Apparatus and method for differentiating antibiotic-resistant klebsiella pneumoniae

Abstract

The application belongs to the technical field of bioengineering and specifically relates to a device and method for distinguishing antibiotic-resistant Klebsiella pneumoniae. The device comprises a nanopore, a molecular probe, a Klebsiella pneumoniae RNA extraction reagent unit and a nanopore electrophysiological signal detection unit. The molecular probe is probe A and probe B. The nanopore electrophysiological signal detection unit contains HEPES, KCl, a double-layer lipid membrane or a polymer membrane and DPHPC.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a device and method for distinguishing antibiotic-resistant Klebsiella pneumoniae. Background Technology

[0002] Klebsiella pneumoniae is one of the most serious opportunistic pathogens in clinical infections. It is commonly found in the intestines of humans and animals and can cause serious clinical consequences, including central nervous system infections and abdominal infections. Currently, antibiotics are the primary treatment for Klebsiella pneumoniae infections. However, the widespread use of broad-spectrum antibiotics has led to increasingly serious bacterial resistance, resulting in prolonged clinical treatment and failure. Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a special type of Klebsiella pneumoniae that is particularly resistant to carbapenems, which were once considered "humanity's last line of defense against bacteria." Therefore, broad-spectrum antibiotics that can treat common drug-resistant Klebsiella pneumoniae may not be effective against carbapenem-resistant Klebsiella pneumoniae. The literature "Essentials of Clinical Application of Carbapenems" discloses that carbapenem-resistant Klebsiella pneumoniae can develop carbapenem resistance through the production of β-lactamases, alterations in porins, and increased efflux pump activity.

[0003] Therefore, accurately and quickly identifying the type of bacteria infecting a patient is crucial for selecting appropriate antibiotics for subsequent treatment. Determining the type of bacterial resistance helps doctors choose the right antibiotic, avoiding antibiotic overuse, shortening the treatment cycle, and improving prognosis. Currently, bacterial resistance phenotype detection, β-lactamase detection, and resistance gene detection are the main methods for detecting antibiotic resistance. However, bacterial resistance phenotype detection requires a long time to culture Klebsiella pneumoniae, making it time-consuming; β-lactamase detection, while fast, has a relatively small detection range, only able to detect a narrow concentration range; and while resistance gene detection has high accuracy, it is very expensive and time-consuming.

[0004] In summary, there is a need to propose a more optimized and efficient device and method for distinguishing whether bacteria are drug-resistant. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide an apparatus and method for distinguishing antibiotic-resistant Klebsiella pneumoniae, the specific technical solution of which is as follows.

[0006] A device for distinguishing between carbapenem-resistant Klebsiella pneumoniae (CRKP) and carbapenem-sensitive Klebsiella pneumoniae (CSKP), the device comprising a nanopore, molecular probes, a Klebsiella pneumoniae RNA extraction reagent unit, and a nanopore electrophysiological signal detection unit; the molecular probes are probe A and probe B, the nucleotide sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively; the nanopore electrophysiological signal detection unit contains HEPES, KCl, a lipid bilayer or polymer membrane, and DPHPC.

[0007] Furthermore, the diameter of the nanopores ranges from 1.0 to 1.5 nm.

[0008] Furthermore, the diameter of the nanopore is 1.3 nm.

[0009] Furthermore, the types of nanopores include Mycobacterium smegmatis porin A, alpha hemolysin, silicon nitride, or graphene nanopores.

[0010] Furthermore, the Klebsiella pneumoniae RNA extraction reagent unit contains TRIZOL, ethanol, DEPC water / RNase-free water, and an RNase inhibitor.

[0011] The method for distinguishing between carbapenem-resistant Klebsiella pneumoniae and carbapenem-sensitive Klebsiella pneumoniae using the above-mentioned device includes the following steps:

[0012] Step 1: In the Klebsiella pneumoniae RNA extraction reagent unit of the device, total RNA of Klebsiella pneumoniae is extracted with TRIZOL, washed with ethanol, dissolved in DEPC water or RNase-free water, and then stored with RNase inhibitor.

