Kit, nucleic acid reagent, and system for detecting st11-type hv-crkp

Abstract

The present disclosure relates to the use of a reagent for detecting a molecular marker in the preparation of a kit for detecting ST11 type high virulence carbapenem-resistant Klebsiella pneumoniae, a kit for detecting ST11 type high virulence carbapenem-resistant Klebsiella pneumoniae, a nucleic acid reagent and a system, wherein the molecular marker comprises a plt-1 gene, and the nucleic acid sequence of the plt-1 gene is shown as SEQ ID NO. 1. The present disclosure takes plt-1 gene as a specific new high virulence gene for ST11 type Hv-CRKp detection, and takes it as a molecular marker for ST11 type carbapenem-resistant Klebsiella pneumoniae high virulence detection. By identifying whether the plt-1 gene exists in the ST11 type carbapenem-resistant Klebsiella pneumoniae, the ST11 type Hv-CRKp can be accurately distinguished from the traditional ST11 type carbapenem-resistant Klebsiella pneumoniae.

Description

Technical Field

[0001] This disclosure relates to the field of biomedical technology, specifically to the use of reagents for detecting molecular markers in the preparation of kits for detecting ST11 highly virulent carbapenem-resistant Klebsiella pneumoniae, kits for detecting ST11 highly virulent carbapenem-resistant Klebsiella pneumoniae, nucleic acid reagents and systems. Background Technology

[0002] ST11 highly virulent carbapenem-resistant Klebsiella pneumoniae (ST11 Hv-CRKp) is a variant of ST11 carbapenem-resistant Klebsiella pneumoniae. Specifically, it is formed after the traditional ST11 carbapenem-resistant Klebsiella pneumoniae acquires a pLVPK-like virulence plasmid of approximately 170 kb, leading to high pathogenicity and mortality. However, current technology lacks a method to rapidly and accurately distinguish ST11 Hv-CRKp from traditional ST11 carbapenem-resistant Klebsiella pneumoniae. Summary of the Invention

[0003] The purpose of this disclosure is to provide the use of reagents for detecting molecular markers in the preparation of kits for detecting ST11 highly virulent carbapenem-resistant Klebsiella pneumoniae, kits for detecting ST11 highly virulent carbapenem-resistant Klebsiella pneumoniae, nucleic acid reagents, and systems.

[0004] To achieve the above objectives, this disclosure provides the use of reagents for detecting molecular markers in the preparation of kits for detecting ST11 highly virulent carbapenem-resistant Klebsiella pneumoniae, wherein the molecular markers include the plt-1 gene, the nucleic acid sequence of which is shown in SEQ ID NO.1.

[0005] Optionally, the reagents for detecting the molecular markers include primers capable of specifically amplifying the plt-1 gene, and / or probes capable of specifically hybridizing with the plt-1 gene.

[0006] Optionally, the primers include primer pairs as shown in SEQ ID NO.2-3.

[0007] This disclosure also provides a kit for detecting ST11 type highly virulent carbapenem-resistant Klebsiella pneumoniae, wherein the kit contains reagents for detecting molecular markers, said molecular markers including the plt-1 gene, the nucleic acid sequence of said plt-1 gene being shown in SEQ ID NO.1.

[0008] Optionally, the reagents for detecting the molecular markers include primers capable of specifically amplifying the plt-1 gene, and / or probes capable of specifically hybridizing with the plt-1 gene.

[0009] Optionally, the primers include primer pairs as shown in SEQ ID NO.2-3.

[0010] This disclosure also provides a nucleic acid reagent for detecting ST11 type highly virulent carbapenem-resistant Klebsiella pneumoniae, wherein the nucleic acid reagent includes primers for detecting the plt-1 gene, the nucleic acid sequence of the plt-1 gene is shown in SEQ ID NO. 1, and the primers include primer pairs as shown in SEQ ID NO. 2 to 3.

[0011] This disclosure also provides a system for detecting ST11 type highly virulent carbapenem-resistant Klebsiella pneumoniae, wherein the system includes a sequencing device, a computing device, and an output device;

[0012] The sequencing device is used to sequence the total nucleic acid of the sample to be tested to obtain the total nucleic acid sequence.

[0013] The computing device includes a memory and a processor. The memory stores a computer program, and the processor is configured to execute the computer program stored in the memory to perform the following discrimination:

[0014] If the total nucleic acid sequence contains the plt-1 gene sequence, it is determined that the sample to be tested contains ST11 type highly virulent carbapenem-resistant Klebsiella pneumoniae, wherein the plt-1 gene sequence is shown in SEQ ID NO.1;

[0015] The output device is used to output the determination result of the computing device.

