CN112280875A - Method, device and system for rapidly detecting bacterial drug resistance by using nanopore - Google Patents
Method, device and system for rapidly detecting bacterial drug resistance by using nanopore Download PDFInfo
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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 specific signals of a compound generated after a nano-pore detection probe is combined with a bacterial biological mark are utilized, and the bacterial growth is detected by quantitative detection of the bacterial biological mark. 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
The application requests the priority of Chinese patent with application number 201910660192.1, application date of 2019, 7 and 22, and named as 'a method, a device and a system for rapid detection of bacterial drug resistance by using nanopore'.
Technical Field
The invention belongs to the technical field of biological engineering, and particularly relates to a method for detecting bacterial drug resistance and application thereof, a 16S rRNA-probe compound and application thereof, and a device and a kit for detecting carbapenem-resistant Klebsiella pneumoniae.
Background
Klebsiella pneumoniae is one of the most serious opportunistic pathogens in clinical infections, and is usually present in the intestines of humans and animals, and can cause serious clinical consequences, including central nervous system infection or abdominal cavity infection, and the like. The use of antibacterial drugs is the primary treatment for klebsiella pneumoniae infection; early use and proper use of antibacterial drugs are critical to cure klebsiella pneumoniae infection. However, the widespread use of broad spectrum antibacterial drugs results in strong resistance to klebsiella pneumoniae, which leads to prolongation and failure of the treatment. Carbapenem resistance of Klebsiella pneumoniae HS11286 (PMID: 26169555) may be caused by biofilm formation, active antimicrobial efflux and beta-lactamase production.
Accurate and rapid diagnosis of the drug resistance of klebsiella pneumoniae of infected patients is very important for treatment because it can help doctors select appropriate kinds of antibacterial drugs, shorten treatment period, and improve prognosis. Bacterial drug resistance phenotype detection, beta-lactamase detection and drug resistance gene detection are the main methods currently used for drug resistance detection. However, detection of bacterial resistance phenotypes requires sufficient time to culture klebsiella pneumoniae, which is often time consuming; the detection speed of the beta-lactamase is high, but the detection range is relatively small, and only a narrow concentration interval can be detected; the detection of drug-resistant genes has high accuracy, but it is also expensive and time consuming.
Therefore, it is necessary to develop a more optimized and efficient method and device for detecting bacterial drug resistance.
Disclosure of Invention
In order to meet clinical requirements, the invention provides a bacterial drug resistance detection method based on a nanopore sensing technology, and the method has the advantages of low time cost, high accuracy, no need of expensive first-stage equipment and the like.
Specifically, the invention provides a method for detecting bacterial drug resistance, which is characterized in that the method is used for detecting the growth of bacteria by using a nanopore detection probe to generate a specific signal of a complex after being combined with a bacterial biological marker and using quantitative detection of the bacterial biological marker.
Further, the bacterial organism was identified as 16S rRNA.
Further, the bacterium is a carbapenem-resistant Klebsiella pneumoniae.
Further, the bacteria are one or more of escherichia coli, klebsiella pneumoniae, klebsiella oxytoca, enterococcus faecalis, and enterococcus faecium.
Furthermore, 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 total RNA of target bacteria; 2) designing a probe, and preparing a 16 SrRNA-probe complex; 3) detecting a nanopore electrophysiological signal.
Further, when the bacteria are cultured in the step 1) at a concentration of about 2MCF to 10MCF, total RNA is extracted.
Further, the concentration of the cultured bacteria in step 1) is about 4 MCF.
Further, the length of the time for culturing the bacteria in the step 1) is about 1 to 8 hours, and total RNA is extracted.
Further, the length of time for culturing the bacteria in step 1) is about 4 hours.
Further, the bacteria in the step 1) are Klebsiella pneumoniae, and imipenem with the final concentration of 16mg/L is added during 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 probes A and B and 16S rRNA of Klebsiella pneumoniae.
Further, the nanopore electrophysiological signal detection in step 3) is performed at a voltage of 50-200 millivolts.
