CN112280875B - Method, device and system for rapidly detecting bacterial drug resistance by utilizing nanopores - Google Patents
Method, device and system for rapidly detecting bacterial drug resistance by utilizing nanopores Download PDFInfo
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- CN112280875B CN112280875B CN202010685189.8A CN202010685189A CN112280875B CN 112280875 B CN112280875 B CN 112280875B CN 202010685189 A CN202010685189 A CN 202010685189A CN 112280875 B CN112280875 B CN 112280875B
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Abstract
The invention relates to a method and a device for detecting bacterial drug resistance and application thereof, which are characterized in that a specific signal of a compound generated after a nanopore detection probe is combined with a bacterial biomarker is utilized, and bacterial growth is detected by utilizing quantitative detection of the bacterial biomarker. Compared with the prior art, the invention has high detection sensitivity and high speed, and has potential application value in the aspect of rapid drug resistance detection of clinical microorganisms.
Description
This application claims priority to chinese patent with application number 201910660192.1 and application date 2019, 7, 22, entitled "a rapid detection method, apparatus and system for bacterial resistance using nanopores".
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a method for detecting bacterial drug resistance and application thereof, a 16S rRNA-probe compound and application thereof, a device for detecting carbapenem-resistant klebsiella pneumoniae and a kit.
Background
Klebsiella pneumoniae is one of the most serious opportunistic pathogens in clinical infections, commonly found in the intestines of humans and animals, and can cause serious clinical consequences, including central nervous system infections or abdominal infections, and the like. The use of antibacterial agents is the primary treatment for klebsiella pneumoniae infection; early and correct use of antibacterial agents is critical for the cure of klebsiella pneumoniae infection. However, the widespread use of broad-spectrum antibacterial drugs leads to strong resistance to klebsiella pneumoniae, which leads to prolonged and failed treatments. Carbapenem resistance of klebsiella pneumoniae HS11286 (PMID: 26169555) may be caused by biofilm formation, active antimicrobial efflux and β -lactamase production.
The accurate and rapid diagnosis of the resistance of the antibacterial drug against Klebsiella pneumoniae in infected patients is very important for treatment, because it can help doctors select appropriate kinds of antibacterial drugs, shorten the treatment period, and improve prognosis. Bacterial resistance phenotype detection, beta-lactamase detection and resistance gene detection are the main methods currently used for resistance detection. However, detection of bacterial drug resistance phenotypes requires sufficient time to culture klebsiella pneumoniae, and is often time consuming; the beta-lactamase has high detection speed, but the detection range is relatively smaller, and only a very narrow concentration interval can be detected; drug-resistant gene detection has high accuracy, but it is also expensive and time-consuming.
There is therefore a need to develop a more optimal, efficient method and apparatus for bacterial resistance detection.
Disclosure of Invention
In order to meet clinical demands, the invention provides a bacterial drug resistance detection method based on a nanopore sensing technology, which has the advantages of low time cost, high accuracy, no need of expensive primary equipment and the like.
Specifically, the invention provides a method for detecting bacterial drug resistance, which is characterized in that the method generates a specific signal of a complex after a nanopore detection probe is combined with a bacterial biomarker, and detects bacterial growth by quantitative detection of the bacterial biomarker.
Further, the bacterial biomarker is 16S rRNA.
Further, the bacteria are carbapenem-resistant klebsiella pneumoniae.
Further, the bacteria are one or more of Escherichia coli, klebsiella pneumoniae, klebsiella oxytoca, enterococcus faecalis and enterococcus faecium.
Further, the 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.
Further, the method for detecting bacterial drug resistance comprises the following steps: 1) Extracting target bacterial total RNA; 2) Designing a probe and preparing a 16 SrRNA-probe complex; 3) And detecting the nanopore electrophysiological signal.
Further, step 1) total RNA was extracted at a bacterial concentration of about 2 to 10 MCF.
Further, the cultured bacteria concentration of step 1) is about 4MCF.
Further, step 1) culturing the bacteria for a period of about 1 hour to 8 hours, and extracting total RNA.
Further, step 1) cultures the bacteria for a period of time of about 4 hours.
Further, the bacteria in the step 1) are klebsiella pneumoniae, and imipenem with a final concentration of 16mg/L is added during the culture.
Further, the probes in the step 2) 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.
