathogen actually exists 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 experiment

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

[0066] Table 4 Strain information

[0067]

[0068] Table 5 Specificity detection results

[0069] Bacterial strain DNA Detection rate of target gene Non-specific amplification rate Hypervirulent Klebsiella pneumoniae 100％(10 / 10) 0％ Common Klebsiella pneumoniae 0％(0 / 5) 0％ Other Enterobacteriaceae 0％(0 / 5) 0％

[0070] As can be seen from the results in Table 5, the multiplex PCR detection method of the present invention can detect all highly virulent Klebsiella pneumoniae, while common Klebsiella pneumoniae and other Enterobacteriaceae bacteria 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), and each concentration was repeated 3 times. The lowest detection limit (LoD) was determined, and a detection rate of ≥95% was considered qualified. The results are shown in Table 6. The capillary electrophoresis diagrams of the amplification products at DNA concentrations of 1×10 6 copies / μL and 1×10 2 copies / μL are shown in Figure 2 and Figure 3 respectively.

[0073] Table 6 Sensitivity detection results

[0074]

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

[0076] 3. Repeatability verification

[0077] Select 2 positive samples of highly virulent Klebsiella pneumoniae and 2 other Enterobacter strains in Table 4, extract nucleic acids and perform PCR amplification according to the conditions of Example 2, and repeat the test 10 times. The results are shown in Table 7, and the test results of each sample are consistent.

[0078] Table 7 Results of repeatability verification

[0079]

[0080] 4. Clinical sample verification

[0081] Twenty clinical samples (sputum, throat swab, bronchoalveolar lavage fluid, pleural effusion, puncture fluid, etc.) containing Klebsiella pneumoniae collected from the Clinical Laboratory of Taian Central Hospital were used. Nucleic acid extraction and PCR amplification were carried out according to the conditions of Example 2. At the same time, next-generation sequencing analysis was performed on them, and the coincidence rate of this method with the "gold standard" (next-generation sequencing method) was calculated (≥90% is qualified). The results are shown in Table 8.

[0082] Table 8 Results of clinical sample verification

[0083]

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

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

[0086] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. A primer combination for simultaneously amplifying the capsular serotype, virulence genes and drug resistance genes of highly virulent Klebsiella pneumoniae, characterized in that, Primers for amplifying the following target fragments: 5 virulence genes, 2 capsular serotype marker genes, and 7 drug resistance genes; The 5 virulence genes are the iucA gene, the iroB gene, the peg344 gene, the rmpA gene, and the rmpA2 gene; The 2 capsular serotypes are the K1 capsular serotype and the K2 capsular serotype; The 7 drug resistance genes are the CTXM gene, the SHV gene, the OXA-23 gene, the MCR gene, the NDM gene, the KPC gene, and the 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; the primer sequences for amplifying the MCR gene are shown in SEQ ID NO.25-26; the primer sequences for amplifying the NDM gene are shown in SEQ ID NO.27-28; the primer sequences for amplifying the KPC gene are shown in SEQ ID NO.31-32; the primer sequences for amplifying the OXA-48 gene are shown in SEQ ID NO.29-30.

2. Use of the primer combination according to claim 1 in the preparation of a kit for detecting hypervirulent Klebsiella pneumoniae.

3. The application according to claim 2, characterized in that The primer combination detects the capsular serotype, virulence, and drug resistance of the hypervirulent Klebsiella pneumoniae.

4. A kit for detecting highly virulent Klebsiella pneumoniae, characterized in that, Comprising the primer combination according to claim 1.

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

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

7. The kit according to claim 4, wherein Also includes a multiplex PCR reaction premix.

8. Use of the primer combination according to claim 1 or the kit according to any one of claims 4-7 in the detection of hypervirulent Klebsiella pneumoniae for non-disease diagnosis purposes.

9. The application according to claim 8, wherein The primer combination or the kit detects the capsular serotype, virulence, and drug resistance of the hypervirulent Klebsiella pneumoniae.

10. The application according to claim 8, characterized in that, The detection is achieved based on multiplex fluorescence PCR amplification and capillary electrophoresis.

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