Detection and application of high-toxicity klebsiella pneumoniae

Abstract

The invention relates to the technical field of gene detection, in particular to a rapid detection product and method for high-toxicity klebsiella pneumoniae hvKp and application. The kit has good specificity for virulence genes (iucA, rmpA, rmpA2, iroB and peg344) of high-virulence klebsiella pneumoniae, can be stably detected within the lowest detection limit of 0.01 copies / ml, can effectively distinguish high-virulence types and low-virulence types, and is suitable for clinical use.

Description

Technical Field

[0001] The present application relates to the field of gene detection technology, and in particular to a detection product, method and application for highly virulent Klebsiella pneumoniae. Background Art

[0002] Klebsiella pneumoniae (Kp) is a clinically important Gram-negative bacterium that has evolved into two different pathological types: hypervirulent Klebsiella pneumoniae (hvKP) and classical Klebsiella pneumoniae (cKP). Hypervirulent Klebsiella pneumoniae is a highly virulent Gram-negative bacillus. Compared with classical Klebsiella pneumoniae (cKP), hypervirulent Klebsiella pneumoniae carries specific virulence genes and can cause higher mortality in animal models even at lower doses, showing stronger pathogenicity.

[0003] At present, the detection of highly virulent Klebsiella pneumoniae in clinical practice mainly includes etiological examination, imaging examination and molecular biological detection. Pathological examination includes direct smear and culture. Direct smear can observe short and thick Gram-negative rods through Gram staining. These bacteria are usually surrounded by capsules and appear as translucent areas. Since highly virulent Klebsiella pneumoniae has a large polysaccharide capsule, its colonies on the culture medium are usually highly viscous. Culture is to isolate pathogens from clinical samples (such as sputum, blood, etc.) to further confirm whether it is highly virulent Klebsiella pneumoniae. Imaging examination uses chest X-ray or CT scan to show characteristic changes of lung infection, such as lobar consolidation, cavity formation, pleural effusion, etc.; the imaging manifestations of highly virulent Klebsiella pneumoniae infection may be similar to other bacterial infections, but it also has its specificity, such as the phenomenon of interlobar fissure sagging caused by gel-like sputum. Molecular biological testing is a popular and relatively convenient method in recent years. Molecular biological methods such as PCR technology are used to detect specific gene sequences, such as rmpA, magA, etc. These genes are associated with the virulence factors of highly virulent Klebsiella pneumoniae and can serve as an important basis for identifying highly virulent Klebsiella pneumoniae.

[0004] In terms of molecular biological detection, reliable hvKp molecular markers are needed. Currently, markers including iucA, iroB, peg344, rmpA and rmpA2 have been proven to have high diagnostic accuracy for identifying hvKp. However, current detection methods are mostly for individual markers, and the accuracy needs to be improved. In addition, methods for simultaneously detecting these five highly toxic genes are rare. The main reason is that when the above five genes are detected at the same time, the sensitivity and specificity of the product are difficult to guarantee. In view of this, this application is proposed. Summary of the invention

[0005] In order to solve the above technical problems, the present application has developed a system, method and application for distinguishing highly virulent Klebsiella pneumoniae with high sensitivity and specificity based on the highly virulent genes iucA, iroB, peg344, rmpA and rmpA2.

[0006] Therefore, the present application includes at least the following objectives: to seek a method and product for distinguishing or detecting highly virulent Klebsiella pneumoniae with high sensitivity and specificity

[0007] To achieve the above purpose, this application specifically adopts the following technical solutions:

[0008] The present application first provides a primer-probe composition for highly virulent Klebsiella pneumoniae, wherein the primer-probe composition targets highly virulent Klebsiella pneumoniae;

[0009] Furthermore, the primer-probe combination further includes genes iucA, iroB, peg344, rmpA and rmpA2 targeting highly virulent Klebsiella pneumoniae.

[0010] Furthermore, the primer sequences are shown as SEQ ID NO.1-10, or have at least 90% homology with SEQ ID NO.1-10; the probe sequences are shown as SEQ ID NO.15-19, or have at least 90% homology with SEQ ID NO.15-19.

[0011] Furthermore, the primer composition further includes primers and probe sequences for genes Khe and GAPDH, the primer sequences are respectively as shown in SEQ ID NO.11-14, or have at least 90% homology with SEQ ID NO.11-14; the probe sequences are respectively as shown in SEQ ID NO.20-21, or have at least 90% homology with SEQ ID NO.20-21.