Further, the 16S rRNA-probe complex of step 2) is formed by annealing the probes A and B and 16S rRNA of Klebsiella pneumoniae.
Further, the nanopore electrophysiological signal detection of step 3) is performed at a voltage of 50-200 millivolts.
Further, step 3) the nanopore electrophysiological signal detection is performed at a voltage of 150 millivolts.
Further, the nanopore is an MspA, alpha hemolysin, silicon nitride or graphene nanopore.
Specifically, the invention also provides a 16S rRNA-probe complex, which is characterized in that the complex is formed by annealing probes A and B and 16S rRNA of Klebsiella pneumoniae, and the nucleotide sequences of the probes A and B are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.
Furthermore, the invention also provides application of the 16S rRNA-probe complex in detection of carbapenem-resistant klebsiella pneumoniae.
Specifically, the invention also provides a device for detecting the carbapenem-resistant klebsiella pneumoniae, which is characterized by comprising a nanopore, a probe, a klebsiella pneumoniae RNA extraction reagent unit and a nanopore electrophysiological signal detection unit.
Further, the 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.
Further, the klebsiella pneumoniae RNA extraction reagent unit contains TRIZOL, ethanol, DEPC water/RNase-free water, and an RNase inhibitor.
Further, the nanopore is an MspA, alpha hemolysin, silicon nitride or graphene nanopore.
Further, the nanopore electrophysiological signal detection cell contains HEPES, KCl, a membrane, and DPHPC.
Further, the membrane is a bilayer lipid membrane or a polymeric membrane.
Specifically, the invention also provides a kit for detecting carbapenem-resistant klebsiella pneumoniae, which is characterized by comprising a nanopore, a probe and an RNA extraction reagent, wherein the probe is a probe A and a probe B, and the nucleotide sequences of the probe A and the probe B are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.
Further, the RNA extraction reagent includes TRIZOL, ethanol, DEPC water/RNase-free water, and an RNase inhibitor.
Further, the nanopore is an MspA, alpha hemolysin, silicon nitride or graphene nanopore.
Specifically, the invention also provides a method for detecting carbapenem-resistant klebsiella pneumoniae by using the device, which is characterized by comprising the following steps:
(1) Extracting total RNA of Klebsiella pneumoniae by TRIZOL in a Klebsiella pneumoniae RNA extraction reagent unit, washing by ethanol, adding DEPC water/RNase-free water for dissolution, and then adding an RNase inhibitor for storage;
(2) Annealing probes a and B to the sample stored in step (1) to form a 16S rRNA-probe complex;
(3) Placing the nanopore and the 16S rRNA-probe complex in the step (2) in a nanopore electrophysiological signal detection unit for detection;
(4) And (3) carrying out data analysis on the detected electrophysiological signals, and carrying out quantitative detection on klebsiella pneumoniae.
Specifically, the invention also provides application of the method in detecting microbial drug resistance.
Further, the microorganism is a bacterium.
Further, the bacteria is klebsiella pneumoniae.
Specifically, in the method of the present invention, we used specific probes to bind to 16S rRNA in klebsiella pneumoniae and record the nucleic acid reading process by nanopore assay. The frequency of the specific signal transported by the target nucleic acid through the nanopore reflects the number of live klebsiella pneumoniae. Thus, residual live Klebsiella pneumoniae of carbapenem can be quantitatively analyzed by this method. Based on the blocking rate and retention time of the specific blocking signal, we could detect 16S rRNA in Klebsiella pneumoniae samples with carbapenem resistance, and the course and time required for the whole detection method are shown in FIG. 6.
Specifically, the invention provides a novel, efficient and rapid detection method based on nanopores, which can distinguish carbapenem-resistant klebsiella pneumoniae from carbapenem-sensitive klebsiella pneumoniae on a single molecular level. These strains identified by MALDI-TOF MS were incubated with imipenem for several hours, respectively. 16S rRNA has high conservation and specificity, can be used as a powerful tool for pathogen detection and identification in gene detection technology, and is therefore selected as a parameter for measuring the amount of active Klebsiella pneumoniae after culturing under antibiotics. In other words, since 16S rRNA-based species identification is the most commonly used method in microbiome research, the detection scheme provided by the present invention is suitable for identifying most bacteria, and can be used for identifying bacterial resistance by combining with a control culture experiment in an antibiotic-containing/antibiotic-free environment.
