WO2021013041A1 - 一种利用纳米孔进行细菌耐药性的快速检测方法、装置和系统 - Google Patents

一种利用纳米孔进行细菌耐药性的快速检测方法、装置和系统 Download PDF

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WO2021013041A1
WO2021013041A1 PCT/CN2020/102381 CN2020102381W WO2021013041A1 WO 2021013041 A1 WO2021013041 A1 WO 2021013041A1 CN 2020102381 W CN2020102381 W CN 2020102381W WO 2021013041 A1 WO2021013041 A1 WO 2021013041A1
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klebsiella pneumoniae
probes
nanopore
rrna
seq
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耿佳
魏于全
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四川大学华西医院
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Definitions

  • the invention belongs to the technical field of bioengineering, and specifically relates to a method for detecting bacterial drug resistance and its application, a 16S rRNA-probe complex and its application, and a method for detecting carbapenem-resistant Klebsiella pneumoniae Devices and kits.
  • Klebsiella pneumoniae is one of the most serious opportunistic pathogens in clinical infections. It usually exists in the intestines of humans and animals and can cause serious clinical consequences, including central nervous system infections or abdominal cavity infections.
  • the use of antibacterial drugs is the main treatment method for Klebsiella pneumoniae infection; early use and correct use of antibacterial drugs are the key to the cure of Klebsiella pneumoniae infection.
  • the widespread use of broad-spectrum antibacterial drugs has led to strong resistance to Klebsiella pneumoniae, which leads to prolonged and failed treatment.
  • the carbapenem resistance of Klebsiella pneumoniae HS11286 may be caused by biofilm formation, active antimicrobial efflux, and ⁇ -lactamase production.
  • Bacterial drug resistance phenotype detection, ⁇ -lactamase detection and drug resistance gene detection are the main methods currently used for drug resistance detection.
  • the detection of bacterial resistance phenotypes requires sufficient time to cultivate Klebsiella pneumoniae, which is usually time-consuming; ⁇ -lactamase is fast in detection, but the detection range is relatively small, and can only detect very narrow concentrations Interval; genetic testing for drug resistance has high accuracy, but it is also expensive and time-consuming.
  • the present invention proposes a method for detecting bacterial drug resistance based on nanopore sensing technology, which has the advantages of low time cost, high accuracy, and no need for expensive first-stage equipment.
  • the present invention provides a method for detecting bacterial drug resistance, which is characterized in that the method generates a complex specific signal by using a nanopore detection probe combined with a bacterial biomarker, and using the Quantitative detection of bacterial biomarkers to detect bacterial growth.
  • the bacterial biomarker is 16S rRNA.
  • bacteria are carbapenem-resistant Klebsiella pneumoniae.
  • the bacteria are one or more of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterococcus faecalis and Enterococcus faecium.
  • probes are probes A and B, and the nucleotide sequences of the probes A and B are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.
  • the method for detecting bacterial drug resistance includes the following steps: 1) extracting total RNA of target bacteria; 2) designing probes and preparing 16SrRNA-probe complexes; 3) detecting nanopore electrophysiological signals.
  • step 1) when the concentration of the cultured bacteria is about 2MCF-10MCF, total RNA is extracted.
  • the concentration of the cultured bacteria in step 1) is about 4MCF.
  • step 1) the length of time for culturing the bacteria is about 1 hour to 8 hours, and the total RNA is extracted.
  • step 1) the length of time for culturing bacteria is about 4 hours.
  • the bacterium in step 1) is Klebsiella pneumoniae, and imipenem with a final concentration of 16 mg/L is added during culture.
  • the probes are probes A and B, and the nucleotide sequences of the probes A and B are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.
  • step 2) the 16S rRNA-probe complex is formed by annealing the probes A and B and the 16S rRNA of Klebsiella pneumoniae.
  • step 3 the nanopore electrophysiological signal detection is performed at a voltage of 50-200 mV.