[0012] Furthermore, the above-mentioned primers are specifically designed by the following method:

[0013] 1) Targeted region design:

[0014] Download the whole genome sequence and internal reference gene GAPDH sequence of Klebsiella strains from NCBI. The number of strains downloaded should not be less than 1,000, including strains existing in China.

[0015] The whole genome sequence of the screened Klebsiella strains was compared to screen out the conserved genes and their sequence regions for the identification of highly pathogenic Klebsiella pneumoniae species;

[0016] Each strain was compared and screened according to the five highly toxic genes iucA, iroB, peg344, rmpA and rmpA2, and the conserved specific sequence regions of the toxicity genes were selected;

[0017] 2) Primer probe design:

[0018] The primer length is 18-24bp, the primer GC content is between 40%-65%, the Tm value is close to 55-65℃, and the Tm value difference between the upstream and downstream primers does not exceed 2℃; the primer itself or between primers forms 4 or more consecutive pairs to avoid the primer itself forming a circular hairpin structure; avoid using A at the 3' end of the primer and avoid 3 or more identical bases; the product length is best between 100-200bp; the primer end (the last 5 nucleotides) cannot have more than 2 G / C; the probe length should be 15bp-30bp to avoid secondary structure; the probe Tm value is 65℃-70℃, usually 5℃-10℃ higher than the primer Tm value, and the GC% content is 40%-70%; avoid using G at the 5' end of the probe, and the C content in the entire probe should be significantly higher than G.

[0019] Based on the above steps, a targeted primer system for Klebsiella pneumoniae and its five highly virulence genes was preliminarily designed, followed by screening and evaluation to select the primer-probe combination with the best multiplex amplification effect.

[0020] In some aspects, the 5' ends of the probes are labeled with a fluorescent reporter group, and the 3' ends of the probes are labeled with a quencher group;

[0021] Preferably, the fluorescence and quenching labels are as follows: the fluorescence reporter group labeled with FAM at the 5' end of the Khe primer, and the quenching group labeled with BHQ1 at the 3' end; the fluorescence reporter group labeled with ROX at the 5' end of the iroB primer, and the quenching group labeled with BHQ2 at the 3' end; the fluorescence reporter group labeled with VIC at the 5' end of the iucA primer, and the quenching group labeled with BHQ1 at the 3' end; the fluorescence reporter group labeled with FAM at the 5' end of the peg344 primer, and the quenching group labeled with BHQ1 at the 3' end; the fluorescence reporter group labeled with VIC at the 5' end of the rmpA primer, and the quenching group labeled with BHQ1 at the 3' end; the fluorescence reporter group labeled with ROX at the 5' end of the rmpA2 primer, and the quenching group labeled with BHQ2 at the 3' end; the fluorescence reporter group labeled with CY5 at the 5' end of the GAPDH primer, and the quenching group labeled with BHQ2 at the 3' end.

[0022] In other aspects, the primer probe composition can be divided into two groups according to the characteristics of the gene sequence: Group 1: primer probes targeting iucA, iroB, Khe and GAPDH; Group 2: primer probes targeting peg344, rmpA, rmpA2 and GAPDH.

[0023] In other aspects, the concentration ratio of the above primers to probes is 2:1.

[0024] The present application also provides a product for detecting highly virulent Klebsiella pneumoniae, wherein the product comprises any of the primer combinations described above.

[0025] Furthermore, the product is in the form of a kit.

[0026] Furthermore, the collection also includes common components such as enzymes, buffers and quality control products for amplification.

[0027] The present application also provides the use of any of the above-mentioned primer combinations in the preparation of a highly virulent Klebsiella pneumoniae detection product.

[0028] The present application also provides the use of any of the above primer combinations in the detection of highly virulent Klebsiella pneumoniae.

[0029] The present application also provides a method for detecting highly virulent Klebsiella pneumoniae, which comprises the step of amplifying a sample to be tested using any of the above-mentioned primer combinations; or the step of amplifying a sample to be tested using any of the above-mentioned products or kits.

