CN116814818A - Device and method for distinguishing antibiotic-resistant klebsiella pneumoniae - Google Patents

Abstract

The invention belongs to the field of bioengineering technology, and specifically relates to a device and method for distinguishing antibiotic-resistant Klebsiella pneumoniae. The device of the present invention includes 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 The detection unit contains HEPES, KCl, double-layer lipid membrane or polymer membrane, and DPHPC.

Description

A device and method for distinguishing antibiotic-resistant Klebsiella pneumoniae

Technical field

The invention belongs to the field of bioengineering technology, and specifically relates to a device and method for distinguishing antibiotic-resistant Klebsiella pneumoniae.

Background technique

Klebsiella pneumoniae is one of the most serious opportunistic pathogens in clinical infections. It usually exists in the intestines of humans and animals and can cause serious clinical consequences including central nervous system infection or abdominal infection. Antimicrobial drugs are currently the treatment for Klebsiella pneumoniae infections. However, the widespread use of broad-spectrum antibacterial drugs has led to increasing bacterial resistance, which in turn leads to prolonged clinical treatment and failure. Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a special type of Klebsiella pneumoniae that is particularly resistant to carbapenem-resistant antibiotics, which were once known as "human The last line of defense against bacteria.” Therefore, broad-spectrum antibiotics that can treat generally drug-resistant Klebsiella pneumoniae may not necessarily be effective against carbapenem-resistant Klebsiella pneumoniae. The literature "Essentials of the Clinical Application of Carbapenems" discloses that carbapenem-resistant Klebsiella pneumoniae can induce carbon dioxide through the production of β-lactamase, changes in porins and an increase in efflux pump activity. Penem resistance.

Based on this, it is very important to accurately and quickly distinguish the types of bacteria infecting patients to help select antibacterial drugs for subsequent treatment, because clarifying the resistance types of bacteria can help doctors choose appropriate types of antibacterial drugs, avoid antibiotic abuse, and shorten the treatment cycle. Improve prognosis. At present, bacterial resistance phenotypic detection, β-lactamase detection and drug resistance gene detection are the main methods used for drug resistance detection. However, the detection of bacterial resistance phenotype requires a long time to culture Klebsiella pneumoniae, so it is usually time-consuming; although β-lactamase detection is fast, the detection range is relatively small and can only detect a few Narrow concentration range; while gene detection of drug resistance, although highly accurate, is very expensive and time-consuming.

In summary, it is necessary to propose a more optimized and efficient device and method for distinguishing whether bacteria are drug-resistant or not.

Contents of the invention

In view of this, the purpose of the present invention is to provide a device and method for distinguishing antibiotic-resistant Klebsiella pneumoniae. The specific technical solutions are as follows.

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

Further, the diameter range of the nanopore is 1.0-1.5nm.

Further, the diameter of the nanopore is 1.3nm.

Further, the types of nanopores include Mycobacterium smegmatis porin A, alpha hemolysin, silicon nitride or graphene nanopores.

Further, the Klebsiella pneumoniae RNA extraction reagent unit contains TRIZOL, ethanol, DEPC water/RNase-free water, and RNase inhibitor.

The method of using the above device to distinguish carbapenem-resistant Klebsiella pneumoniae from carbapenem-sensitive Klebsiella pneumoniae includes the following steps:

Step 1: In the Klebsiella pneumoniae RNA extraction reagent unit of the device, use TRIZOL to extract the total RNA of Klebsiella pneumoniae, then wash it with ethanol, add DEPC water or RNase-free water to dissolve, and then add RNase inhibitor to store;

Step 2: anneal probe A and probe B of the device with the sample stored in step 1 to form a 16S rRNA-probe complex;

Step 3: Place the nanopore of the device and the 16S rRNA-probe complex in step 2 into the nanopore electrophysiological signal detection unit of the device, and conduct the electrophysiological signal of the 16S rRNA-probe complex through the nanopore. detection;

Step 4: Analyze the detected electrophysiological signals to differentiate between carbapenem-resistant Klebsiella pneumoniae and carbapenem-susceptible Klebsiella pneumoniae.