  • step 3 said nanopore electrophysiological signal detection is performed at a voltage of 150 mV.
  • nanopores are MspA, alpha hemolysin, silicon nitride or graphene nanopores.
  • the present invention also provides a 16S rRNA-probe complex, which is characterized in that the complex is formed by annealing the probes A and B and the 16S rRNA of Klebsiella pneumoniae, and the probe A
  • the nucleotide sequences of and B are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.
  • the present invention also provides the application of the 16S rRNA-probe complex in the detection of carbapenem-resistant Klebsiella pneumoniae.
  • the present invention also provides a device for detecting carbapenem-resistant Klebsiella pneumoniae, characterized in that the device includes a nanopore, a probe, a Klebsiella pneumoniae RNA extraction reagent unit, and a nanopore. Hole electrophysiological signal detection unit.
  • probes are probes A and B, and the nucleotide sequences of the probes A and B are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.
  • the Klebsiella pneumoniae RNA extraction reagent unit contains TRIZOL, ethanol, DEPC water/RNase-free water, and an RNase inhibitor.
  • nanopores are MspA, alpha hemolysin, silicon nitride or graphene nanopores.
  • the nanopore electrophysiological signal detection unit contains HEPES, KCl, membrane and DPHPC.
  • the membrane is a double-layer lipid membrane or a polymer membrane.
  • the present invention also provides a kit for detecting carbapenem-resistant Klebsiella pneumoniae, which is characterized in that the kit is composed of a nanopore, a probe and an RNA extraction reagent, and the probe They are probes A and B, and the nucleotide sequences of the probes A and B are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.
  • RNA extraction reagent includes TRIZOL, ethanol, DEPC water/RNase-free water, and RNase inhibitor.
  • nanopores are MspA, alpha hemolysin, silicon nitride or graphene nanopores.
  • the present invention also provides a method for detecting carbapenem-resistant Klebsiella pneumoniae by using the device, which is characterized in that it comprises the following steps:
  • RNA extraction reagent unit extract the total RNA of Klebsiella pneumoniae with TRIZOL, then wash with ethanol, add DEPC water/RNase-free water to dissolve it, and then add RNase inhibitor for storage;
  • step (3) Place the nanopore and the 16S rRNA-probe complex of step (2) in the nanopore electrophysiological signal detection unit for detection;
  • the present invention also provides the application of the method of the present invention in detecting microbial drug resistance.
  • microorganisms are bacteria.
  • the bacterium is Klebsiella pneumoniae.
  • the method of the present invention we use a specific probe to bind to the 16S rRNA in Klebsiella pneumoniae, and record the nucleic acid reading process through a nanopore assay.
  • the frequency of the specific signal transported by the target nucleic acid through the nanopore reflects the number of live Klebsiella pneumoniae. Therefore, this method can be used to quantitatively analyze the remaining live Klebsiella pneumoniae of carbapenem.
  • the blocking rate and residence time of the specific blocking signal we can detect the 16S rRNA in the carbapenem-resistant Klebsiella pneumoniae sample. The process and the required time of the entire detection method are shown in Figure 6.
  • the present invention provides a novel, efficient and rapid detection method based on nanopores, which distinguishes carbapenem-resistant Klebsiella pneumoniae from carbapenem-sensitive Klebsiella pneumoniae at a single molecular level bacteria.
  • These strains identified by MALDI-TOF MS were incubated with imipenem for several hours.
  • 16S rRNA is highly conservative and specific, and can be used as a powerful tool for pathogen detection and identification in genetic testing technology. Therefore, it was selected as a parameter for measuring the amount of live Klebsiella pneumoniae after being cultured under antibiotics.
  • the detection scheme provided by the present invention is suitable for the identification of most bacteria, combined with the control culture experiment of antibiotic-containing environment/antibiotic-free environment , You can realize the identification of bacterial resistance.