[0030] Compared with the prior art, this application has at least the following advantages:

[0031] Currently, detection of highly virulent Klebsiella pneumoniae rarely covers all five highly virulent genes. This application achieves comprehensive detection by analyzing, designing and optimizing the detection system for five highly virulent genes (iucA, iroB, peg344, rmpA and rmpA2). This method not only improves the reliability of clinical detection, but also ensures very high sensitivity and specificity. Compared with existing methods, it has higher clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1 , target sequence selection and primer probe design;

[0034] Figure 2 , primer and probe structure analysis and screening;

[0035] Figure 3 , Melting curves of primers targeting highly virulent Pneumococcal strain Y8;

[0036] Figure 4 , GAPDH primer melting curve for sample NA12878;

[0037] Figure 5 , the individual test results of each primer probe targeting the Y8 core of the highly virulent Pneumococcal strain;

[0038] Figure 6 , GAPDH primer probe test results for sample NA12878;

[0039] Figure 7 , the detection limit results of Khe, iroB, iucA and GAPDH at different dilution gradients for the highly toxic sample Y8;

[0040] Figure 8 , the detection limit results of peg344, rmpA, rmpA2 and GAPDH at different dilution gradients for the highly toxic sample Y8;

[0041] Fig. 9 , the detection limit results of Khe, iroB and iucA at different dilution concentrations for non-high virulence sample 329;

[0042] Fig.10, the detection limit results of peg344, rmpA and rmpA2 at different dilution concentrations for non-high-virulence sample 329;

[0043] Fig.11 , Khe, iroB and iucA detection results for 8 clinical samples;

[0044] Fig.12 , detection results of peg344, rmpA and rmpA2 for 8 clinical samples. DETAILED DESCRIPTION

[0045] The technical solution of the present application will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present application.

[0046] Experimental Example 1: Design, screening and feasibility evaluation of primer probes

[0047] This application designs primers based on the highly toxic genes iucA, iroB, peg344, rmpA and rmpA2 of highly toxic Klebsiella pneumoniae, specifically:

[0048] 1) Selection and design of targeted areas:

[0049] Download the whole genome sequence and internal reference gene GAPDH sequence of Klebsiella strains from NCBI. The number of strains downloaded should not be less than 1,000, mainly covering strains existing in China.

[0050] The whole genome sequence of the screened Klebsiella strains was compared to screen out the conserved genes and their sequence regions for the identification of highly pathogenic Klebsiella pneumoniae species;

[0051] Then, the strains were compared and screened according to the five highly virulence genes (iucA, iroB, peg344, rmpA and rmpA2), and the conserved specific sequence regions of the virulence genes were selected;

[0052] 2) Primer and probe design:

[0053] The primer length is 18-24bp, the primer GC content is between 40%-65%, the Tm value is close to 55-65℃, and the Tm value difference between the upstream and downstream primers does not exceed 2℃; the primer itself or between primers forms 4 or more consecutive pairs to avoid the primer itself forming a circular hairpin structure; avoid using A at the 3' end of the primer and avoid 3 or more identical bases; the product length is best between 100-200bp; there cannot be more than 2 G / C at the end of the primer (the last 5 nucleotides); the probe length should be 15bp-30bp to avoid secondary structure; the probe Tm value is 65℃-70℃, usually 5℃-10℃ higher than the primer Tm value, and the GC% content is 40%-70%; avoid using G at the 5' end of the probe, and the C content in the entire probe should be significantly higher than G (high G content will reduce reaction efficiency).

[0054] Based on the above steps, a targeted primer system for Klebsiella pneumoniae and its five highly virulence genes was preliminarily designed, followed by screening and evaluation to select the primer-probe combination with the best multiplex amplification effect.

[0055] 3) Tm value and combination of primer and probe targeting sequence:

[0056] The fluorescence quantitative PCR instrument has 4 effective fluorescence channels, namely FAM, VIC, ROX and Cy5. It is impossible to amplify more than 4 target sequences in one tube. It is necessary to divide the 6 targets including Klebsiella pneumoniae and its 5 high virulence factors into two tubes for detection. In the process of combination, the sequence specificity, sequence GC content and TM value are used to ensure that the primers in the combined single tube are not prone to dimers. At the same time, it is also necessary to ensure that the GC content in the single tube does not differ by more than 25% to ensure that the amplification efficiency in the same amplification tube is consistent without bias. In order to ensure the simplicity of the experiment, the PCR annealing temperature was explored to 61°C in the actual experiment. The amplification efficiency will decrease with a higher Tm value, and non-specific amplification will occur if the Tm value is too low, resulting in non-target signals. Clinical samples contain gDNA. In order to control the quality of the extraction and PCR amplification process, each tube must contain the internal reference gene GAPDH detection as the internal quality control of the product.