Furthermore, the voltage condition for electrophysiological signal detection in step 3 is 150 millivolts.

Further, the nanopore of the device is inserted into the bilayer lipid membrane or polymer membrane of the device.

Further, in step 4, the transport signal in the nanopore is analyzed, and the transport signal with a blocking rate of 0.6 to 0.8 and a residence time in the range of 100 milliseconds to 400 milliseconds is selected as the specific signal.

Furthermore, f=0.1·min ^-1 was used as the target signal transfer frequency threshold to distinguish carbapenem-resistant Klebsiella pneumoniae from carbapenem-susceptible Klebsiella pneumoniae.

Beneficial technical effects

The present invention provides a novel, efficient and rapid device and method for distinguishing carbapenem-resistant Klebsiella pneumoniae based on nanopores, realizing the distinction between carbapenem-resistant Klebsiella pneumoniae and carbon-resistant Klebsiella pneumoniae at a single molecular level. Penem-susceptible Klebsiella pneumoniae. Since the species identification of 16S rRNA is a commonly used method in microbiome research and has species specificity, the detection scheme provided by the present invention can also be applied to the identification of other bacteria, combined with the control of antibiotic-containing environment/antibiotic-free environment. Culture experiments and specific electrical signal recognition can distinguish and identify multiple types of drug-resistant bacteria.

Compared with the conventional disk diffusion method and PCR method, the detection method provided by the invention has the advantages of high sensitivity, real-time operation, low cost and less time consumption. It has been verified that the bacterial culture time only takes 4 hours and the accuracy is 90%.

The device provided by the invention contains a complete set of necessary reagents and specific probes for distinguishing carbapenem-resistant Klebsiella pneumoniae from carbapenem-sensitive Klebsiella pneumoniae. It is easy to operate and has high detection result accuracy. Therefore, it has great application value in clinical rapid detection.

Description of the drawings

In order to more clearly explain the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to describe the embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without exerting any creative effort.

Figure 1 shows the structure of the nanopore and an example of single-channel recording setup for nanopore measurement (a is a schematic diagram of the MspA nanopore structure, b is a schematic diagram of the signal transport to be measured in the MspA nanopore);

Figure 2 shows the 16S rRNA-probe complex formed by probe A and probe B and its nanopore electrical signal (a is a schematic diagram of the 16S rRNA-probe complex formed by probe A and probe B; b is 16S rRNA -Probe complex agarose electrophoresis results; 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);

Figure 3 shows the translocation electrical signal of the probe set;

Figure 4 shows the distinction between carbapenem-resistant Klebsiella pneumoniae and carbapenem-susceptible Klebsiella pneumoniae and the single-channel recording signal (a is the translocation of the carbapenem-resistant Klebsiella pneumoniae group Frequency, b is the total RNA of two kinds of Klebsiella pneumoniae incubated with probes A and B and then detected through the MspA nanopore respectively, c is the scatter plot drawn by the blocking rate and residence time of the signal measured by the nanopore. );

Figure 5 is an example of double-blind testing of clinical samples and evaluation of measurement accuracy (a is the target signal transfer frequency threshold for the detection of two Klebsiella pneumoniae, b is a statistical chart of the result correctness of the detection samples);

Figure 6 is an example of the detection flow chart and total time cost;

Figure 7 is an example of the protocol flow for nanopore detection of carbapenem-resistant Klebsiella pneumoniae.

Detailed ways

In order to make the purpose, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

As used herein, "and/or" includes any and all combinations of one or more of the associated listed items.

"Plural" in this article means two or more, that is, it includes two, three, four, five, etc.