[0013] Step 2: Anneal the probes A and B of the device with the sample stored in Step 1 to form a 16S rRNA-probe complex.

[0014] Step 3: Place the nanopore of the device and the 16S rRNA-probe complex from Step 2 into the nanopore electrophysiological signal detection unit of the device, and detect the electrophysiological signal of the 16S rRNA-probe complex passing through the nanopore.

[0015] Step 4: Analyze the detected electrophysiological signals to distinguish between carbapenem-resistant Klebsiella pneumoniae and carbapenem-sensitive Klebsiella pneumoniae.

[0016] Furthermore, the voltage condition for detecting electrophysiological signals in step 3 is 150 millivolts.

[0017] Furthermore, the nanopores of the device are inserted into the bilayer lipid membrane or polymer membrane of the device.

[0018] Further, in step 4, the transport signals within the nanopores are analyzed, and transport signals with a blockage rate of 0.6 to 0.8 and a residence time in the range of 100 ms to 400 ms are selected as specific signals.

[0019] Furthermore, using f = 0.1·min -1 The target signal transport frequency threshold was used to distinguish between carbapenem-resistant Klebsiella pneumoniae and carbapenem-sensitive Klebsiella pneumoniae.

[0020] Beneficial technical effects

[0021] This invention provides a novel, efficient, and rapid device and method for distinguishing drug-resistant Klebsiella pneumoniae based on nanopores, achieving the differentiation of carbapenem-resistant and carbapenem-sensitive Klebsiella pneumoniae at the single-molecule level. Since 16S rRNA species identification is a commonly used method in microbiome research and is species-specific, the detection scheme provided by this invention can also be applied to the identification of other bacteria. Combined with antibiotic-containing / antibiotic-free control culture experiments and specific electrical signal recognition, it is possible to distinguish and identify multiple types of drug-resistant bacteria.

[0022] Compared with conventional paper disc diffusion and PCR methods, the detection method provided by this invention has the advantages of high sensitivity, real-time operation, low cost and short time consumption. It has been verified that the bacterial culture time is only 4 hours and the accuracy is 90%.

[0023] The device provided by this invention includes a complete set of necessary reagents and specific probes for distinguishing between carbapenem-resistant and carbapenem-sensitive Klebsiella pneumoniae. It is easy to operate and provides highly accurate results. Therefore, it has significant application value in rapid clinical detection. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0025] Figure 1 Example of setting up a single-channel recording for the structure of nanopores and the measurement of nanopores (a is a schematic diagram of the structure of MspA nanopores, b is a schematic diagram of the signal transport of the MspA nanopores to be measured).

[0026] Figure 2The diagram shows the 16S rRNA-probe complex formed by probe A and probe B and its nanopore electrophoresis signals (a is a schematic diagram of the formation of the 16S rRNA-probe complex by probe A and probe B; b is the agarose gel electrophoresis result of the 16S rRNA-probe complex; c is the retention time and peak value of the transport signal of the 16S rRNA-probe complex; d is the retention time and peak value of the single-stranded DNA transport signal).

[0027] Figure 3 This is the electrical signal indicating the displacement of the probe group;

[0028] Figure 4 To distinguish between carbapenem-resistant and carbapenem-sensitive Klebsiella pneumoniae and to record signals in a single channel (a is the translocation frequency of the carbapenem-resistant Klebsiella pneumoniae group, b is the total RNA of the two Klebsiella pneumoniae groups after incubation with probes A and B and detection through MspA nanopores, and c is a scatter plot of the blocking rate and retention time of the signals measured by the nanopores);

[0029] Figure 5 Examples of double-blind testing and accuracy assessment for clinical samples (a is the target signal transport frequency threshold for the detection of two types of Klebsiella pneumoniae, and b is a statistical graph of the accuracy of the test results for the samples).

[0030] Figure 6 Example of a testing flowchart and total time cost;

[0031] Figure 7 Example of a protocol for detecting carbapenem-resistant Klebsiella pneumoniae using nanopores. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0033] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0034] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0035] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0036] As used in this specification, the term "about" typically means + / - 5% of the value, more typically + / - 4%, more typically + / - 3%, more typically + / - 2%, even more typically + / - 1%, even more typically + / - 0.5% of the value.