Further, the nanopore electrophysiological signal detection of step 3) is performed at a voltage of 150 mv.
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.
Further, the invention also provides application of the 16S rRNA-probe compound in detecting the carbapenem-resistant Klebsiella pneumoniae.
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.
Furthermore, 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 unit contains HEPES, KCl, membrane, and DPHPC.
Further, the membrane is a bilayer lipid membrane or a polymer membrane.
Specifically, the invention also provides a kit for detecting the carbapenem-resistant Klebsiella pneumoniae, which is characterized by comprising a nanopore, probes and an RNA extraction reagent, wherein 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 RNA extraction reagent comprises 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 the 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 with ethanol, adding DEPC water/RNase-free water for dissolving, and adding an RNase inhibitor for storage;
(2) forming 16S rRNA-probe complexes by annealing probes A and B to the stored sample of step (1);
(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 carrying out data analysis on the detected electrophysiological signals, and carrying out quantitative detection on the Klebsiella pneumoniae.
Specifically, the invention also provides application of the method in detecting the drug resistance of the microorganism.
Further, the microorganism is a bacterium.
Further, the bacterium is klebsiella pneumoniae.
Specifically, in the methods of the invention, we used specific probes to bind to 16S rRNA in klebsiella pneumoniae and recorded the nucleic acid read process by nanopore assay. The frequency of the specific signal transported by the target nucleic acid through the nanopore reflects the number of viable klebsiella pneumoniae. Therefore, the residual viable Klebsiella pneumoniae of carbapenems can be quantitatively analyzed by the method. Based on the blocking rate and retention time of specific blocking signals, we can detect 16S rRNA in the sample of Klebsiella pneumoniae with carbapenem resistance, and the process and required time of the whole detection method are shown in FIG. 6.
Specifically, the invention provides a novel, efficient and rapid detection method based on a nanopore, which distinguishes carbapenem-resistant Klebsiella pneumoniae and carbapenem-sensitive Klebsiella pneumoniae on a single molecular level. These strains, identified by MALDI-TOF MS, were each incubated with imipenem for several hours. The 16S rRNA has high conservation and specificity and can be used as a powerful tool for detecting and identifying pathogens in a gene detection technology, so that the 16S rRNA is selected as a parameter for measuring the quantity of live Klebsiella pneumoniae after being cultured under antibiotics. In other words, since species identification based on 16S rRNA is the most commonly used method in microbiome research, the detection scheme provided by the present invention is applicable to identification of most bacteria, and identification of bacterial resistance can be achieved by combining with control culture experiments with/without antibiotic 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 biomarkers with high conservation and specificity may be used in the detection methods and devices provided by the present invention, depending on the actual requirements, for example: rpoB, SodA, gyrB, groEL, recN, etc. are also bacterial biomarkers that can be used in the present invention; the invention can also be used for detecting the growth of bacteria such as Escherichia coli, Klebsiella oxytoca, enterococcus faecalis, and enterococcus faecium, and detecting 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 examples, experimental data and results of the present invention, and it is obvious for those skilled in the art to derive other examples and perform other experiments according to the contents of the drawings to obtain other experimental results without any creative effort.
Figure 1 is a single channel recording setup for nanopore architecture and nanopore assays.
FIG. 2 is a 16S rRNA-probe complex and its nanopore signal.
FIG. 3 shows translocation signals of probe sets.
Fig. 4 shows a single channel recording signal for distinguishing carbapenem-resistant klebsiella pneumoniae from carbapenem-sensitive klebsiella pneumoniae.
Fig. 5 is a double-blind test of clinical samples and evaluation of assay accuracy.
FIG. 6 is a detection flow chart and total time cost.
Figure 7 is a protocol procedure 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 have the drug resistance or not by the amount of the 16S rRNA in the bacteria;
compared with the conventional paper diffusion method and PCR method, the method for rapidly detecting the drug resistance of the bacteria has the advantages of high sensitivity, real-time operation, low cost and less time consumption, and the bacteria culture time is only 4 hours and the accuracy is 90 percent by verification;
if the method for rapidly detecting the drug resistance of the bacteria eliminates the reason of RNA degradation in the sample storage or transfer process, the accuracy is higher, 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 clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless otherwise specified, the terms "comprise" and "comprise," as well as grammatical variations thereof, are used to denote "open" or "including" language such that they include the recited features but also allow for the inclusion of additional, non-recited features.