It should be noted that, in this document, the terms "comprising", "comprising" or any other variations thereof are intended to cover a non-exclusive inclusion, such that a process, method, article or device that includes a series of elements not only includes those elements, It also includes other elements not expressly listed or inherent in the process, method, article or apparatus. Without further limitation, an element defined by the statement "comprises a..." does not exclude the presence of additional identical elements in a process, method, article, or device that includes that element.

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

In this specification, certain embodiments may be disclosed in a format that falls within a range. It should be understood that this "within a range" description is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, descriptions of ranges should be considered to have specifically disclosed all possible subranges and individual numerical values within such ranges. For example, range The description of Individual numbers such as 1, 2, 3, 4, 5 and 6. The above rules apply regardless of the breadth of the scope.

Carbapenem-resistant Klebsiella pneumoniae has rapidly become epidemic worldwide in recent decades, posing a huge challenge to today's clinical practice. Rapid detection of carbapenem-resistant Klebsiella pneumoniae could reduce inappropriate antimicrobial treatment and save lives. Traditional detection methods for carbapenem-resistant Klebsiella pneumoniae are very time-consuming, and PCR and other sequencing methods are too expensive and technically demanding to meet clinical needs. Nanopore detection has the advantages of high sensitivity, real-time operation and low cost, and has been applied to the screening of disease biomarkers. In this study, the amount of 16S rRNA in nucleic acid extracts after short-term culture of bacteria with the antibiotic imipenem was detected to reflect the growth of bacteria, thereby distinguishing carbapenem-sensitive Klebsiella pneumoniae from resistant Klebsiella pneumoniae. Carbapenems Klebsiella pneumoniae. Nanopores can be used to record the specific signal generated after the probe binds to 16S rRNA, thereby completing ultra-sensitive and rapid quantitative detection of 16S rRNA. The present invention proves that the nanopore detection method can distinguish carbapenem-resistant Klebsiella pneumoniae from carbapenem-sensitive Klebsiella pneumoniae in only 4 hours of incubation time. The time cost of this method is about 5% of that of the paper diffusion method, and at the same time it achieves an accuracy similar to that of the paper diffusion method.

Specifically, nanopore sensing technology contributes to its widespread application in third-generation DNA single-molecule sequencing. Nanosized protein pores are embedded in a phospholipid membrane that divides the protein pore chamber into two parts (cis and trans). When a voltage is applied across a chamber containing a concentration of ionic solutions, charged detection species in the system are driven through the pores to another chamber. The patch-clamp sensor detects the current change signal of the nanopore. Different molecules transported through the nanopore can cause corresponding current blocking signals. Through specific transport signals and transport frequencies, qualitative and quantitative analysis of the detected molecules can be achieved. This nanopore sensing technology has the advantages of label-free, fast, real-time operation and high sensitivity, requiring only a small amount of sample. Therefore, these features are suitable for biomarker detection.

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, as shown in Figure 1, and a diameter of 1.3 nm. By efficiently incorporating nanopores into bilayer lipid membranes and allowing single-stranded nucleic acid transport through the pore, MspA nanopores are well suited for nanopore sequencing due to their short and narrow channels. Of course, in addition to MspA nanopores, other common nanopores, such as alpha hemolysin, silicon nitride and graphene nanopores, can be suitable for nanopore sequencing. In addition, in addition to bilayer lipid membranes, polymer membranes can also be applied to the present invention.

Specifically, 16S rRNA, present in all bacteria, is a component of the 30S subunit of prokaryotic ribosomes, and its function does not change over time. 16S rRNA can be used to identify bacterial species because it contains highly conserved regions common to all bacteria and hypervariable regions that differ among different bacteria. 16S rRNA has been shown to be a reliable genetic marker that is commonly used for bacterial classification and has documented utility in identifying clinical pathogens.

