CN116814818A - Device and method for distinguishing antibiotic-resistant klebsiella pneumoniae - Google Patents
Device and method for distinguishing antibiotic-resistant klebsiella pneumoniae Download PDFInfo
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Abstract
The invention belongs to the technical field of bioengineering, and particularly relates to a device and a 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 probes are a probe A and a probe B; the nanopore electrophysiological signal detection unit contains HEPES, KCl, a bilayer lipid membrane or a macromolecule membrane and DPHPC.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a device and a method for distinguishing antibiotic-resistant klebsiella pneumoniae.
Background
Klebsiella pneumoniae is one of the most serious opportunistic pathogens in clinical infections, and is commonly present in the intestinal tract of humans and animals, and can cause serious clinical consequences including central nervous system infections, abdominal infections, and the like. The current use of antibacterial agents is a method of treating klebsiella pneumoniae infection. However, the widespread use of broad-spectrum antibacterial drugs has led to increasingly severe bacterial resistance, which in turn has led to prolonged and failed clinical treatments. Carbapenem-resistant klebsiella pneumoniae (CRKP) is a special type of klebsiella pneumoniae that is particularly resistant to carbapenem-resistant antibiotics, which have been referred to as the "last line of defense against bacteria in humans". Therefore, a broad-spectrum antibiotic capable of treating general drug-resistant klebsiella pneumoniae is not necessarily effective against carbapenem-resistant klebsiella pneumoniae. Literature data, the clinical application essence of carbapenems, discloses that carbapenem-resistant klebsiella pneumoniae can cause carbapenem resistance through the generation of beta-lactamase, the change of porin and the increase of efflux pump activity.
Based on this, accurate and rapid differentiation of the bacterial species of a patient's infection is very important to help select antibacterial drugs for subsequent treatment, since a physician can be helped to select appropriate species of antibacterial drugs after specifying the type of bacterial resistance, avoiding antibiotic abuse, shortening the treatment cycle, improving prognosis. Currently, bacterial drug resistance phenotype detection, beta-lactamase detection and drug resistance gene detection are the primary means of methods for drug resistance detection. However, detection of bacterial drug resistance phenotypes requires a long time to culture klebsiella pneumoniae and is therefore often time consuming; the beta-lactamase detection has high speed, but the detection range is relatively small, and only a narrow concentration interval can be detected; while drug-resistant gene detection, while highly accurate, is very expensive and time-consuming.
In view of the foregoing, there is a need for a more optimal and efficient device and method for distinguishing whether bacteria are resistant to drugs.
Disclosure of Invention
In view of the above, the present invention aims to provide a device and a method for differentiating antibiotic-resistant klebsiella pneumoniae, which specifically adopts the following technical scheme.
A device for distinguishing carbapenem-resistant klebsiella pneumoniae (CRKP) from carbapenem-sensitive klebsiella pneumoniae (CSKP), the device comprising a nanopore, a molecular probe, a klebsiella pneumoniae RNA extraction reagent unit, and a nanopore electrophysiological signal detection unit; the molecular probes are probes A and B, and the nucleotide sequences of the probes A and B are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2; the nanopore electrophysiological signal detection unit contains HEPES, KCl, a bilayer lipid membrane or a macromolecule membrane and DPHPC.
Further, the diameter of the nanopore ranges from 1.0 to 1.5nm.
Further, the diameter of the nanopore is 1.3nm.
Further, the class of nanopores includes 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 an RNase inhibitor.