[0057] Through the above design logic and preliminary screening, the optimal primer and probe sequences are finally obtained as shown in Table 1, where the targeting evaluation and structure screening processes of the primers and probes are as follows: Figure 1 and Figure 2As shown. When the optimal primer and probe sequences are used, they are divided into two tubes A+B to prepare primer probe premixes. Tube A includes primer probe premixes of iucA, iroB, Khe and GAPDH (including upstream and downstream primers and probes); Tube B includes primer probe premixes of peg344, rmpA, rmpA2 and GAPDH (including upstream and downstream primers and probes) and qPCR premixes. In terms of probe labeling, the 5' end of the probes contained are labeled with a fluorescent reporter group, and the 3' end is labeled with a quencher group (see Table 2 for probe labeling groups).

[0058] Table 1 Primer and probe sequence information

[0059]

[0060] Table 2. Division and probe labeling information

[0061]

[0062]

[0063] Example 2: Further evaluation of primer and probe sequences and system adjustment

[0064] In order to further improve the performance of the primer-probe system to meet the high standards of subsequent clinical detection, this example further determines the feasibility of the primers, the ratio system and the reaction system for analysis.

[0065] Specifically, the basic plan includes the following:

[0066] 1) The specificity of primers for Klebsiella pneumoniae Khe gene and its virulence genes iucA, iroB, peg344, rmpA and rmpA2 was evaluated using nucleic acid from the highly virulent Klebsiella pneumoniae strain Y8. The results showed that the single peak shape of the melting curve indicated good primer specificity (e.g. Figure 1 shown).

[0067] 2) NA12878 was used to evaluate the specificity of GAPDH primers. The results showed that the single peak of the melting curve indicated good primer specificity (e.g. Figure 2 shown).

[0068] 3) The highly virulent Klebsiella pneumoniae strain Y8 nucleic acid was used to detect and evaluate the primer and probe monoplex system of Klebsiella pneumoniae Khe gene and its virulence genes iucA, iroB, peg344, rmpA and rmpA2. The results showed that the target genes (such as Figure 3 shown).

[0069] 4) NA12878 was used to detect and evaluate the primers and probes of the internal reference gene GAPDH. The results showed that the GAPDH gene could be correctly detected (e.g. Figure 4shown).

[0070] 5) Using highly virulent Pneumococcal strain Y8-4 (1000 copies / mL) nucleic acid diluted 10-fold and 100-fold, the primers and probes of Khe, iroB and iucA were mixed in different proportions for detection to determine the optimal ratio of primers and probes.

[0071] Experimental setup 1 is as follows:

[0072]

[0073] The results are shown below:

[0074]

[0075]

[0076] Experimental setup 2 is as follows:

[0077]

[0078] The results are shown below:

[0079]

[0080] Experimental setup 3 is as follows:

[0081]

[0082] The results are shown below:

[0083]

[0084] Experimental setup 4 is as follows

[0085]

[0086] The results are shown below:

[0087]

[0088] The experimental setup 5 is as follows:

[0089]

[0090]

[0091] The results are shown below:

[0092]

[0093] 6) Using highly virulent Pneumococcal strain Y8-4 (1000 copies / ml) nucleic acid diluted 10-fold and 100-fold, the primers and probes of peg344, rmpA and rmpA2 were mixed in different proportions for detection to determine the feasibility of the primers and probes.

[0094] Experimental setup 1 is as follows:

[0095]

[0096] The results are shown below:

[0097]

[0098] Experimental setup 2 is as follows:

[0099]

[0100] The results are shown below:

[0101]

[0102] Experimental setup 3 is as follows:

[0103]

[0104]

[0105] The results are shown below:

[0106]

[0107] Experimental setup 4 is as follows:

[0108]

[0109] The results are shown below:

[0110]

[0111] 7) Based on the determined primer-probe ratio, the feasibility of each gene primer probe and the internal reference GAPDH primer probe was determined using nucleic acid of highly virulent Pneumococcus strain diluted 10 times and 100 times + 10 ng NA12878 and Pneumococcus without the above virulence gene + 10 ng NA12878.