[0037] In this specification, certain embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered as having specifically disclosed all possible subranges and independent numerical values ​​within those ranges. For example, range The description should be considered as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0038] Carbapenem-resistant Klebsiella pneumoniae (CRP) has rapidly spread globally in recent decades, posing a significant challenge to current clinical practice. Rapid detection of CRP can reduce inappropriate antibiotic treatment and save lives. Traditional CRP detection methods are time-consuming, and PCR and other sequencing methods are too expensive and technically demanding to meet clinical needs. Nanopore assays, with their advantages of high sensitivity, real-time operation, and low cost, have been applied to the screening of disease biomarkers. In this study, the amount of 16S rRNA in the nucleic acid extract after short-term bacterial culture with the antibiotic imipenem was used to differentiate between carbapenem-sensitive and carbapenem-resistant KRP. Nanopores allow for the recording of specific signals generated after probe binding to 16S rRNA, enabling ultrasensitive and rapid quantitative detection of 16S rRNA. This invention demonstrates that the nanopore detection method requires only 4 hours of incubation time to distinguish between carbapenem-resistant and carbapenem-sensitive Klebsiella pneumoniae. The time cost of this method is approximately 5% of that of the disc diffusion method, while achieving similar accuracy.

[0039] Specifically, nanopore sensing technology facilitates its widespread application in third-generation DNA single-molecule sequencing. Nanoscale protein pores are embedded in phospholipid membranes, which divide the pore chambers into two parts (cis and trans). When a voltage is applied to the chamber containing a specific concentration of ion solution, charged detectable substances in the system are driven through the pores to the other chamber. A patch-clamp sensor detects the current change signal in the nanopore. Different molecules transported through the nanopore can induce corresponding current blocking signals; through specific transport signals and frequencies, qualitative and quantitative analysis of the detected molecules can be achieved. This nanopore sensing technology offers advantages such as label-free operation, speed, real-time operation, and high sensitivity, requiring only a small amount of sample. Therefore, these characteristics are suitable for the detection of biomarkers.

[0040] Specifically, taking Mycobacterium smegmatis porin A (MspA) as an example, it is one of the outer membrane proteins of mycobacteria, with a length of 9.6 nm, such as... Figure 1 As shown, the diameter is 1.3 nm. The nanopores are efficiently integrated into a lipid bilayer and allow single-stranded nucleic acid transport through the pores. Due to their short and narrow channels, MspA nanopores are ideally suited for nanopore sequencing. Of course, besides MspA nanopores, other common nanopores, such as alpha-hemolysin, silicon nitride, and graphene nanopores, are also suitable for nanopore sequencing. Furthermore, in addition to lipid bilayers, polymer membranes can also be used in this invention.

[0041] Specifically, 16S rRNA, present in all bacteria, is a component of the 30S subunit of the prokaryotic ribosome, and its function remains unchanged over time. 16S rRNA can be used to identify bacterial species because it contains highly conserved regions common to all bacteria and highly variable regions that differentiate between different bacteria. 16S rRNA has been proven to be a reliable genetic marker, commonly used in bacterial classification, and there is literature demonstrating its potential for identifying clinical pathogens.

[0042] Material

[0043] The reagents 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES, purity >99.5%, CAS#7365-45-9), potassium chloride (KCl, purity >99.0%, CAS#7447-40-7), agarose (purity >99.0%, CAS#: 9012-36-6), chloroform (purity >99.0%, CAS: 67-66-3), isopropanol (purity >99.0%, CAS#: 67-63-0) and ethanol (purity >99.0%, CAS#: 64-17-5) were purchased from Sigma-Aldrich. RNase inhibitor (5KU), pET-28b plasmid and all DNA were provided by Sangon Biotech, 1,2-diacetyl-sn-glycerol-3-phosphocholine (DPHPC) was purchased from Avanti, PrimeSTAR HS DNA polymerase was purchased from TaKaRa, and imipenem (CAS#: 64221-86-9) was purchased from MSD.