Material

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), isopropyl alcohol (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 SangonBiotech, 1,2-diacetyl-sn-glycero-3-phosphocholine (DPHPC) was purchased from Avanti, and PrimeSTAR HS DNA polymerase was purchased from TaKaRa , Imipenem (CAS#: 64221-86-9) was purchased from MSD.

Clinical specimens:

Blood samples from 2 patients with Klebsiella pneumoniae infection were provided by the Laboratory Department of West China Hospital of Sichuan University. The present study was conducted in accordance with the recommendations of the Chinese National Biomedical Research Involving Human Ethics Review and the WMA Declaration of Helsinki. The protocol was approved by the Biomedical Ethics Committee of West China Hospital of Sichuan University. This inventive study used leftover specimens, i.e., the remnants of specimens used for routine clinical care or analysis, which would have been discarded and met the criteria for a waiver of informed consent. A waiver of informed consent was granted by the Biomedical Ethics Committee of West China Hospital of Sichuan University.

Example 1

This embodiment provides a device for distinguishing carbapenem-resistant Klebsiella pneumoniae from carbapenem-susceptible Klebsiella pneumoniae. The device includes nanopores, molecular probes, and Klebsiella pneumoniae RNA. Extraction reagent unit, nanopore electrophysiological signal detection unit; the molecular probes are probe A and probe B, and the nucleotide sequences of probe A and probe B are as SEQ ID NO. 1 and SEQ ID respectively. As shown in NO.2; the nanopore electrophysiological signal detection unit contains HEPES, KCl, bilayer lipid membrane or polymer membrane, and DPHP. This Klebsiella pneumoniae RNA extraction reagent unit contains TRIZOL, ethanol, DEPC water/RNase-free water, and RNase inhibitor.

Among them, probe A and probe B were designed by the inventor team. Since the target 16S rRNA is 932bp long, it is difficult to distinguish the 16S rRNA-probe complex without a probe or a single probe, so the inventor team designed two probes to combine the specific expression of Klebsiella pneumoniae of 16S rRNA. The nucleotide sequences of the two probes are shown in Table 1. The probes A and B were annealed to the stored sample and the formation of the probe 16SrRNA-probe complex was verified using agarose gel electrophoresis (Fig. 2a).

Table 1 Probe sequences

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

Example 2

This embodiment provides a method for distinguishing carbapenem-resistant Klebsiella pneumoniae from carbapenem-susceptible Klebsiella pneumoniae using the device of Embodiment 1.

1. Preparation of Bacterial Extracts

Two sets of K. pneumoniae samples from clinical patients were provided by West China Hospital of Sichuan University. Klebsiella pneumoniae samples were cultured to two different concentrations, the concentration of the first group was 0.5MCF and the concentration of the second group was 4MCF. At the beginning of culture, the final concentration of imipenem used in both groups was 16 mg/L, and the total RNA of Klebsiella pneumoniae was extracted by the TRIZOL method. First, collect 100 μL of bacterial solution. After centrifugation, take out the supernatant (8000g, 4°C, 2 minutes). Precipitate with lysozyme and incubate at 37 °C for 10 min. Klebsiella pneumoniae was lysed, and total RNA was extracted and washed with ethanol. Remove the cap of the centrifuge tube, dry it at room temperature for 5-10 minutes, add DEPC water or dissolve it in RNAs-free water. Add RNase inhibitor to the dissolved solution to a final concentration of 20 U/μL for storage.

2.Detection

1) Anneal probes A and B with the above extracted sample to form a 16S rRNA-probe complex.

2) Place the nanopore and 16S rRNA-probe complex in the nanopore electrophysiological signal detection unit for detection, apply a voltage of 150 millivolts, and detect the electrophysiological signal of the 16S rRNA-probe complex passing through the nanopore.

3) Analyze the detected electrophysiological signals to differentiate between carbapenem-resistant Klebsiella pneumoniae and carbapenem-sensitive Klebsiella pneumoniae.