It will be apparent to those skilled in the art that the application of the present invention is not limited to klebsiella pneumoniae and 16S rRNA, and that other highly conserved and specific biomarkers are equally applicable to the detection methods and devices provided by the present invention, for example: rpoB, sodA, gyrB, groEL, recN, etc. are also bacterial biomarkers that can be used in the invention; the invention can also be used for detecting the growth conditions of bacteria such as escherichia coli, klebsiella oxytoca, enterococcus faecalis, enterococcus faecium and the like, and detecting the drug resistance based on the same
(e.g., using a control culture with/without antibiotic environment). In addition, common nanopores may be suitable for use in the present invention: for example, mspA, alpha hemolysin, silicon nitride, and graphene nanopores.
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 should be apparent that the drawings in the following description are some embodiments of the present invention, experimental data and results, and it is possible for one of ordinary skill in the art to derive other embodiments from the contents of these drawings and perform other experiments to obtain other experimental results without inventive effort.
FIG. 1 is a single channel recording setup of the structure of the nanopore and nanopore assay.
FIG. 2 shows 16S rRNA-probe complexes and nanopore signals thereof.
FIG. 3 is a translocation signal of a probe set.
FIG. 4 shows the signals recorded on a single channel for distinguishing between Klebsiella pneumoniae resistant to carbapenem and Klebsiella pneumoniae sensitive to carbapenem.
Fig. 5 is an evaluation of double blind testing and assay accuracy for clinical samples.
FIG. 6 is a detection flow chart and total time cost.
FIG. 7 is a protocol for nanopore detection of carbapenem-resistant Klebsiella pneumoniae.
The invention has the beneficial effects that:
the method for rapidly detecting the drug resistance of the bacteria can quantitatively detect the amount of 16S rRNA in the bacteria, and judge whether the bacteria are drug resistant or not by the amount of the 16S rRNA in the bacteria;
compared with the conventional paper sheet diffusion method and PCR method, the method for rapidly detecting the bacterial drug resistance has the advantages of high sensitivity, real-time operation, low cost and less time consumption, and the accuracy is 90% because the culture time of the verified bacteria is only 4 hours;
the method for rapidly detecting the bacterial drug resistance has higher accuracy if the reason of RNA degradation in the sample storage or transfer process is eliminated, so that the method has great potential application value in the aspect of clinical rapid drug resistance detection.
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.
Unless otherwise specified, the terms "comprise" and "comprising" and grammatical variants thereof are used to denote "open" or "comprising" languages such that they include recited features but also allow for inclusion of additional non-recited features.
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. Accordingly, the description of a range should be considered to have specifically disclosed all possible sub-ranges and individual numerical values within that range. 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, we distinguished carbapenem-sensitive and carbapenem-resistant Klebsiella pneumoniae by detecting the amount of 16S rRNA in a nucleic acid extract of bacteria after a short-term culture with the antibiotic imipenem to reflect the growth of the bacteria. Specific signals generated after the probes are combined with the 16S rRNA can be recorded by using the MspA nanopore, so that ultrasensitive and rapid quantitative detection of the 16S rRNA can be completed. We prove that the nanopore detection method can distinguish the carbapenem-resistant klebsiella pneumoniae from the carbapenem-sensitive klebsiella pneumoniae only by a culture time of 4 hours. 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. The novel method has potential application value in the aspect of rapid drug resistance detection of clinical microorganisms.
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 using 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 suitable for rapid diagnosis of disease and detection of biomarkers.
Specifically, mycobacterium smegmatis (Mycobacterium smegmatis) porin AMspA) The nanopore protein is one of outer membrane proteins of mycobacteria, with a length of 9.6nm and a diameter of 1.3nm as shown in FIG. 1. Nanopores incorporate efficiently into bilayer lipid membranes and allow transport of single stranded nucleic acids through the pore, mspA nanopores are well suited for nanopore sequencing due to their short, narrow channels. 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. It has proven to be a reliable genetic marker, commonly used in bacterial classification and has been documented to be useful in identifying clinical pathogens. Of course, in addition to using 16S rRNA as a bacterial biomarker, other bacterial biomarkers, such as rpoB, sodA, gyrB, groEL, recN, are equally applicable to the present invention to detect bacterial growth, and to drug resistance detection based thereon (e.g., using control cultures in an antibiotic-containing/antibiotic-free environment).