[0016] Optionally, the system further includes a nucleic acid extraction device for extracting nucleic acids from the sample to be tested to obtain total nucleic acids from the sample.

[0017] Preferably, the nucleic acid extraction device includes a nucleic acid extractor and / or a nucleic acid extraction kit.

[0018] Optionally, the sequencing device includes at least one of the Sanger sequencing platform, Illumina Novaseq, HiSeq Xten, and HiSeq 2500 / 2000 / 4000 sequencing platforms.

[0019] Through the above technical solution, this disclosure uses the plt-1 gene as a novel and specific high virulence gene for the detection of ST11 type Hv-CRKp, and uses it as a molecular marker for the detection of high virulence of ST11 type carbapenem-resistant Klebsiella pneumoniae. By detecting whether the plt-1 gene exists in ST11 type carbapenem-resistant Klebsiella pneumoniae, ST11 type Hv-CRKp can be accurately identified.

[0020] Other features and advantages of this disclosure will be described in detail in the following detailed description section. Attached Figure Description

[0021] The accompanying drawings are provided to further illustrate the present disclosure and form part of the specification. They are used together with the following detailed description to explain the present disclosure, but do not constitute a limitation thereof. In the drawings:

[0022] Figure 1 This is a second-generation sequencing result diagram of 9 ST11 carbapenem-resistant Klebsiella pneumoniae strains in the embodiments of this disclosure;

[0023] Figure 2 This is a line graph showing the change in the survival rate of *Hemiberlesia lataniae* larvae treated with plt-1 positive strains, plt-1 negative strains, and control strains over time in the embodiments of this disclosure. Detailed Implementation

[0024] The following provides a detailed description of specific embodiments of this disclosure. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit this disclosure.

[0025] The first aspect of this disclosure provides the use of reagents for detecting molecular markers in the preparation of a kit for detecting ST11 highly virulent carbapenem-resistant Klebsiella pneumoniae, wherein the molecular markers include the plt-1 gene, the nucleic acid sequence of which is shown in SEQ ID NO.1.

[0026] The inventors of this disclosure discovered that plt-1 positive ST11 carbapenem-resistant Klebsiella pneumoniae exhibits higher virulence than plt-1 negative ST11 carbapenem-resistant Klebsiella pneumoniae. Therefore, the plt-1 gene can be used as a molecular marker of high virulence in ST11 carbapenem-resistant Klebsiella pneumoniae. In other words, the plt-1 gene is a novel high virulence gene unique to ST11 highly virulent carbapenem-resistant Klebsiella pneumoniae (Hv-CRKp). Therefore, identifying and detecting the plt-1 gene in ST11 carbapenem-resistant Klebsiella pneumoniae can accurately identify ST11 Hv-CRKp.

[0027] The sequence of the plt-1 gene is as follows:

[0028] .

[0029] According to this disclosure, the reagents used for detecting molecular markers can be selected within a certain range, and any reagents capable of detecting the plt-1 gene can be used in this disclosure. Exemplarily, the reagents used for detecting molecular markers may include primers capable of specifically amplifying the plt-1 gene, and / or probes capable of specifically hybridizing with the plt-1 gene. In a preferred embodiment of this disclosure, the primers may, for example, include primer pairs as shown in SEQ ID NO. 2-3.

[0030] The primer sequence shown in SEQ ID NO.2 can be: GGCGGCCCTAATATATCCAA; the primer sequence shown in SEQ ID NO.3 can be: AAATATTGTGCCCGCGAAAA.

[0031] The second aspect of this disclosure provides a kit for detecting ST11 type highly virulent carbapenem-resistant Klebsiella pneumoniae, wherein the kit contains reagents for detecting molecular markers, said molecular markers including the plt-1 gene, the nucleic acid sequence of said plt-1 gene being shown in SEQ ID NO.1.

[0032] Optionally, the reagents for detecting molecular markers may include primers capable of specifically amplifying the plt-1 gene, and / or probes capable of specifically hybridizing with the plt-1 gene.

[0033] Optionally, the primers may include primer pairs as shown in SEQ ID NO.2-3.

[0034] Specifically, in this disclosure, the kit may further contain at least one of the following components: reaction system buffer, blank control, negative control, positive control, and water. These components may be stored individually or in a mixture within the kit. The kit may have various specifications; for example, it may be a kit for a single test sample or a kit for multiple test samples.