[0044] Clinical specimens:

[0045] Blood samples from two patients with Klebsiella pneumoniae infection were provided by the Department of Laboratory Medicine, West China Hospital, Sichuan University. This invention study was conducted in accordance with the recommendations of the Chinese National Biomedical Research Ethics Review and the Declaration of the Helsinki WMA. The protocol was approved by the Biomedical Ethics Committee of West China Hospital, Sichuan University. This invention study used residual specimens, i.e., specimen remnants intended for routine clinical care or analysis, which would otherwise be discarded, and met the criteria for waiving informed consent. The Biomedical Ethics Committee of West China Hospital, Sichuan University, granted an exemption from informed consent.

[0046] Example 1

[0047] This embodiment provides a device for distinguishing between carbapenem-resistant and carbapenem-sensitive Klebsiella pneumoniae. The device includes a nanopore, molecular probes, a Klebsiella pneumoniae RNA extraction reagent unit, and a nanopore electrophysiological signal detection unit. The molecular probes are probe A and probe B, whose nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The nanopore electrophysiological signal detection unit contains HEPES, KCl, a lipid bilayer or polymer membrane, and DPHP. The Klebsiella pneumoniae RNA extraction reagent unit contains TRIZOL, ethanol, DEPC-treated water / RNase-free water, and an RNase inhibitor.

[0048] Probes A and B were designed by the inventors' team. Because the target 16S rRNA is 932 bp long, it is difficult to distinguish the 16S rRNA-probe complex without a probe or with a single probe. Therefore, the inventors' team designed two probes to bind to the specifically expressed 16S rRNA of Klebsiella pneumoniae. The nucleotide sequences of the two probes are shown in Table 1. Probes A and B were annealed with stored samples, and the formation of the 16S rRNA-probe complex was verified using agarose gel electrophoresis. Figure 2 a).

[0049] Table 1 Probe Sequence

[0050] Furthermore, agarose gel electrophoresis results showed that the 16S rRNA-probe complex was successfully obtained. Figure 2 b). In carbapenem-resistant Klebsiella pneumoniae samples, the retention time of the 16S rRNA-probe complex transport signal ranged from 100 to 400 ms, with a peak value of 196.98 ms. The retention time of single-stranded DNA transport ranged from 0 to 100 ms, with a peak value of 12.03 ms. Figure 2 c and Figure 2 d). The residence times of probes A and B are in the range of 0-70 ms. Figure 3 These results indicate that the long-retention signal is caused by the 16S rRNA-probe complex.

[0051] Example 2

[0052] This embodiment provides a method for distinguishing between carbapenem-resistant Klebsiella pneumoniae and carbapenem-sensitive Klebsiella pneumoniae using the apparatus of Embodiment 1.

[0053] 1. Preparation of bacterial extract

[0054] Two groups of Klebsiella pneumoniae samples from clinical patients were provided by West China Hospital of Sichuan University. The Klebsiella pneumoniae samples were cultured to two different concentrations: 0.5 MCF in the first group and 4 MCF in the second. At the start of culture, the final concentration of imipenem used in both groups was 16 mg / L. Total RNA was extracted from Klebsiella pneumoniae using the TRIZOL method. First, 100 μL of bacterial solution was collected. After centrifugation, the supernatant was collected (8000 g, 4 °C, 2 min). Lysozyme was used for precipitation, and the solution was incubated at 37 °C for 10 min. Klebsiella pneumoniae was lysed, and total RNA was extracted and washed with ethanol. The centrifuge tube caps were removed, and the tubes were dried at room temperature for 5–10 min. DEPC water or water free of RNAs was added. RNase inhibitors were added to the dissolved solution to a final concentration of 20 U / μL for storage.

[0055] 2. Detection

[0056] 1) The probes A and B were annealed with the extracted sample to form a 16S rRNA-probe complex.

[0057] 2) The nanopore and the 16S rRNA-probe complex were placed in the nanopore electrophysiological signal detection unit for detection. A voltage of 150 mV was applied to detect the electrophysiological signal of the 16S rRNA-probe complex passing through the nanopore.