Example 3

Optimization of bacterial concentration and testing of standard samples

1. Expression and purification of MspA nanopore

The MspA nanopore gene was cloned into the pET-28b plasmid, and the pET-28b plasmid carrying the MspA gene was transferred into the engineering strain BL21 Escherichia coli competent cells. The successfully transferred E. coli was cultured in LB medium at 37°C, and kanamycin was added to 50 μg/ml. When the optical density (600nm) is close to 0.8, 0.8mM IPTG is added to the LB (lysogenic fermentation broth) medium, and the induction temperature is 15°C. After 12 hours of induction, E. coli were collected by centrifugation. After breaking E. coli with an ultrasonic generator, the supernatant was collected and further purified using an anion exchange column (Q-Sepharose) and molecular sieve (Superdex 200 16/90). Purified proteins were analyzed by 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). Purified MspA nanopore protein can be aliquoted and stored at -80°C. The aliquoted specimens remained stable for years, and the nanopores remained structurally intact upon thawing.

2. Nanopore electrophysiological signal detection experiment to determine the optimal sample concentration and optimal bacterial culture time

2.1 Determine the best sample concentration

Nanopore electrophysiological signal detection experiments were performed on two different concentrations of bacterial extract samples in Example 2. The experimental method is:

The experiments were performed in chambers provided by Warner Instrument. Nanopore electrophysiological signal detection experiments were performed at a voltage of 150 millivolts. The conductive buffer solution for the cis and trans sides is a 400mM KCl solution containing 10mM HEPES, pH 7.0. Bilayer lipid membranes (BLM) coated on both sides of 150 μm pores are formed from 1,2-dihydroxyformyl-sn-glycero-3-phosphocholine (DPHPC). Adding MspA to the solution in the cis chamber allows for MspA protein insertion and faster BLM formation. Embedding a single MspA nanohole will lead to an increase in current, corresponding to a conductance of 1.2 nS. After recording current signals inserted into a single MspA nanopore while passing through a Heka EPC-10 patch clamp (HEKA), the sample was added to the cis side.

Two concentrations of K. pneumoniae were used to optimize detection efficiency. In the 0.5MCF sample, the frequency of the target RNA transfer signal in the control group was 0.02±0.02/min (n=3), and the frequency of the target RNA transfer signal in the carbapenem-resistant Klebsiella pneumoniae group was 0.13±0.05/min. minutes (n=3). In the 4MCF sample, the target RNA translocation signal frequency in the control group was 0 per minute (n=3), and the translocation frequency in the carbapenem-resistant Klebsiella pneumoniae group was 0.33±0.07 per minute (n= 3) (Figure 4a). Compared with the 0.5MCF sample, the 4MCF sample can be better detected in the nanopore assay.

2.2. Determine the optimal bacterial culture time

Total RNA extracted from carbapenem-resistant Klebsiella pneumoniae and carbapenem-susceptible Klebsiella pneumoniae was incubated with probe A and probe B, and the incubated RNA was detected through the MspA nanopore respectively. solution (Fig. 4b). The two parameters of the signal obtained by measuring the sample through the nanopore, blocking rate and retention time, were plotted into a scatter plot (Figure 4c). Obvious differences in retention time between different groups can be observed, especially when the blocking rate is 0.6 to 0.6. 0.8, residence time in the range of 100 ms to 400 ms. Therefore, signals within this range are selected as specific signals for diagnosis. After comparing the number of 16S rRNA-probe signals within a given range from blank, control, carbapenem-resistant Klebsiella pneumoniae and carbapenem-susceptible Klebsiella pneumoniae samples, using f =0.1·min ^-1 is used as the target signal transfer frequency threshold to distinguish carbapenem-resistant Klebsiella pneumoniae from carbapenem-susceptible Klebsiella pneumoniae. When it is higher than 0.1·min ^-1, it is judged to be carbapenem-resistant Klebsiella pneumoniae, and when it is lower than 0.1·min ^-1 , it is judged to be carbapenem-susceptible Klebsiella pneumoniae. Further, in order to determine the minimum bacterial culture time required to distinguish carbapenem-resistant Klebsiella pneumoniae from carbapenem-susceptible Klebsiella pneumoniae, samples with different bacterial culture times were detected through the MspA nanopore, Including 2 hours, 4 hours and 8 hours, the experimental results show that 4 hours is the optimal bacterial culture time that takes into account sensitivity and efficiency.