The method for distinguishing the carbapenem-resistant klebsiella pneumoniae from the carbapenem-sensitive klebsiella pneumoniae by using the device comprises the following steps:
step 1: extracting total RNA of Klebsiella pneumoniae by TRIZOL in a Klebsiella pneumoniae RNA extraction reagent unit of the device, washing by ethanol, adding DEPC water or RNase-free water for dissolution, and then adding an RNase inhibitor for storage;
step 2: annealing probe a and probe B of the device to the sample stored in step 1 to form a 16S rRNA-probe complex;
step 3: placing the nanopore of the device and the 16S rRNA-probe complex of the step 2 in a nanopore electrophysiological signal detection unit of the device, and detecting the electrophysiological signal of the 16S rRNA-probe complex passing through the nanopore;
step 4: the detected electrophysiological signals are analyzed to distinguish between carbapenem-resistant klebsiella pneumoniae and carbapenem-sensitive klebsiella pneumoniae.
Further, the voltage condition for the electrophysiological signal detection in step 3 is 150 millivolts.
Further, the nanopore of the device is inserted into a bilayer lipid membrane or a polymeric membrane of the device.
Further, in step 4, the transport signal in the nanopore is analyzed, and a transport signal with an occlusion rate of 0.6 to 0.8 and a residence time ranging from 100ms to 400ms is selected as the specific signal.
Further, f=0.1·min was used -1 As a target signal transfer frequency threshold, the carbapenem-resistant klebsiella pneumoniae and the carbapenem-sensitive klebsiella pneumoniae are distinguished.
Beneficial technical effects
The invention provides a novel, efficient and quick device and method for distinguishing drug-resistant klebsiella pneumoniae based on nanopores, which realize distinguishing carbapenem-resistant klebsiella pneumoniae from carbapenem-sensitive klebsiella pneumoniae on a single molecular level. As the 16S rRNA species identification is a common method in microbiome research and has species specificity, the detection scheme provided by the invention can be also suitable for identifying other bacteria, and can realize the differentiation and identification of various drug-resistant bacteria by combining a control culture experiment containing an antibiotic environment/an antibiotic-free environment and specific electric signal identification.
Compared with the conventional paper sheet diffusion method and the PCR method, the detection method provided by the invention has the advantages of high sensitivity, real-time operation, low cost and less time consumption, and the accuracy is 90% when the culture time of the verified bacteria is only 4 hours.
The device provided by the invention comprises the whole set of necessary reagents and specific probes for distinguishing the carbapenem-resistant klebsiella pneumoniae from the carbapenem-sensitive klebsiella pneumoniae, and is convenient to operate and high in accuracy of detection results. Therefore, the method has great application value in the aspect of clinical rapid detection.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It will be apparent to those of ordinary skill in the art that the drawings in the following description are of some embodiments of the invention and that other drawings may be derived from these drawings without inventive faculty.
Fig. 1 is a single-channel recording setup example of nanopore structure and nanopore measurement (a is a schematic diagram of MspA nanopore structure, b is a schematic diagram of MspA nanopore signal transport to be measured);
FIG. 2 is a graph showing the electrical signals of the 16S rRNA-probe complex formed by the probe A and the probe B and the nanopore thereof (a is a schematic diagram showing the formation of the 16S rRNA-probe complex by the probe A and the probe B; B is a graph showing the agarose 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 transport signal of the single-stranded DNA);
FIG. 3 is a translocation electrical signal for a probe set;
FIG. 4 is a scatter plot of signals recorded in a single channel for distinguishing between carbapenem-resistant and carbapenem-sensitive Klebsiella pneumoniae (a is translocation frequency of carbapenem-resistant Klebsiella group, b is total RNA of two Klebsiella pneumoniae bacteria after incubation with probe A, B and then detected by MspA nanopores, c is obstruction rate and residence time of the signals measured by the nanopores;
FIG. 5 is an example of evaluation of double-blind testing and measurement accuracy of clinical samples (a is a threshold value of the transit frequency of target signals for two Klebsiella pneumoniae tests, b is a statistical diagram of the correctness of the results of the test samples);
FIG. 6 is a detection flow chart and total time cost example;
FIG. 7 is an example of a scheme for nanopore detection of carbapenem-resistant Klebsiella pneumoniae.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Herein, "and/or" includes any and all combinations of one or more of the associated listed items.