[0112] Experimental setup 1 is as follows:

[0113]

[0114] The results are shown below

[0115]

[0116] From the above results, we can know that in tube A, the primer mixing ratio is Khe:iroB:iucA:GAPDH=1:1:1:1, that is, all are 0.5μM, and the probe mixing ratio is Khe:iroB:iucA:GAPDH=1:1:1:1, that is, all are 0.25μM; tube B, peg344:rmpA:rmpA2:GAPDH=1:1:1:1, that is, all are 0.5μM, and the probe mixing ratio is peg344:rmpA:rmpA2:GAPDH=1:1:1:1, that is, all are 0.25μM. It can be concluded that the amplification detection effect is best when the primer and probe are 2:1 as a whole.

[0117] 8) Study on PCR amplification reaction procedure:

[0118] The experimental design is as follows:

[0119]

[0120] The results are shown below:

[0121]

[0122]

[0123]

[0124] By comparing the detection results of the above different cycle numbers (40 and 45 cycles), there is no difference in ct value, and the 40-cycle PCR program time is shorter. The optimal reaction program is determined as follows:

[0125]

[0126] Experimental Example 3 Establishment of the detection system of this application

[0127] The present invention is based on a QPCR reaction system for highly virulent Klebsiella pneumoniae, and a system and method suitable for detecting highly virulent Klebsiella pneumoniae is established based on the primer-probe system obtained above. The specific steps are as follows:

[0128] 1) Nucleic acid extraction

[0129] A commercial DNA extraction kit was used to extract nucleic acid and measure the DNA concentration. In the application examples, the experimental methods used are conventional methods unless otherwise specified, and the materials and reagents used are as follows:

[0130]

[0131]

[0132] 2) Prepare QPCR reaction system:

[0133] Using the DNA extracted in step 1) as a template, add the two tubes (A, B) of primer probe premix and QPCR premix in the above-mentioned kit to prepare the reaction system. The configuration system is as follows:

[0134]

[0135]

[0136] 3) Add sample

[0137] The sample was vortexed and mixed, then centrifuged at 3000 rpm for 30 seconds, 2 μL of nucleic acid was added, and after checking for bubbling, the sample was centrifuged and transferred to the amplification zone.

[0138] 4) QPCR reaction on the machine

[0139] Use the Hongshi SLAN-96S instrument, put the reagents into the PCR amplification instrument, set the reaction program, and start amplification. The amplification reaction program is as follows:

[0140]

[0141] 5) Data interpretation

[0142] The test results are judged by the CT value and analyzed.

[0143] Example 4: System performance verification

[0144] This example evaluates the performance of the above system in terms of detection limit, specificity and precision.

[0145] 1. Minimum detection limit test

[0146] 1. For the detection of highly virulent Pneumococcus: Use Y8 nucleic acid: gradient dilution into five gradient standards of 100 copies / ml, 10 copies / ml, 1 copy / ml, 0.1 copies / ml and 0.01 copies / ml, add 10 ng NA12878 to each, and determine the detection limit. The specific test results are shown in the following table and Figure 5 .

[0147]

[0148]

[0149] 2. For the detection of Lung infection that does not carry the above virulence genes: use 329 nucleic acid: 1000 copies / ml, 100 copies / ml, 50 copies / ml, 10 copies / ml and 0 copies / ml to determine the detection limit. The specific results are shown in the following table and Figure 6 .

[0150]

[0151]

[0152] Through the above evaluation, the present application can stably detect the five virulence genes (iucA, rmpA, rmpA2, iroB and peg344) of highly virulent Pneumococcus at a minimum of 0.01 copies / ml, which can effectively distinguish highly virulent types, with high overall sensitivity, which is significantly better than all current products.

[0153] 2. Specificity Detection

[0154] In terms of specificity, three non-pulmonary nucleic acid samples, sample 20 Klebsiella aerogenes, sample 21 Klebsiella oxytoca and sample 22 Klebsiella variicola, were used for specific cross-evaluation. The specific results are shown in the following table and attached. Figure 7 shown.