Example 4

Double-blind trial of MspA nanopore detection in clinical samples

Bacteria in blood samples from 20 patients with Klebsiella pneumoniae infection provided by West China Hospital were cultured, and total RNA was extracted and used in a double-blind experiment. Each sample was probed at least three times with the MspA nanopore. After analysis, the number of 16S rRNA probe signals with a blocking rate of 0.6 to 0.8 and a residence time of 100ms to 400ms was collected and compared with the target signal transport frequency threshold fthreshold.

Among the 20 samples, as shown in Table 2, 9 were above the threshold (0.1·min ^-1 ) and were judged to be carbapenem-resistant Klebsiella pneumoniae. As shown in Table 3, the other 11 samples were lower than the threshold 0.1·min ^-1 , and these clinical samples were determined to be carbapenem-susceptible Klebsiella pneumoniae samples (Fig. 5a). Compared with assay results obtained from standard clinical methods (paper disk diffusion method 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 through the nanopore were correct (Figure 5b), and there were two false negative results.

Table 2 Clinical sample information of carbapenem-resistant Klebsiella pneumoniae

Note: The sample ID is the patient ID in the hospital, and the sample number is the corresponding number in the study of this invention.

Table 3 Clinical sample information of carbapenem-susceptible 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 twenty four burden - 17008404-1 11 6 KPC No 17012837-3 12 6 KPC KPC-2 17020362-3 20 6 KPC KPC-2

Note: The sample ID is the patient ID in the hospital, and the sample number is the corresponding number in the study of this invention.

Table 4 Comparison of detection methods for different carbapenem-resistant Klebsiella pneumoniae

The above examples used software Clampfit 10.6 and Origin Pro 8.0 for data analysis. The blocking current is defined as ΔI/I ₀ , where I ₀ is the current of a fully open pore and ΔI is the amplitude of the blocking current caused by the transport molecule. Residence times are collected by the single channel search function of Clampfit10.6. These two parameters were used for quantitative analysis of target 16S rRNA from viable carbapenem-resistant Klebsiella pneumoniae. All data are from 20 minutes of electrophysiological recording, and the experimental group was independently repeated three times.

Summarize

In the present invention, the inventor team designed two DNA molecular probes to specifically bind to the 16S rRNA of Klebsiella pneumoniae with carbapenem resistance, and the 16S rRNA-probe complex translocated through the MspA nanopore. This will result in a dwell time between 100ms and 400ms. Based on the blocking rate and residence time of the specific blocking signal, 16S rRNA in carbapenem-resistant Klebsiella pneumoniae samples can be detected (Figure 6). Through the detection of carbapenem-resistant Klebsiella pneumoniae standard samples and carbapenem-sensitive Klebsiella pneumoniae standard samples, it was confirmed that this method can be used to distinguish carbapenem-resistant Klebsiella pneumoniae standard samples. pneumoniae samples and the bacterial sample culture time is only 4 hours.

In addition, the inventor team used MspA nanopores to measure 20 clinical samples provided by West China Hospital. Among 11 clinical samples of carbapenem-sensitive Klebsiella pneumoniae, 9 samples obtained correct diagnostic results, and 2 samples were detected as false negative; in 9 cases of carbapenem-resistant Klebsiella pneumoniae, Among the B. burgdorferi samples, 9 samples all received correct diagnostic results. The nanopore diagnostic method is 90% accurate. Analysis suggests that RNA degradation during sample storage or transfer is the main cause of 10% of false-negative diagnoses. The transportation process of clinical samples from hospital to laboratory and the time lag between sample processing and nanopore assay increase the possibility of RNA degradation, resulting in reduced amounts of 16S rRNA and specific blocking signals.