Herein, "plurality" means two or more, i.e., it includes two, three, four, five, etc.
It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.
As used in this specification, the term "about" is typically expressed as +/-5% of the value, more typically +/-4% of the value, more typically +/-3% of the value, more typically +/-2% of the value, even more typically +/-1% of the value, and even more typically +/-0.5% of the value.
In this specification, certain embodiments may be disclosed in a format that is within a certain range. It should be appreciated that such a description of "within a certain range" is merely for convenience and brevity and should not be construed as a inflexible limitation on the disclosed ranges. The description of the range should therefore be taken as having specifically disclosed all possible sub-ranges and the ranges thereinAn independent numerical value within. For example, a rangeThe description of (c) should be taken as having specifically disclosed sub-ranges 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 such ranges, e.g., 1,2,3,4,5, and 6. The above rule applies regardless of the breadth of the range.
Klebsiella pneumoniae resistant to carbapenems has rapidly become popular worldwide over the last decades, and presents a great challenge to current clinical practice. The rapid detection of carbapenem-resistant klebsiella pneumoniae can reduce improper antimicrobial treatment and save lives. The traditional carbapenem-resistant klebsiella pneumoniae detection method is very time-consuming, and the PCR and other sequencing methods are too expensive and have higher technical requirements, so that the clinical requirements are difficult to meet. Nanopore detection has the advantages of high sensitivity, real-time operation and low cost, and has been applied to screening of disease biomarkers. In this study, the growth of bacteria was reflected by detecting the amount of 16S rRNA in the nucleic acid extract after a short-term culture of the bacteria and the antibiotic imipenem, thereby distinguishing carbapenem-sensitive Klebsiella pneumoniae from carbapenem-resistant Klebsiella pneumoniae. The specific signal generated after the probe is combined with the 16S rRNA can be recorded by utilizing the nanopore, so that the ultrasensitive and rapid quantitative detection of the 16S rRNA is completed. The invention proves that the nano-pore detection method can distinguish the carbapenem-resistant klebsiella pneumoniae from the carbapenem-sensitive klebsiella pneumoniae only by 4 hours of culture time. The time cost of the method is about 5% of that of the paper sheet diffusion method, and the accuracy similar to that of the paper sheet diffusion method is achieved.
In particular, nanopore sensing technology facilitates its wide application in third generation DNA single molecule sequencing. The nano-sized 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 solution, the charged detection species in the system is driven through the aperture to another chamber. The patch clamp sensor detects a current change signal of the nanopore. Different molecules transported through the nanopore can cause corresponding current blocking signals, and qualitative and quantitative analysis of the detected molecules can be achieved through specific transport signals and transport frequencies. The nanopore sensing technology has the advantages of no marking, rapidness, real-time operation and high sensitivity, and only needs a small amount of sample. Thus, these features are applicable to the detection of biomarkers.
Specifically, taking Mycobacterium smegmatis (Mycobacterium smegmatis) porin A (MspA) as an example, which is one of the outer membrane proteins of Mycobacteria, has a length of 9.6nm and a diameter of 1.3nm as shown in FIG. 1. The nanopore is efficiently incorporated into a bilayer lipid membrane and allows single stranded nucleic acid to be transported through the pore, which is well suited for nanopore sequencing due to its short, narrow channel. Of course, other common nanopores, such as alpha hemolysin, silicon nitride, and graphene nanopores, in addition to MspA nanopores, may be suitable for nanopore sequencing. In addition, a polymer membrane may be applied to the present invention in addition to a bilayer lipid membrane.
Specifically, 16S rRNA present in all bacteria is a component of the 30S subunit in the prokaryotic ribosome, whose 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 of different bacterial differences. 16S rRNA has proven to be a reliable genetic marker, is commonly used in bacterial classification and has been documented to identify clinical pathogens.