[0058] 3) Analyze the detected electrophysiological signals to distinguish between carbapenem-resistant Klebsiella pneumoniae and carbapenem-sensitive Klebsiella pneumoniae.

[0059] Example 3

[0060] Optimization of bacterial concentration and testing of standard samples

[0061] 1. Expression and purification of MspA nanopores

[0062] The MspA nanopore gene was cloned into the pET-28b plasmid, and the pET-28b plasmid carrying the MspA gene was transferred into engineered E. coli competent cells (BL21). Successfully transferred E. coli were cultured in LB medium at 37°C with kanamycin added to a concentration of 50 μg / mL. When the optical density (600 nm) approached 0.8, 0.8 mM IPTG was added to LB (lysogenic fermentation broth) medium, and the induction temperature was 15°C. After 12 hours of induction, E. coli were collected by centrifugation. The supernatant was collected after the E. coli were disrupted using an ultrasonic generator and further purified using an anion exchange column (Q-Sepharose) and molecular sieves (Superdex 200 16 / 90). The purified protein was analyzed by 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). The purified MspA nanopore protein can be aliquoted and stored at -80°C. The aliquoted samples remain stable for many years, and the nanopores retain their structural integrity upon thawing.

[0063] 2. Nanopore electrophysiological signal detection experiments determined the optimal sample concentration and the best bacterial culture time.

[0064] 2.1 Determining the optimal sample concentration

[0065] Nanopore electrophysiological signal detection experiments were performed on two bacterial extract samples of different concentrations from Example 2. The experimental method was as follows:

[0066] The experiments were conducted in a chamber provided by Warner Instruments. Electrophysiological signal detection experiments of the nanopores were performed at 150 mV. The conductive buffer solutions for both the cis and trans sides were 400 mL M KCl solutions containing 10 mM HEPES at pH 7.0. A bilayer lipid membrane (BLM) coated on both sides of the 150 μm pore was formed from 1,2-dihydroxyformyl-sn-glycerol-3-phosphocholine (DPHPC). Adding MspA to the solution in the cis chamber allowed for MspA protein insertion, resulting in faster BLM formation. Insertion of a single MspA nanopore resulted in an increase in current, corresponding to a conductance of 1.2 nS. After recording the current signal of a single MspA nanopore insertion via a Heka EPC-10 patch clamp (HEKA), the sample was added to the cis side.

[0067] Detection efficiency was optimized using two concentrations of Klebsiella pneumoniae. In 0.5 MCF samples, the target RNA transport signal frequency was 0.02 ± 0.02 / min (n = 3) in the control group and 0.13 ± 0.05 / min (n = 3) in the carbapenem-resistant Klebsiella pneumoniae group. In 4 MCF samples, the target RNA transport signal frequency was 0 / min (n = 3) in the control group and 0.33 ± 0.07 / min (n = 3) in the carbapenem-resistant Klebsiella pneumoniae group. Figure 4 a) Compared with the 0.5 MCF sample, the 4 MCF sample can be detected better in nanopore assays.

[0068] 2.2. Determining the optimal bacterial culture time

[0069] Total RNA extracted from carbapenem-resistant and carbapenem-sensitive Klebsiella pneumoniae was incubated with probes A and B, and the incubated solutions were detected using MspA nanopores. Figure 4 b). The two parameters of the signal obtained from the sample passing through the nanopore, the blockage rate and the residence time, were plotted as a scatter plot. Figure 4 c) Significant differences in retention time were observed between different groups, especially in the range of 100 ms to 400 ms with a blockage rate of 0.6 to 0.8. Therefore, signals within this range were selected as specific diagnostic signals. After comparing the number of 16S rRNA probe signals within a given range from blank, control, carbapenem-resistant Klebsiella pneumoniae, and carbapenem-sensitive Klebsiella pneumoniae samples, f = 0.1·min -1 The target signal transport frequency threshold was used to distinguish between carbapenem-resistant and carbapenem-sensitive Klebsiella pneumoniae. A threshold higher than 0.1 min... -1 The bacteria were identified as carbapenem-resistant Klebsiella pneumoniae, with a mortality rate below 0.1 mmol / L. -1 The bacteria were identified as carbapenem-sensitive Klebsiella pneumoniae. Further, to determine the minimum bacterial culture time required to differentiate between carbapenem-resistant and carbapenem-sensitive Klebsiella pneumoniae, samples with different culture times (2 hours, 4 hours, and 8 hours) were analyzed using MspA nanopores. The results showed that 4 hours was the optimal bacterial culture time, balancing sensitivity and efficiency.