[0155]

[0156] The test results show that the system of the present application tested negative for the five virulence genes iucA, rmpA, rmpA2, iroB and peg344 as well as the Khe gene for other Klebsiella samples, and the system has good specificity and no cross-problems.

[0157] Example 5: Clinical sample testing

[0158] Clinical samples with clinical gold standards were collected through the hospital. Specifically, 5 clinically highly virulent Lung samples were collected (sample 1059, sample 1045, sample 782, sample 1090, and sample 1084), and 3 common Lung samples that did not carry virulence genes were also collected (sample 7-A, sample 8-A, and sample 14-A), as shown in the following table.

[0159]

[0160] These standard samples are used to verify the ability of this application system to distinguish actual samples (clinical requirement, detection consistency is not less than 95%). Figure 9-10As shown, it can be seen that the total compliance rate of the method of the present application for clinical sample detection is 100%, the positive compliance rate is 100%, and the negative compliance rate is also 100%, indicating its reliability in actual clinical sample detection.

[0161] The foregoing description of the specific exemplary embodiments of the present application is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present application to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present application and its practical application, so that those skilled in the art can realize and utilize the various exemplary embodiments of the present application and various selections and changes. The scope of the present application is intended to be limited by the claims and their equivalents.

Claims

1. A primer-probe combination for highly virulent Klebsiella pneumoniae, characterized in that: The primer-probe combination is directed against highly virulent Klebsiella pneumoniae; preferably, the primer-probe combination is further directed against genes iucA, iroB, peg344, rmpA and rmpA2 of highly virulent Klebsiella pneumoniae.

2. The primer-probe combination according to claim 1, characterized in that The sequences of the primers are shown in SEQ ID NO.1-10, or have at least 90% homology to SEQ ID NO.1-10; the sequences of the probes are shown in SEQ ID NO.15-19, or have at least 90% homology to SEQ ID NO.15-19.

3. The primer-probe composition according to claim 4, characterized in that: The primer composition further includes primers and probes for genes Khe and GAPDH, wherein the sequences of the primers are shown in SEQ ID NOs.11-14, or have at least 90% homology with SEQ ID NOs.11-14; the sequences of the probes are shown in SEQ ID NOs.20-21, or have at least 90% homology with SEQ ID NOs.20-21.

4. The primer-probe combination according to claim 3, characterized in that: The 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end of the probe is labeled with a quencher group; specifically: the fluorescent reporter group labeled with FAM at the 5' end of the Khe primer, and the quencher group labeled with BHQ1 at the 3' end; the fluorescent reporter group labeled with ROX at the 5' end of the iroB primer, and the quencher group labeled with BHQ2 at the 3' end; the fluorescent reporter group labeled with VIC at the 5' end of the iucA primer, and the quencher group labeled with BHQ1 at the 3' end; the fluorescent reporter group labeled with FAM at the 5' end of the peg344 primer, and the quencher group labeled with BHQ1 at the 3' end; the fluorescent reporter group labeled with VIC at the 5' end of the rmpA primer, and the quencher group labeled with BHQ1 at the 3' end; the fluorescent reporter group labeled with ROX at the 5' end of the rmpA2 primer, and the quencher group labeled with BHQ2 at the 3' end; the fluorescent reporter group labeled with CY5 at the 5' end of the GAPDH primer, and the quencher group labeled with BHQ2 at the 3' end.

5. The primer-probe combination according to claim 4, characterized in that: The primer and probe combinations were further divided into two groups: Group 1: the primer probes were directed against iucA, iroB, Khe and GAPDH; Group 2: the primer probes were directed against peg344, rmpA, rmpA2 and GAPDH.

6. A detection product for highly virulent Klebsiella pneumoniae, characterized in that: The product comprises the primer composition according to any one of claims 1 to 5.

7. The detection product according to claim 6, characterized in that: The product is a test kit.

8. The product according to claim 7, characterized in that Also included are enzymes, buffers, and controls for amplification.

9. Use of the primer combination described in any one of claims 1 to 5 in preparing a highly virulent Klebsiella pneumoniae detection product, or in detecting highly virulent Klebsiella pneumoniae.

10. A method for detecting highly virulent Klebsiella pneumoniae, characterized in that: The method comprises the step of amplifying the sample to be tested by using the primer combination described in any one of claims 1 to 5.

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