Material
Reagent 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 (5 KU), pET-28b plasmid and all DNA were supplied by Sangon Biotech, 1, 2-diacetyl-sn-glycero-3-phosphorylcholine (DPHPC) from Avanti, primeSTAR HSDNA polymerase from TaKaRa, imipenem (CAS#: 64221-86-9) from MSD.
Clinical specimen:
blood samples of 2 cases of klebsiella pneumoniae infected patients were provided by the department of clinical laboratory at the department of western medicine, university of si. The study according to the invention was carried out according to the recommendations related to human ethical examination and to the declaration of helsinki WMA in the chinese national biomedical study. The protocol was approved by the biomedical ethics committee of the university of Sichuan Huaxi hospital. The inventive study used the remaining specimens, i.e., specimen residues for routine clinical care or analysis, which were discarded and met the criteria for giving up informed consent. The biomedical ethics committee of the Huaxi hospital at the university of Sichuan gave exemptions of informed consent.
Example 1
The embodiment provides a device for distinguishing carbapenem-resistant klebsiella pneumoniae from carbapenem-sensitive klebsiella pneumoniae, which comprises a nanopore, a molecular probe, a klebsiella pneumoniae RNA extraction reagent unit and a nanopore electrophysiological signal detection unit; the molecular probes are probes A and B, and the nucleotide sequences of the probes A and B are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2; the nanopore electrophysiological signal detection unit contains HEPES, KCl, a bilayer lipid membrane or a macromolecule membrane and DPHP. The Klebsiella pneumoniae RNA extraction reagent unit comprises TRIZOL, ethanol, DEPC water/RNase-free water, and an RNase inhibitor.
Wherein, the probe A and the probe B are designed by the group of the inventors. 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, and thus the inventors have devised 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. The probes a and B were annealed to the stored samples and the formation of probe 16S rRNA-probe complexes was verified using agarose gel electrophoresis (fig. 2 a).
TABLE 1 probe sequences
Further, agarose gel electrophoresis results showed that 16S rRNA-probe complex was successfully obtained (FIG. 2 b). The retention time of the transport signal of the 16S rRNA-probe complex in the sample of Klebsiella pneumoniae resistant to carbapenems was in the range of 100-400ms, the peak value was 196.98ms, the retention time of single-stranded DNA transport was in the range of 0-100ms, and the peak value was 12.03ms (FIGS. 2c and 2 d). The residence time of probe A and probe B was in the range of 0-70ms (FIG. 3). These results indicate that the long residence time signal is caused by the 16S rRNA-probe complex.
Example 2
This example provides a method for differentiating between carbapenem-resistant klebsiella pneumoniae and carbapenem-sensitive klebsiella pneumoniae using the apparatus of example 1.
1. Preparation of bacterial extracts
Two sets of klebsiella pneumoniae samples from clinical patients were provided by the university of four-channel department of western medicine. Samples of klebsiella pneumoniae were incubated to two different concentrations, the first set at a concentration of 0.5MCF and the second set at a concentration of 4MCF. At the beginning of the culture, the final concentration of imipenem used in both groups was 16mg/L, and total RNA of Klebsiella pneumoniae was extracted by TRIZOL method. First, 100. Mu.L of the bacterial solution was collected. After centrifugation, the supernatant (8000 g,4 ℃ C., 2 minutes) was removed. The pellet was precipitated with lysozyme and incubated at 37℃for 10 minutes. Klebsiella pneumoniae was lysed, total RNA was extracted and washed with ethanol. The centrifuge tube cap was removed, dried at room temperature for 5-10min, and DEPC water was added or dissolved in RNAs-free water. RNase inhibitor was added to the dissolved solution to a final concentration of 20U/. Mu.L for storage.
2. Detection of
1) Probes A and B were annealed to the extracted samples to form 16S rRNA-probe complexes.
2) The nanopore and the 16S rRNA-probe complex are placed in a nanopore electrophysiological signal detection unit for detection, a voltage of 150 mV is applied, and the 16S rRNA-probe complex passes through the nanopore electrophysiological signal for detection.