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 HS DNA 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 one detection of 16S rRNA-Probe Complex
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 unnormas water. RNase inhibitor was added to the dissolved solution to a final concentration of 20U/. Mu.L for storage.
2. Designing probes and incubating with samples
Probes were designed to bind to specific fragments of 16S rRNA so that the inventors team could recognize specific signals for target nucleic acids through nanopores. 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 A and B are shown as SEQ ID NO.1 and SEQ ID NO. 2. 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 (a in fig. 2).
3. Results
The results of agarose gel electrophoresis showed that the 16S rRNA-probe complex (B in FIG. 2) was successfully obtained. The retention time of the transport signal of the 16S rRNA-probe complex in the sample of carbapenem-resistant Klebsiella pneumoniae was in the range of 100 to 400ms, the peak value was 196.98ms, the retention time of single-stranded DNA transport was in the range of 0 to 100ms, and the peak value was 12.03ms (C and D in FIG. 2). 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 two 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 concentrations of bacterial extract samples in example one. 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 (a in fig. 4). 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 (B in FIG. 4). 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 (C in FIG. 4), and the residence time between the different groups can be observedParticularly in the range of 100ms to 400ms for occlusion rates of 0.6 to 0.8. 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 Carbapenem resistance of klebsiella pneumoniae is distinguished as a target signal transit frequency threshold. 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 tested through MspA nanopores, and experimental results showed that 4 hours was the best bacterial culture time for both sensitivity and efficiency.
Example three MspA nanopore double blind test for detecting clinical samples
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 20 samples, 9 were above the threshold (0.1 min, as shown in Table 1 -1 ) And is judged to be resistant to carbapenem pneumonia klebsiella. As shown in Table 2, the other 11 samples were below the threshold 0.1 min -1 These clinical samples were judged as carbapenem-sensitive klebsiella pneumoniae samples (a in fig. 5). 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 3). The results of 18 samples measured by nanopores were correct (B in fig. 5), with two false negative results.
TABLE 1 clinical sample information of carbapenem-sensitive 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 2 clinical sample information of Klebsiella pneumoniae resistant to carbapenems
| 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 3 comparison of the 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 of technical problems and solutions of the invention
The rapid and accurate detection of carbapenem resistance by klebsiella pneumoniae is very important in the clinical treatment process. However, the current detection technology cannot fully meet the clinical requirements. Our aim is to develop a novel detection method for carbapenem resistance of klebsiella pneumoniae based on nanopore sensing technology to solve the problems faced in clinic.
Protein nanopore expression purification and electrophysiological detection techniques have been rapidly developed over the last 20 years, and nucleic acid detection methods based on various types of protein nanopores have become very mature. In the present invention, the team of inventors designed two DNA probes to specifically bind to 16S rRNA,16S rRNA-probe complex translocation of carbapenem-resistant klebsiella pneumoniae 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-resistant klebsiella pneumoniae clinical samples, 9 cases of samples obtain correct diagnosis results, and 2 cases of samples are detected as false negatives; of the 9 carbapenem-sensitive klebsiella pneumoniae samples, all 9 samples gave the correct diagnostic results. The accuracy of the nanopore diagnostic method is 90%. 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.
The above results are experimental studies based on novel nanopore single molecule diagnostic techniques, and further validation studies aimed at improving sensitivity can be developed around the following aspects: optimizing bacterial culture conditions and RNA extraction technology; modification and reconstruction of protein nanopores for 16S rRNA-probe complex detection; a statistical method for processing data is improved by testing with a large number of clinical samples. Stable storage of the dispensed MspA protein at-80 ℃ enables mass production of nanopores for subsequent multiple testing.
In summary, the studies of the group of inventors have demonstrated that the nanopore single molecule detection technique can be used for rapid clinical diagnosis of carbapenem-resistant klebsiella pneumoniae. Compared with the most widely used methods in two clinical diagnoses, namely a paper sheet diffusion method or a PCR method, the nanopore detection method has the advantages of low cost, high efficiency and easy operation. The method can be applied to clinical laboratory diagnosis as a complement to existing diagnostic methods. With the development of nanopore chip technology, multiple clinical sample detection based on nanopore arrays will be further applied to clinical instant diagnosis.
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.
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.