As used in this specification, the term "about" typically means +/-5% of the stated value, more typically +/-4% of the stated value, more typically +/-3% of the stated value, more typically +/-2% of the stated value, even more typically +/-1% of the stated value, and even more typically +/-0.5% of the stated value.
In this specification, certain embodiments may be disclosed in a range of formats. It should be understood that this description of "within a certain range" is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, the range
The description should be read as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, e.g., 1,2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.
The carbapenem-resistant Klebsiella pneumoniae rapidly prevails in the world in recent decades, and poses a great challenge to the clinical practice at present. Rapid detection of carbapenem-resistant klebsiella pneumoniae can reduce inappropriate antibacterial therapy and save lives. The traditional detection method for the carbapenem-resistant Klebsiella pneumoniae is time-consuming, and 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 the carbapenem-sensitive and carbapenem-resistant Klebsiella pneumoniae by measuring the amount of 16S rRNA in nucleic acid extracts from bacteria after short-term culture with the antibiotic imipenem to reflect bacterial growth. Specific signals generated after the probe is combined with 16S rRNA can be recorded by using the MspA nanopore, so that the ultra-sensitive and rapid quantitative detection of the 16S rRNA is completed. We prove that the nanopore 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 percent of that of the paper diffusion method, and the accuracy similar to that of the paper diffusion method is achieved. The new 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 cavity 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 mark, high speed, real-time operation and high sensitivity, and only needs a small amount of samples. Thus, these features are useful for rapid diagnosis of disease and detection of biomarkers.
Specifically, Mycobacterium smegmatis (Mycobacterium smegmatis) porin A: (A), (B), (C), (MspA) The nanopore protein is one of outer membrane proteins of mycobacteria, 9.6nm in length, as shown in fig. 1, and 1.3nm in diameter. Nanopores bind efficiently into bilayer lipid membranes and allow single-stranded nucleic acid transport through the pore, MspA nanopores are well-suited for nanopore sequencing due to their short, narrow channels. Of course, in addition to MspA nanopores, other common nanopores, such as alpha hemolysin, silicon nitride, and graphene nanopores, may be suitable for nanopore sequencing. In addition, except for bilayer lipidsMembranes, polymeric membranes may also be suitable for use in the present invention.
Specifically, 16S rRNA present in all bacteria is a component of the 30S subunit in 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 from bacteria to bacteria. It has been shown to be a reliable genetic marker commonly used in bacterial classification and it 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 suitable for use in the present invention to detect bacterial growth, and resistance detection based thereon (e.g., using antibiotic-containing/antibiotic-free control cultures).
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), isopropanol (purity > 99.0%, CAS #: 67-63-0) and ethanol (purity > 99.0%, CAS #: 64-17-5) were purchased from Sigma-Aldrich. RNase inhibitor (5KU), pET-28b plasmid and all DNA were supplied by Sangon Biotech, 1, 2-diacetyl-sn-glycerol-3-phosphocholine (DPHPC) was purchased from Avanti, 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 western hospital clinical laboratory, university of Sichuan. The present study was conducted in accordance with the recommendations of Chinese national biomedical research relating to human ethical review and the declaration of Helsinki WMA. The protocol was approved by the ethical committee of biomedical science of western hospital, Sichuan university. The inventive study used the remaining specimens, i.e., specimen residues for routine clinical care or analysis, which were discarded and met the criteria of giving up informed consent. The biomedical ethical committee of the western hospital, university of Sichuan, was exempted from the grant of informed consent.