3) The detected electrophysiological signals are analyzed to distinguish between carbapenem-resistant klebsiella pneumoniae and carbapenem-sensitive klebsiella pneumoniae.
Example 3
Optimization of bacterial concentration and Standard sample testing
Expression and purification of MspA nanopores
Cloning the MspA nanopore gene into a pET-28b plasmid, and transferring the pET-28b plasmid carrying the MspA gene into engineering bacterium BL21 escherichia coli competent cells. Successfully transferred E.coli was cultured in LB medium at 37℃and kanamycin was added to 50. Mu.g/ml. When the optical density (600 nm) approaches 0.8, 0.8mM IPTG was added to LB (lysogenic broth) medium and the induction temperature was 15 ℃. After 12 hours of induction, E.coli was collected by centrifugation. The supernatant was collected after disruption of E.coli with an ultrasonic generator, and further purified with an anion exchange column (Q-Sepharose) and a molecular sieve (Superdex 200 16/90). Purified proteins were analyzed by 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). Purified MspA nanopore proteins can be split and stored at-80 ℃. The dispensed samples can remain stable for many years and the nanopores remain structurally intact when thawed.
2. Nanopore electrophysiological signal detection experiments determine the optimal sample concentration and optimal bacterial culture time
2.1 determination of preferred sample concentration
Nanopore electrophysiological signal detection experiments were performed on two different concentration bacterial extract samples in example 2. The experimental method comprises the following steps:
the experiment was performed in a chamber provided by Warner Instrument. The nanopore electrophysiological signal detection experiment was performed at a voltage of 150 millivolts. The cis-and trans-side conductive buffer solutions were 400mM KCl solution containing 10mM HEPES, pH 7.0. Double-layered lipid films (BLM) smeared on both sides of 150 μm wells were formed from 1, 2-dihydroxyformyl-sn-glycerol-3-phosphorylcholine (DPHPC). Addition of MspA to the solution in the cis chamber allows MspA protein insertion and faster formation of BLM. Single MspA nanopore embedding will result in an increase in current, corresponding to a conductance of 1.2nS. After recording the current signal inserted into a single MspA nanopore when passing through Heka EPC-10 patch clamp (Heka), the sample was added to the cis side.
Two concentrations of klebsiella pneumoniae were used to optimize detection efficiency. In the sample of 0.5MCF, the frequency of the target RNA transfer signal of the control group was 0.02±0.02/min (n=3), and the frequency of the target RNA transfer signal of the carbapenem-resistant klebsiella pneumoniae group was 0.13±0.05/min (n=3). In the 4MCF sample, the target RNA transfer signal frequency of the control group was 0 (n=3) per minute, and the translocation frequency of the carbapenem-resistant klebsiella pneumoniae group was 0.33±0.07 (n=3) per minute (fig. 4 a). The 4MCF sample was better detected in the nanopore assay than the 0.5MCF sample.
2.2. Determination of optimal bacterial culture time
Total RNA extracted from carbapenem-resistant Klebsiella pneumoniae and carbapenem-sensitive Klebsiella pneumoniae were incubated with probe A and probe B, and the incubated solutions were detected through MspA nanopores, respectively (FIG. 4B). Two parameters of the signal obtained by measuring the sample through the nanopore, the blocking rate and the residence time are plotted in a scatter plot (fig. 4 c), and a significant difference in residence time between the different groups can be observed, especially in the range of blocking rates 0.6 to 0.8, residence times 100ms to 400 ms. Thus, signals within this range are selected as diagnostic specific signals. After comparing the number of 16S rRNA-probe signals within a given range from a blank, control, carbapenem-resistant klebsiella pneumoniae and carbapenem-sensitive klebsiella pneumoniae sample, f=0.1·min was used -1 As a target signal transfer frequency threshold, the carbapenem-resistant klebsiella pneumoniae and the carbapenem-sensitive klebsiella pneumoniae are distinguished. Higher than 0.1 min -1 The carbapenem-resistant klebsiella pneumoniae is less than 0.1 min -1 And judging that the carbapenem-sensitive klebsiella pneumoniae is the carbapenem-sensitive klebsiella pneumoniae. Further, in order to determine the minimum bacterial culture time required to differentiate between carbapenem-resistant klebsiella pneumoniae and carbapenem-sensitive klebsiella pneumoniae, samples with different bacterial culture times, including 2 hours, 4 hours and 8 hours, were examined through MspA nanopores, and experimental results showed that 4 hours was the optimal bacterial culture time for both sensitivity and efficiency.