[0035] The third aspect of this disclosure provides a nucleic acid reagent for detecting ST11 type highly virulent carbapenem-resistant Klebsiella pneumoniae, wherein the nucleic acid reagent may include primers for detecting the plt-1 gene, the nucleic acid sequence of the plt-1 gene being shown in SEQ ID NO.1, and the primers may, for example, include primer pairs as shown in SEQ ID NO.2-3.

[0036] This disclosure provides a fourth aspect of a system for detecting ST11 highly virulent carbapenem-resistant Klebsiella pneumoniae, wherein the system includes a sequencing device, a computing device, and an output device; the sequencing device is used to sequence the total nucleic acid of the sample to be tested to obtain a total nucleic acid sequence; the computing device includes a memory and a processor, the memory storing a computer program, and the processor being configured to execute the computer program stored in the memory to perform the following judgment: if the total nucleic acid sequence contains a plt-1 gene sequence, then it is determined that the sample to be tested contains ST11 highly virulent carbapenem-resistant Klebsiella pneumoniae, wherein the plt-1 gene sequence is shown in SEQ ID NO.1; the output device is used to output the judgment result of the computing device.

[0037] Optionally, the system may further include a nucleic acid extraction device for extracting nucleic acids from the sample to be tested to obtain total nucleic acids from the sample; preferably, the nucleic acid extraction device may include a nucleic acid extractor and / or a nucleic acid extraction kit.

[0038] Optionally, the sequencing device may be a second-generation sequencing device, for example, the sequencing device may include at least one of the Sanger sequencing platform, Illumina Novaseq, HiSeq Xten, and HiSeq2500 / 2000 / 4000 sequencing platforms.

[0039] The present disclosure is further illustrated below by means of examples, but the present disclosure is not limited thereto. Unless otherwise specified, the raw materials, reagents, instruments and equipment involved in the embodiments of the present disclosure can all be obtained by purchase.

[0040] Example 1

[0041] This example illustrates that the plt-1 gene exists in ST11 type Hv-CRKp.

[0042] Nine ST11 carbapenem-resistant Klebsiella pneumoniae strains were collected, and these strains were then used to conduct infection experiments on the larvae of the giant wax moth. Based on the experimental results, these strains were divided into ST11 highly virulent carbapenem-resistant Klebsiella pneumoniae (Hv-CRKp) and conventional ST11 carbapenem-resistant Klebsiella pneumoniae (CRKp).

[0043] The infection experiment with the larvae of the large wax moth was conducted as follows:

[0044] Using each strain, a concentration of 1×10⁻⁶ was prepared. 5 CFU / mL bacterial suspensions were prepared, and then, using a microsyringe, each bacterial suspension was injected into the body cavity of the larvae of the large wax moth via the right hind leg, with an injection volume of 10 μl per larva. After injection, the virulence of each strain was characterized by the survival rate of the large wax moth larvae; the lower the survival rate of the large wax moth larvae, the greater the virulence of the strain.

[0045] Based on the results of the infection experiment of the large wax moth larvae, the strains were divided into two groups. Group 1 contained 8 ST11 type Hv-CRKp strains, and Group 2 contained 1 traditional ST11 type CRKp strain.

[0046] DNA was extracted from the two groups of strains using a DNA extraction kit to obtain the total DNA for each strain. The total DNA from each strain was then subjected to next-generation HiSeq sequencing. The sequencing results are shown below. Figure 1 As shown.

[0047] Sequencing results showed that the total DNA sequencing results of each strain in group 1 contained the sequence shown in SEQ ID NO.1, while the total DNA sequencing results of each strain in group 2 did not contain the sequence shown in SEQ ID NO.1.

[0048] Therefore, it can be concluded that the plt-1 gene exists in ST11 type Hv-CRKp. The plt-1 gene can serve as a high virulence gene for ST11 type Hv-CRKp. Using it as a molecular marker to detect whether ST11 type carbapenem-resistant Klebsiella pneumoniae has high virulence can effectively improve the accuracy of distinguishing between ST11 type Hv-CRKp and traditional ST11 type CRKp.

[0049] Example 2

[0050] This embodiment is used to verify that the plt-1 positive ST11 carbapenem-resistant Klebsiella pneumoniae has higher virulence than the plt-1 negative ST11 carbapenem-resistant Klebsiella pneumoniae.