  • the application of the present invention is not limited to Klebsiella pneumoniae and 16S rRNA. According to actual needs, other highly conservative and specific biomarkers are also suitable for use in the present invention. Detection methods and devices, such as rpoB, SodA, gyrB, groEL, recN, etc., can also be used for the bacterial biomarkers of the present invention; the present invention can also be used to detect Escherichia coli and Klebsiella oxytoca The growth of bacteria such as Enterococcus faecalis and Enterococcus faecium, and drug resistance testing based on this
  • nanopores can be applied to the present invention: for example, MspA, alpha hemolysin, silicon nitride and graphene nanopores.
  • Figure 1 shows the structure of the nanopore and the single-channel recording setup for nanopore measurement.
  • Figure 2 shows the 16S rRNA-probe complex and its nanopore signal.
  • Figure 3 shows the translocation signal of the probe set.
  • Figure 4 shows the distinction between carbapenem-resistant Klebsiella pneumoniae and carbapenem-sensitive Klebsiella pneumoniae and single-channel recording signals.
  • Figure 5 shows the double-blind testing of clinical samples and the evaluation of measurement accuracy.
  • Figure 6 shows the detection flow chart and total time cost.
  • Figure 7 is the protocol procedure for the nanopore detection of carbapenem-resistant Klebsiella pneumoniae.
  • the method for rapidly detecting bacterial drug resistance of the present invention can quantitatively detect the amount of 16S rRNA in the bacteria, and judge whether the bacteria are drug resistant through the amount of 16S rRNA in the bacteria;
  • the method for rapidly detecting bacterial resistance of the present invention has the advantages of high sensitivity, real-time operation, low cost and less time-consuming. It has been verified that the bacterial culture time only needs 4 hours , The accuracy is 90%;
  • the method for rapid detection of bacterial drug resistance of the present invention has higher accuracy if it eliminates the cause of RNA degradation during sample storage or transfer, and therefore has great potential application value in clinical rapid drug resistance detection .
  • the term "about” is typically expressed as +/-5% of the stated value, more typically +/-4% of the stated value, and more typically ++ of the stated value +/- 3%, more typically +/- 2% of the stated value, even more typically +/- 1% of the stated value, even more typically +/- 0.5% of the stated value.
  • Carbapenem-resistant Klebsiella pneumoniae has spread rapidly around the world in recent decades, which poses a huge challenge to today's clinical practice. Rapid detection of carbapenem-resistant pneumonia Klebsiella can reduce inappropriate antibacterial treatment and save lives. Traditional carbapenem-resistant Klebsiella pneumoniae detection methods are very time-consuming. PCR and other sequencing methods are too expensive and require high technical requirements to meet clinical needs. Nanopore detection has the advantages of high sensitivity, real-time operation and low cost, and has been applied to the screening of disease biomarkers.
  • Nanopore sensing technology helps its wide application in third-generation DNA single molecule sequencing.
  • Nano-sized protein pores are embedded in a phospholipid membrane, which divides the protein pore chamber into two parts (cis and trans).
  • the patch clamp sensor detects the current change signal of the nanopore.
  • Different molecules transmitted through the nanopore can cause corresponding current blocking signals.
  • This nanopore sensing technology has the advantages of label-free, fast, real-time operation and high sensitivity, requiring only a small amount of samples. Therefore, these features are suitable for rapid diagnosis of diseases and detection of biomarkers.
  • MspA Mycobacterium smegmatis porin A
  • MspA nanoporin is one of the outer membrane proteins of Mycobacterium, with a length of 9.6 nm, as shown in FIG. 1, and a diameter of 1.3 nm.
  • the nanopore efficiently binds to the double-layer lipid membrane and allows single-stranded nucleic acid to be transported through the pore. Due to its short and narrow channel, the MspA nanopore is very suitable for nanopore sequencing.
  • other common nanopores such as alpha hemolysin, silicon nitride and graphene nanopores, can all be suitable for nanopore sequencing.
  • polymer membranes can also be applied to the present invention.