Example one detection of 16S rRNA-Probe complexes
1. Preparation of bacterial extracts
Two groups of klebsiella pneumoniae specimens from clinical patients were provided by western hospital, university of Sichuan. Klebsiella pneumoniae samples were cultured to two different concentrations, the first at 0.5MCF and the second at 4 MCF. At the beginning of the culture, imipenem was used in both groups at a final concentration of 16mg/L, and total RNA of Klebsiella pneumoniae was extracted by the TRIZOL method. First, 100. mu.L of the bacterial solution was collected. After centrifugation, the supernatant was removed (8000g, 4 ℃ C., 2 minutes). Precipitated with lysozyme and incubated at 37 ℃ for 10 min. Klebsiella pneumoniae was lysed and total RNA was extracted and washed with ethanol. Taking off the centrifugal tube cap, drying at room temperature for 5-10min, adding DEPC water or dissolving in rnas-free water. RNase inhibitor was added to the dissolved solution to a final concentration of 20U/. mu.L for storage.
2. Design probes and incubate with samples
Probes were designed to bind to specific fragments of 16S rRNA so that groups of inventors could recognize specific signals about the target nucleic acid through the nanopore. Since the target 16S rRNA is 932bp long, it is difficult to distinguish 16S rRNA-probe complexes without probes or a single probe, so the team of inventors designed two probes to bind the specifically expressed 16S rRNA of klebsiella pneumoniae. The nucleotide sequences of the two probes, namely 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 sample and the formation of the probe 16S rRNA-probe complex was verified by 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 resistant to Klebsiella pneumoniae of carbapenems was in the range of 100-400ms, with a peak of 196.98ms, and the retention time of single-stranded DNA transport was in the range of 0-100ms, with a peak of 12.03ms (C and D in FIG. 2). The residence time of the probe A and the probe B was in the range of 0 to 70ms (FIG. 3). These results indicate that the long retention time signal is caused by the 16S rRNA-probe complex.
Example two optimization of bacterial concentration and Standard sample testing
MspA nanopore expression and purification
The gene of the MspA nanopore is cloned into a pET-28b plasmid, and the pET-28b plasmid carrying the MspA gene is transferred into an engineering bacterium BL21 escherichia coli competent cell. Successfully transferred E.coli were cultured in LB medium and kanamycin was added to 50. mu.g/ml at 37 ℃. When the optical density (600nm) was close to 0.8, 0.8mM IPTG was added to LB (lysogenic fermentation broth) medium at an induction temperature of 15 ℃. After 12 hours of induction, E.coli was collected by centrifugation. The supernatant was collected after disruption of E.coli by a sonicator and further purified by anion exchange column (Q-Sepharose) and molecular sieves (Superdex 20016/90). The purified protein was analyzed by 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). The purified MspA nanoporous protein can be dispensed and stored at-80 ℃. The dispensed samples can remain stable for years and the nanopores remain structurally intact upon thawing.
2. Determination of better sample concentration and optimal bacteria culture time by using nanopore electrophysiological signal detection experiment
2.1 determination of preferred sample concentrations
The nanopore electrophysiological signal detection experiment was performed on two different concentrations of the bacterial extract samples of example one. The experimental method comprises the following steps:
the experiments were performed in the room provided by the Warner Instrument. The nanopore electrophysiological signal detection experiment was performed at a voltage of 150 mv. The conductive buffer solution for cis-side and trans-side was 400mM KCl solution containing 10mM HEPES, pH 7.0. The Bilayer Lipid Membrane (BLM) smeared on both sides of the 150 μm pores was formed from 1, 2-dihydroxyformyl-sn-glycerol-3-phosphocholine (DPHPC). MspA was added to the solution in the cis chamber, allowing MspA protein insertion, BLM formation faster. Single MspA nanopore intercalation will result in an increase in current, corresponding to a conductance of 1.2 nS. The sample was added to the cis side after recording the insertion of a single MspA nanopore current signal while passing through a Heka EPC-10 patch clamp (Heka).