Example 4
Double-blind test for detecting clinical samples by MspA nano-pores
Bacteria from blood samples of 20 klebsiella pneumoniae infected patients supplied from the national institute of advanced western medicine were cultured, total RNA was extracted and used for double-blind experiments. Each sample was tested at least three times with MspA nanopores. After analysis, the number of 16S rRNA probe signals with an occlusion rate of 0.6 to 0.8 and a residence time of 100ms to 400ms were collected and compared with the target signal transit frequency threshold fthreshold.
Of the 20 samples, 9 were above the threshold (0.1 min, as shown in Table 2 -1 ) And is judged to be resistant to carbapenem pneumonia klebsiella. As shown in Table 3, the other 11 samples were below the threshold 0.1 min -1 These clinical samples were judged as carbapenem-sensitive klebsiella pneumoniae samples (fig. 5 a). The nanopore assay of the present invention has the advantage of low cost and short time consumption compared to assay results obtained from standard clinical methods (paper disc diffusion or PCR) (table 4). The results of 18 samples measured by nanopores were correct (fig. 5 b), with two false negative results.
TABLE 2 clinical sample information of carbapenem-resistant Klebsiella pneumoniae
Note that: the sample ID is the patient ID in the hospital and the sample number is the corresponding number in the study of the invention.
TABLE 3 clinical sample information of carbapenem-sensitive Klebsiella pneumoniae
| Sample ID | Sample #) | SCIM(mm) | Drug resistance gene | Drug resistance gene |
| 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 | Negative pole | - |
| 17008404-1 | 11 | 6 | KPC | Without any provision for |
| 17012837-3 | 12 | 6 | KPC | KPC-2 |
| 17020362-3 | 20 | 6 | KPC | KPC-2 |
Note that: the sample ID is the patient ID in the hospital and the sample number is the corresponding number in the study of the invention.
TABLE 4 comparison of detection methods of different carbapenem-resistant Klebsiella pneumoniae
The above examples used software Clampfit10.6 and Origin Pro 8.0 for data analysis. The blocking current is defined as DeltaI/I 0 Wherein I 0 Is the current of a well open pore and Δi is the amplitude of the blocking current caused by the transport molecule. The residence time was collected by the single channel search function of clampfit10.6. These two parameters were used to quantitatively analyze the target 16S rRNA from surviving carbapenem-resistant klebsiella pneumoniae. All data were from 20 min electrophysiological recordings, and the experimental group was independently repeated 3 times.
Summary
In the present invention, the team of inventors designed two DNA molecular probes to specifically bind to carbapenem-resistant klebsiella pneumoniae 16S rRNA,16S rRNA-probe complex translocation through MspA nanopores would result in residence times between 100ms and 400 ms. Based on the blocking rate and residence time of the specific blocking signal, 16S rRNA in Klebsiella pneumoniae samples with carbapenem resistance could be detected (FIG. 6). Through the detection of the carbapenem-resistant klebsiella pneumoniae standard sample and the carbapenem-sensitive klebsiella pneumoniae standard sample, the method is proved to be applicable to distinguishing carbapenem-resistant from carbapenem-sensitive klebsiella pneumoniae samples, and the culture time of the bacterial samples is only 4 hours.