[0070] Example 4

[0071] Double-blind trial of MspA nanopore assay in clinical samples

[0072] Bacteria from blood samples of 20 patients with Klebsiella pneumoniae infection provided by West China Hospital were cultured, and total RNA was extracted for a double-blind experiment. Each sample was analyzed at least three times using MspA nanopores. After analysis, the number of 16S rRNA probe signals with a blockage rate of 0.6 to 0.8 and a retention time of 100 ms to 400 ms were collected and compared with a target signal transport frequency threshold f.

[0073] Of the 20 samples, as shown in Table 2, 9 were above the threshold (0.1 min). -1 It was also identified as carbapenem-resistant Klebsiella pneumoniae. As shown in Table 3, the other 11 samples were below the threshold of 0.1 min. -1 These clinical samples were identified as carbapenem-sensitive Klebsiella pneumoniae samples. Figure 5 a). Compared with the results obtained from standard clinical methods (paper disc diffusion or PCR), the nanopore assay method of the present invention has the advantages of low cost and short time consumption (Table 4). The results of 18 samples measured by nanopore assay are correct. Figure 5 b) Two of the results were false negatives.

[0074] Table 2. Clinical sample information of carbapenem-resistant Klebsiella pneumoniae

[0075] Note: Sample ID is the patient ID in the hospital, and sample number is the corresponding number in this invention study.

[0076] Table 3. Clinical sample information of carbapenem-sensitive Klebsiella pneumoniae Sample ID sample# SCIM (mm) Drug resistance genes Drug resistance genes 17012889-3 1 6 KPC KPC-2 17019349-3 3 6 KPC KPC-2 1810143046 4 6 KPC KPC-2 15043287-1 5 6 KPC KPC-2 15057156-1 6 6 KPC KPC-2 15083593-1 7 6 KPC KPC-2 1807191036 8 6 KPC KPC-2 1807271015 9 24 burden - 17008404-1 11 6 KPC No 17012837-3 12 6 KPC KPC-2 17020362-3 20 6 KPC KPC-2

[0077] Note: Sample ID is the patient ID in the hospital, and sample number is the corresponding number in this invention study.

[0078] Table 4 Comparison of different carbapenem-resistant Klebsiella pneumoniae detection methods

[0079] The above examples used Clampfit 10.6 and Origin Pro 8.0 software for data analysis. Blocking current was defined as ΔI / I0, where I0 is the current of a fully open pore, and ΔI is the amplitude of the blocking current caused by transported molecules. Retention time was collected using the single-channel search function of Clampfit 10.6. These two parameters were used to quantitatively analyze the target 16S rRNA from surviving carbapenem-resistant Klebsiella pneumoniae. All data were obtained from 20-minute electrophysiological recordings, with each experimental group independently replicated three times.

[0080] Summarize

[0081] In this invention, the inventors designed two DNA molecular probes to specifically bind to the 16S rRNA of carbapenem-resistant Klebsiella pneumoniae. The 16S rRNA-probe complex translocates through MspA nanopores, resulting in a retention time between 100 ms and 400 ms. Based on the blocking rate and retention time of the specific blocking signal, the 16S rRNA in carbapenem-resistant Klebsiella pneumoniae samples can be detected. Figure 6 The method was demonstrated to be effective in differentiating between carbapenem-resistant and carbapenem-sensitive Klebsiella pneumoniae samples by testing carbapenem-resistant and carbapenem-sensitive Klebsiella pneumoniae samples, with a bacterial sample incubation time of only 4 hours.