In conclusion, the research by the inventors' team confirmed that nanopore single-molecule detection technology can be used to rapidly differentiate carbapenem-resistant Klebsiella pneumoniae. Compared with the disk diffusion method or the PCR method, the two most widely used methods in clinical practice, the nanopore detection method has the advantages of low cost, high efficiency, and easy operation.

The embodiments of the present invention have been described above in conjunction with the accompanying drawings. However, the present invention is not limited to the above-mentioned specific implementations. The above-mentioned specific implementations are only illustrative and not restrictive. Those of ordinary skill in the art will Under the inspiration of the present invention, many forms can be made without departing from the spirit of the present invention and the scope protected by the claims, and these all fall within the protection of the present invention.

Claims (10)

1. A device for distinguishing carbapenem-resistant Klebsiella pneumoniae from carbapenem-susceptible Klebsiella pneumoniae, characterized in that the device includes nanopores, molecular probes, and Klebsiella pneumoniae. Bacterial RNA extraction reagent unit, nanopore electrophysiological signal detection unit; the molecular probes are probe A and probe B, and the nucleotide sequences of probe A and probe B are as SEQ ID NO. 1 and SEQ respectively As shown in ID NO. 2; the nanopore electrophysiological signal detection unit contains HEPES, KCl, bilayer lipid membrane or polymer membrane, and DPHPC. 2. The device of claim 1, wherein the diameter of the nanopore ranges from 1.0 to 1.5 nm. 3. The device of claim 2, wherein the diameter of the nanopore is 1.3 nm. 4. The device of claim 2, wherein the types of nanopores include Mycobacterium smegmatis porin A, alpha hemolysin, silicon nitride or graphene nanopores. 5. The device of claim 1, wherein the Klebsiella pneumoniae RNA extraction reagent unit contains TRIZOL, ethanol, DEPC water/RNase-free water, and RNase inhibitor. 6. A method for distinguishing carbapenem-resistant Klebsiella pneumoniae and carbapenem-sensitive Klebsiella pneumoniae using the device according to any one of claims 1 to 5, characterized in that it includes the following steps: Step 1: In the Klebsiella pneumoniae RNA extraction reagent unit of the device, use TRIZOL to extract the total RNA of Klebsiella pneumoniae, then wash it with ethanol, add DEPC water or RNase-free water to dissolve, and then add RNase inhibitor to store; Step 2: anneal probe A and probe B of the device with the sample stored in step 1 to form a 16S rRNA-probe complex; Step 3: Place the nanopore of the device and the 16S rRNA-probe complex in step 2 into the nanopore electrophysiological signal detection unit of the device, and conduct the electrophysiological signal of the 16S rRNA-probe complex through the nanopore. detection; Step 4: Analyze the detected electrophysiological signals to differentiate between carbapenem-resistant Klebsiella pneumoniae and carbapenem-susceptible Klebsiella pneumoniae. 7. The method of claim 6, wherein the voltage condition for electrophysiological signal detection in step 3 is 150 millivolts. 8. The method of claim 6, wherein the nanopores of the device are inserted into the bilayer lipid membrane or polymer membrane of the device. 9. The method of claim 6, wherein in step 4, the transport signal in the nanopore is analyzed, and a transport signal with a blocking rate of 0.6 to 0.8 and a residence time in the range of 100 milliseconds to 400 milliseconds is selected as the specific signal. 10. The method of claim 9, wherein f=0.1·min ^-1 is further used as the target signal transfer frequency threshold to distinguish carbapenem-resistant Klebsiella pneumoniae from carbapenem-sensitive Klebsiella pneumoniae. Klebsiella pneumoniae.