In addition, the inventors team measured 20 clinical specimens provided by the Huaxi hospital using MspA nanopores. Among 11 cases of carbapenem-sensitive klebsiella pneumoniae clinical samples, 9 cases of samples obtain correct diagnosis results, and 2 cases of samples are detected as false negative; among 9 carbapenem-resistant klebsiella pneumoniae samples, all 9 samples gave the correct diagnosis results. The accuracy of the nanopore diagnostic method is 90%. Analysis suggests that RNA degradation during sample storage or transfer is the primary cause of 10% false negative diagnosis. The transport of clinical samples from the hospital to the laboratory and the time interval between sample processing and nanopore measurement increases the likelihood of RNA degradation, resulting in a reduced number of 16S rRNA and specific blocking signals.
In summary, the studies of the inventors team confirm that nanopore single molecule detection techniques can be used to rapidly differentiate carbapenem-resistant klebsiella pneumoniae. Compared with two methods which are the most widely used in clinic, namely a paper sheet diffusion method and a PCR method, 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 with reference to the accompanying drawings, but the present invention is not limited to the above-described embodiments, which are merely illustrative and not restrictive, and many forms may be made by those having ordinary skill in the art without departing from the spirit of the present invention and the scope of the claims, which are to be protected by the present invention.
Claims (10)
1. The device for distinguishing the carbapenem-resistant klebsiella pneumoniae from the carbapenem-sensitive klebsiella pneumoniae is characterized by comprising a nanopore, a molecular probe, a klebsiella pneumoniae RNA extraction reagent unit and a nanopore electrophysiological signal detection unit; the molecular probes are probes A and B, and the nucleotide sequences of the probes A and B are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2; the nanopore electrophysiological signal detection unit contains HEPES, KCl, a bilayer lipid membrane or a macromolecule membrane and DPHPC.
2. The device of claim 1, wherein the nanopore has a diameter in the range of 1.0-1.5nm.
3. The device of claim 2, wherein the nanopore has a diameter of 1.3nm.
4. The device of claim 2, wherein the class of nanopores comprises mycobacterium smegmatis porin A, alpha hemolysin, silicon nitride, or graphene nanopores.
5. The apparatus of claim 1, wherein the klebsiella pneumoniae RNA extraction reagent unit comprises TRIZOL, ethanol, DEPC water/RNase-free water, and an RNase inhibitor.
6. A method for distinguishing between carbapenem-resistant klebsiella pneumoniae and carbapenem-sensitive klebsiella pneumoniae by using the device of any one of claims 1-5, comprising the steps of:
step 1: extracting total RNA of Klebsiella pneumoniae by TRIZOL in a Klebsiella pneumoniae RNA extraction reagent unit of the device, washing by ethanol, adding DEPC water or RNase-free water for dissolution, and then adding an RNase inhibitor for storage;
step 2: annealing probe a and probe B of the device to the sample stored in step 1 to form a 16S rRNA-probe complex;
step 3: placing the nanopore of the device and the 16S rRNA-probe complex of the step 2 in a nanopore electrophysiological signal detection unit of the device, and detecting the electrophysiological signal of the 16S rRNA-probe complex passing through the nanopore;
step 4: the detected electrophysiological signals are analyzed to distinguish between carbapenem-resistant klebsiella pneumoniae and carbapenem-sensitive 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 nanopore of the device is inserted into a bilayer lipid membrane or a polymeric membrane of the device.
9. The method of claim 6, wherein the transport signal within the nanopore is analyzed in step 4, and a transport signal having an occlusion rate of 0.6 to 0.8 and a residence time in the range of 100ms to 400ms is selected as the specific signal.
10. The method of claim 9, further using f = 0.1-min -1 As a target signal transfer frequency threshold, the carbapenem-resistant klebsiella pneumoniae and the carbapenem-sensitive klebsiella pneumoniae are distinguished.
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