[0082] Furthermore, the inventors' team used MspA nanopores to analyze 20 clinical samples provided by West China Hospital. In 11 carbapenem-sensitive Klebsiella pneumoniae samples, 9 samples received correct diagnoses, while 2 samples were false negatives. In 9 carbapenem-resistant Klebsiella pneumoniae samples, all 9 samples received correct diagnoses. The accuracy rate of the nanopore diagnostic method was 90%. Analysis suggests that RNA degradation during sample storage or transfer is the main cause of the 10% false negative rate. The transportation of clinical samples from the hospital to the laboratory, as well as the time interval between sample processing and nanopore assay, increases the likelihood of RNA degradation, leading to a reduction in the amount of 16S rRNA and specific blocking signals.

[0083] In summary, the inventors' research has demonstrated that nanopore single-molecule detection technology can be used for rapid differentiation of carbapenem-resistant Klebsiella pneumoniae. Compared to the two most widely used clinical methods, disk diffusion and PCR, the nanopore detection method offers advantages such as lower cost, higher efficiency, and ease of operation.

[0084] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A device for distinguishing between carbapenem-resistant Klebsiella pneumoniae and carbapenem-sensitive Klebsiella pneumoniae, characterized in that, The device includes a Mycobacterium smegmatis lactin A nanopore, a molecular probe, a Klebsiella pneumoniae RNA extraction reagent unit, and a Mycobacterium smegmatis lactin A nanopore electrophysiological signal detection unit; the molecular probe is probe A and probe B, and the nucleotide sequences of probe A and probe B are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively; The Mycobacterium smegmatis lactin A nanopore electrophysiological signal detection unit contains HEPES, KCl, a lipid bilayer membrane, and DPHPC; the diameter of the Mycobacterium smegmatis lactin A nanopore ranges from 1.0 to 1.5 nm.

2. The apparatus as claimed in claim 1, characterized in that, The diameter of the Mycobacterium smegmatis lactin A nanopore is 1.3 nm.

3. The apparatus as described in claim 1, characterized in that, The Klebsiella pneumoniae RNA extraction reagent unit contains TRIZOL, ethanol, DEPC water / RNase-free water, and an RNase inhibitor.

4. A method for in vitro differentiation of carbapenem-resistant Klebsiella pneumoniae and carbapenem-sensitive Klebsiella pneumoniae using the device according to any one of claims 1-3 for non-diagnostic purposes, characterized in that, Includes the following steps: Step 1: In the Klebsiella pneumoniae RNA extraction reagent unit of the device, total RNA of Klebsiella pneumoniae is extracted with TRIZOL, washed with ethanol, dissolved in DEPC water or RNase-free water, and then stored with RNase inhibitor. Step 2: Anneal the probes A and B of the device with the sample stored in Step 1 to form a 16S rRNA-probe complex. Step 3: Place the Mycobacterium smegmatis lactin A nanopore and the 16S rRNA-probe complex from Step 2 into the Mycobacterium smegmatis lactin A nanopore electrophysiological signal detection unit of the device, and detect the electrophysiological signal of the 16S rRNA-probe complex passing through the Mycobacterium smegmatis lactin A nanopore; the Mycobacterium smegmatis lactin A nanopore of the device is inserted into the bilayer lipid membrane of the device; Step 4: Analyze the detected electrophysiological signals to distinguish between carbapenem-resistant Klebsiella pneumoniae and carbapenem-sensitive Klebsiella pneumoniae.

5. The method as described in claim 4, characterized in that, The voltage condition for electrophysiological signal detection in step 3 is 150 millivolts.

6. The method as described in claim 4, characterized in that, In step 4, the transport signal within the mycobacterium smegmatis lactin A nanopore was analyzed, and the transport signal with a blockage rate of 0.6 to 0.8 and a retention time of 100 ms to 400 ms was selected as the specific signal.

7. The method as described in claim 6, characterized in that, Further use f = 0.1•min -1 The target signal transport frequency threshold was used to distinguish between carbapenem-resistant Klebsiella pneumoniae and carbapenem-sensitive Klebsiella pneumoniae.

Images