The detection efficiency was optimized using two concentrations of klebsiella pneumoniae. In the sample of 0.5MCF, the target RNA transport signal frequency of the control group was 0.02 ± 0.02/min (n ═ 3), and the target RNA transport signal frequency of the carbapenem-resistant klebsiella pneumoniae group was 0.13 ± 0.05/min (n ═ 3). In the 4MCF sample, the target RNA transport signal frequency in the control group was 0(n ═ 3) per minute, and the translocation frequency in 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 was 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 the sample measurement through the nanopore, the occlusion rate and the residence time, are plotted as a scatter plot (C in fig. 4), and a significant difference in residence time between the different groups can be observed, especially in the range of the occlusion rate of 0.6 to 0.8 and the residence time of 100 to 400 milliseconds. Therefore, signals in this range are selected as specific signals for diagnosis. After comparing the number of 16S rRNA-probe signals in a given range from blank, control, carbapenem-resistant Klebsiella pneumoniae and carbapenem-sensitive Klebsiella pneumoniae samples, f ═ 0.1. min was used-1The threshold of the target signal transport frequency is used for distinguishing the carbapenem resistance of the Klebsiella pneumoniae. In order to determine the minimum bacterial culture time required for distinguishing carbapenem-resistant Klebsiella pneumoniae from carbapenem-sensitive Klebsiella pneumoniae, samples with different bacterial culture times including 2 hours, 4 hours and 8 hours are detected through the MspA nanopore, and experimental results show that 4 hours is the optimal bacterial culture time considering both sensitivity and efficiency.
Example double-blind assay for three MspA nanopore detection clinical samples
Bacteria from blood samples from 20 klebsiella pneumoniae infected patients provided in western china hospital 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 blocking rates of 0.6 to 0.8 and retention times of 100ms to 400ms was collected and compared to the target signal transit frequency threshold value fmhreshold.
Of the 20 samples, 9 were above the threshold (0.1. min), as shown in Table 1-1) And is judged to be carbapenem-resistant Klebsiella pneumoniae. As shown in Table 2, the other 11 samples were below the threshold of 0.1. min-1These clinical specimens were judged as carbapenem-sensitive Klebsiella pneumoniae samples (A in FIG. 5). The nanopore assay method of the present invention has the advantages of low cost and short time consumption compared to the assay results obtained from standard clinical methods (paper disc diffusion or PCR) (table 3). The results of 18 samples assayed by nanopore are correct (B in fig. 5), with two false negative results.
TABLE 1 clinical sample information of carbapenem-sensitive 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 the invention.
TABLE 2 clinical sample information of carbapenem-resistant 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 | Is not provided with |
| 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 the invention.
TABLE 3 comparison of detection methods for different carbapenem-resistant Klebsiella pneumoniae
The data analysis was performed in the above examples using the software campfit 10.6 and Origin Pro 8.0. The blocking current is defined as Δ I/I0In which I0Is the current of a fully open pore and Δ I is the amplitude of the blocking current caused by the transport molecule. The residence time is collected by the single channel search function of campfit 10.6. 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 experimental groups were independently repeated 3 times.
Summary of the invention technical problem and solution
The rapid and accurate detection of the drug resistance of the Klebsiella pneumoniae to the carbapenems is very important in the clinical treatment process. However, the current detection technology cannot fully meet the clinical requirement. Our aim was to develop a novel detection method for the carbapenem resistance of klebsiella pneumoniae based on nanopore sensing technology to solve the problems facing the clinic.
Protein nanopore expression purification and electrophysiological detection techniques have evolved rapidly over the past 20 years, and nucleic acid detection methods based on various types of protein nanopores are well established. In the present invention, the team of inventors designed two DNA probes to specifically bind to the 16S rRNA of klebsiella pneumoniae resistant to carbapenem, translocation of the 16S rRNA-probe complex through the MspA nanopore will result in a residence time between 100ms and 400 ms. From the blocking rate and the retention time of the specific blocking signal, 16S rRNA in the carbapenem-resistant klebsiella pneumoniae sample was detected (fig. 6). Through the detection of the Klebsiella pneumoniae standard sample with carbapenem resistance and the Klebsiella pneumoniae standard sample sensitive to carbapenem, the method can be used for distinguishing the Klebsiella pneumoniae sample with carbapenem resistance and carbapenem sensitivity, and the culture time of the bacterial sample is only 4 hours.