[0051] Sixty ST11 carbapenem-resistant Klebsiella pneumoniae strains were collected. DNA was extracted from each strain using a DNA extraction kit to obtain total DNA for each strain. The total DNA of each strain was then amplified using primers shown in SEQ ID NO. 2–3, and the amplification results were detected by gel electrophoresis, yielding gel electrophoresis images for each strain. The presence of a band around 900 bp in the gel electrophoresis image indicated the presence of the plt-1 gene in the corresponding strain. Based on the presence of the plt-1 gene, the strains were divided into a plt-1 positive group and a plt-1 negative group. In this example, the plt-1 positive group consisted of 43 strains, and the plt-1 negative group consisted of 17 strains.

[0052] Total DNA from plt-1 positive and plt-1 negative strains was subjected to next-generation sequencing, and the sequencing results were screened to further determine whether the plt-1 gene was present in each strain. The sequencing results showed that the plt-1 gene was present in all plt-1 positive strains, while it was absent in all plt-1 negative strains. This indicates that the primer pairs shown in SEQ ID NO. 2–3 can specifically amplify the plt-1 gene.

[0053] Highly virulent Klebsiella pneumoniae K2044 was selected as the control strain. Then, the above-mentioned plt-1 positive group strain, plt-1 negative group strain and control strain were used to conduct infection experiments on the larvae of the large wax moth. The survival rate of the large wax moth larvae treated by each group of strains was recorded over time.

[0054] The procedure for the infection experiment with *Hemiberlesia lataniae* larvae was the same as in Example 1. The survival rate of *Hemiberlesia lataniae* larvae treated with different strains over time is as follows: Figure 2 As shown.

[0055] Depend on Figure 2 It can be seen that the survival rate of *Klebsiella pneumoniae* larvae treated with plt-1 positive strains was significantly lower than that of plt-1 negative strains. Moreover, as the experiment progressed, the survival rate of *Klebsiella pneumoniae* larvae treated with plt-1 positive strains gradually became the same as that of the control group strains. This indicates that *Klebsiella pneumoniae* ST11 carbapenem-resistant strains (plt-1 positive) have higher virulence than *Klebsiella pneumoniae* ST11 carbapenem-resistant strains (plt-1 negative), and its virulence is comparable to that of highly virulent *Klebsiella pneumoniae* K2044.

[0056] The preferred embodiments of this disclosure have been described in detail above with reference to the accompanying drawings. However, this disclosure is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this disclosure, various simple modifications can be made to the technical solutions of this disclosure, and these simple modifications all fall within the protection scope of this disclosure.

[0057] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this disclosure will not describe the various possible combinations separately.

[0058] Furthermore, various different embodiments of this disclosure can be combined in any way, as long as they do not violate the spirit of this disclosure, they should also be regarded as the content disclosed in this disclosure. sequence list <110> Peking Union Medical College Hospital, Chinese Academy of Medical Sciences <120> Kits, nucleic acid reagents, and systems for detecting ST11 type Hv-CRKp <130> 17949PUMC <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 648 <212> DNA <213> Artificial Sequence <400> 1 atgattcaaa aagcaaatca gcaaaaactt gtttttgcgt ggggtgtggt gcttttggtg 60 gtgagaacca ccaacctgtt gagccttttt gtggagtggg ttaaattatt tacggataaa 120 gtcaccagag gtggaaaaat gaaaaaatgg atgttagcaa tctgcctgat gtttataaat 180 gggatctgcg aagccgccga ttgctttgat cttgcaggtc gggattacaa aatagacccg 240 gatttactga gagcgatatc atggaaagaa tcccgttacc gggttaatgc catcggtatt 300 aatccggtaa cgggatatgg cagcggactg atgcaggtag attcccagca ttttaacgaa 360 ctggcccgct atggaattaa gccggaacat ctgacaacag atccctgtat gaacatttat 420 accggtgctt attatctggc aatagccttt aaaaaatggg gcgtctcctg ggaggccgtt 480 ggtgcataca atgccggatt caggaagacc gaacgccaga accagagacg tcttgcctac 540 gcatcagagg tttaccggat ttatacctgg ataaagagca gtaaaggcat ccgggttccg 600 accacgaaga aatcactttc ccaaattaac agtgtgcaga aaaattaa 648 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 ggcggcccta atatatccaa 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 aaatattgtg cccgcgaaaa 20

Claims

1. The use of reagents for detecting molecular markers in the preparation of a kit for detecting ST11 type highly virulent carbapenem-resistant Klebsiella pneumoniae, wherein, The molecular marker includes the plt-1 gene, the nucleic acid sequence of which is shown in SEQ ID NO. 1; The reagents used to detect the molecular markers include primers capable of specifically amplifying the plt-1 gene, and / or probes capable of specifically hybridizing with the plt-1 gene; The primers include primer pairs as shown in SEQ ID NO. 2-3.

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