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Claims (12)
1. A method for distinguishing carbapenem-resistant klebsiella pneumoniae from carbapenem-sensitive klebsiella pneumoniae for non-diagnostic purposes, characterized by utilizing a specific signal of a complex generated after a nanopore detection probe is combined with a klebsiella pneumoniae biomarker, and distinguishing carbapenem-resistant klebsiella pneumoniae from carbapenem-sensitive klebsiella pneumoniae by utilizing the detection of the specific signal, comprising the following steps:
1) Extracting total RNA of klebsiella pneumoniae;
2) Designing double probes, and preparing a 16 SrRNA-probe complex, wherein the complex is formed by annealing probes A and B and 16S rRNA of Klebsiella pneumoniae respectively, and the nucleotide sequences of the probes A and B are shown as SEQ ID NO.1 and SEQ ID NO.2 respectively;
3) Detecting a transport signal of the 16 SrRNA-probe complex through a nanopore, the nanopore being MspA;
4) Analyzing the transport signal passing through the MspA nanopore, and selecting the transport signal with the blocking rate of 0.6 to 0.8 and the residence time of 100 milliseconds to 400 milliseconds as a specific signal;
5) Use f=0.1. Min -1 Different Klebsiella pneumoniae are distinguished by being used as target signal transfer frequency threshold value, and the time is higher than 0.1.min -1 The judgment result shows that 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.
2. The method of claim 1, wherein step 1) extracts total RNA when the bacteria are cultured to a concentration of 2 to 10 MCF.
3. The method of claim 2, wherein the cultured bacteria of step 1) are at a concentration of 4MCF.
4. The method according to claim 1, wherein step 1) is performed for a period of time ranging from 1 hour to 8 hours, and total RNA is extracted.
5. The method of claim 4, wherein step 1) is performed for a period of 4 hours.
6. The method according to claim 1, wherein the klebsiella pneumoniae of step 1) is cultivated by adding imipenem at a final concentration of 16 mg/L.
7. The method of claim 1, wherein the 16S rRNA-probe complex of step 2) is formed by annealing of probes a and B and 16S rRNA of klebsiella pneumoniae.
8. The method of claim 1, wherein step 3) the nanopore electrophysiological signal detection is performed at a voltage of 50-200 millivolts.
9. The method of claim 8, wherein step 3) the nanopore electrophysiological signal detection is performed at a voltage of 150 millivolts.
10. The application of the 16S rRNA-probe complex in distinguishing carbapenem-resistant klebsiella pneumoniae from carbapenem-sensitive klebsiella pneumoniae for non-diagnosis purpose is characterized in that the 16S rRNA-probe complex is formed by annealing probes A and B and 16S rRNA of the klebsiella pneumoniae, and the nucleotide sequences of the probes A and B are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2; the 16S rRNA-probe complex generates a transport signal through an MspA nanopore under electric drive; selecting a transport signal with an occlusion rate of 0.6 to 0.8 and a residence time in the range of 100ms to 400ms as a specific signal; use f=0.1. Min -1 Different Klebsiella pneumoniae are distinguished by being used as target signal transfer frequency threshold value, and the time is higher than 0.1.min -1 The judgment result shows that 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.
11. The device for distinguishing the carbapenem-resistant klebsiella pneumoniae from the carbapenem-sensitive klebsiella pneumoniae is characterized by comprising an MspA nanopore, a probe, a klebsiella pneumoniae RNA extraction reagent unit and a nanopore electrophysiological signal detection unit; the 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.
12. The apparatus of claim 11, wherein the klebsiella pneumoniae RNA extraction reagent unit contains TRIZOL, ethanol, DEPC water/RNase-free water, and an RNase inhibitor.
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| CA2986921A1 (en) * | 2015-06-02 | 2016-12-08 | Nanopore Diagnostics, Llc | Nucleic acid detection |
| WO2018093976A1 (en) * | 2016-11-16 | 2018-05-24 | Nanopore Diagnostics, Llc | Analysis of nucleic acids using probe with non-linear tag |
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| EP3190193A1 (en) * | 2010-07-14 | 2017-07-12 | The Curators Of The University Of Missouri | Nanopore-facilitated single molecule detection of nucleic acids |
| CN105372303B (en) * | 2015-09-15 | 2018-10-26 | 西北大学 | A kind of single molecule analysis method of detection methylate DNA |
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