In addition, the inventor team used MspA nanopores to assay 20 clinical specimens provided by western hospital. Of the 11 carbapenem-resistant clinical samples of Klebsiella pneumoniae, 9 samples obtained correct diagnosis results, and 2 samples were detected as false negatives; of the 9 carbapenem-sensitive Klebsiella pneumoniae samples, all 9 samples gave correct diagnosis results. The accuracy of the nanopore diagnostic method was 90%. RNA degradation during sample storage or transfer is the main cause of a 10% false negative diagnosis. The transport process of clinical samples from hospital to laboratory and the time interval between sample handling and nanopore assays increases the likelihood of RNA degradation, resulting in a decrease in the number of 16S rRNA and specific blocking signals.
The above results are experimental studies based on a novel nanopore single molecule diagnostic technique, and further validation studies aimed at improving sensitivity can be developed around several aspects: optimizing bacterial culture conditions and RNA extraction technology; modification and alteration of protein nanopores for 16S rRNA-probe complex detection; a large number of clinical samples were used for testing and to improve statistical methods of data processing. The stable storage of the packaged MspA proteins at-80 ℃ enables the mass production of nanopores for subsequent multiple tests.
In conclusion, research of the inventor team proves that the nanopore single molecule detection technology can be used for rapid clinical diagnosis of carbapenem drug-resistant klebsiella pneumoniae. Compared with the two most widely used methods in clinical diagnosis, namely a paper diffusion method or a PCR method, the nanopore detection method has the advantages of low cost, high efficiency and easiness in operation. The method can be applied to clinical laboratory diagnosis as a supplement to the existing diagnostic methods. With the development of nanopore chip technology, the nanopore array-based multiplex clinical sample detection 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 an … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.
While the present invention has been described with reference to the embodiments shown in the drawings, the present invention is not limited to the embodiments, which are illustrative and not restrictive, and it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention as defined in the appended claims.
<110> Sichuan university Hospital in western China
<120> method, device and system for rapidly detecting bacterial drug resistance by using nanopore
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Claims (10)
1. A method for detecting bacterial drug resistance by detecting the specific signal of a complex generated by binding a nanopore detection probe to a bacterial biomarker and detecting bacterial growth by quantitative detection of the bacterial biomarker.
2. The method of claim 1, wherein the bacterial organism is 16S rRNA.
3. The method according to claim 2, characterized in that it comprises the following steps:
1) extracting total RNA of target bacteria;
2) designing a probe, and preparing a 16 SrRNA-probe complex;
3) detecting a nanopore electrophysiological signal.
4. The method of claim 3, wherein the total RNA is extracted at a concentration of about 2MCF to about 10MCF in step 1) of culturing the bacteria.
5. The method according to claim 3, wherein the probes in step 2) are probes A and B, and the nucleotide sequences of the probes A and B are shown as SEQ ID NO.1 and SEQ ID NO.2 respectively.
6. The method of claim 3, wherein the nanopore electrophysiological signal detection of step 3) is performed at a voltage of 50 to 200 millivolts.
7. A16S rRNA-probe complex, which is formed by annealing probes A and B and 16S rRNA of Klebsiella pneumoniae, wherein the nucleotide sequences of the probes A and B are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.
8. The use of the 16S rRNA-probe complex of claim 7 for detecting carbapenem-resistant klebsiella pneumoniae.
9. The device for detecting the carbapenem-resistant Klebsiella pneumoniae is characterized by comprising a nanopore, a probe, a Klebsiella pneumoniae RNA extraction reagent unit and a nanopore electrophysiological signal detection unit.
10. A kit for detecting carbapenem-resistant Klebsiella pneumoniae is characterized by comprising a nanopore, probes and an RNA extraction reagent, wherein 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.
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| CN116814818A (en) | 2023-09-29 |
| WO2021013041A1 (en) | 2021-01-28 |
| CN112280875B (en) | 2023-07-18 |
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