  • the 16S rRNA present in all bacteria is a component of the 30S subunit in the prokaryotic ribosome, and its function does not change over time.
  • 16S rRNA can be used to identify bacterial species because it contains a highly conserved region shared by all bacteria and a hypervariable region that differs from different bacteria. It has been proven to be a reliable genetic marker, often used in bacterial classification and there are documents that prove it can be used to identify clinical pathogens.
  • bacterial biomarkers such as rpoB, SodA, gyrB, groEL, recN, are also applicable to the present invention to detect the growth of bacteria, and drug resistance detection based on this (such as , Use a control culture with antibiotics/no antibiotics).
  • 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 (5KU), pET-28b plasmid and all DNA were provided by Sangon Biotech, 1,2-diacetyl-sn-glycero-3-phosphocholine (DPHPC) was purchased from Avanti, PrimeSTAR HS DNA polymerase From TaKaRa, imipenem (CAS#: 64221-86-9) was purchased from MSD.
  • the blood samples of 2 patients with Klebsiella pneumoniae infection were provided by the Laboratory Department of West China Hospital of Sichuan University.
  • the research of the present invention was conducted in accordance with the recommendations of the Chinese National Biomedical Research involving human ethics review and the Helsinki WMA Declaration.
  • the protocol was approved by the Biomedical Ethics Committee of West China Hospital of Sichuan University.
  • the research of the invention uses the remaining specimens, that is, specimen residues used for routine clinical care or analysis. These specimens will be discarded and meet the criteria for giving up informed consent.
  • the Biomedical Ethics Committee of West China Hospital of Sichuan University granted an exemption for informed consent.
  • the Klebsiella pneumoniae samples were cultured to two different concentrations, the concentration of the first group was 0.5 MCF, and the concentration of the second group was 4 MCF.
  • the final concentration of imipenem used in the two groups was 16 mg/L, and the total RNA of Klebsiella pneumoniae was extracted by the TRIZOL method.
  • Klebsiella pneumoniae was lysed, total RNA was extracted and washed with ethanol. Remove the cap of the centrifuge tube, dry it at room temperature for 5-10 minutes, add DEPC water or dissolve it in rnas-free water. The RNase inhibitor was added to the dissolved solution to a final concentration of 20U/ ⁇ L for storage.
  • the probe is designed to bind to specific fragments of 16S rRNA, so that the inventor team can identify specific signals about target nucleic acids through the nanopore. Since the target 16S rRNA is 932bp long, it is difficult to distinguish 16S rRNA-probe complexes without a probe or a single probe. Therefore, the inventor team designed two probes to bind to the specific expression of Klebsiella pneumoniae 16S rRNA.
  • the two probes are probes A and B.
  • the nucleotide sequences of the probes are shown in SEQ ID NO. 1 and SEQ ID NO. 2.
  • the probes A and B were annealed with the stored samples, and agarose gel electrophoresis was used to verify the formation of the probe 16S rRNA-probe complex (A in Figure 2).
  • the results of agarose gel electrophoresis showed that the 16S rRNA-probe complex was successfully obtained (B in Figure 2).
  • the retention time of the transport signal of the 16S rRNA-probe complex is in the range of 100-400ms, with a peak value of 196.98ms, and the retention time of single-stranded DNA transport is 0- In the range of 100ms, the peak value is 12.03ms (C and D in Figure 2).
  • the residence time of probe A and probe B is in the range of 0-70 ms ( Figure 3).
  • the gene of the MspA nanopore was cloned into the pET-28b plasmid, and the pET-28b plasmid carrying the MspA gene was transferred to the engineering bacteria BL21 E. coli competent cells.
  • the successfully transferred Escherichia coli was cultured in LB medium, and kanamycin was added to 50 ⁇ g/ml.
  • the optical density (600nm) is close to 0.8
  • 0.8mM IPTG is added to the LB (lysogenic fermentation broth) medium, and the induction temperature is 15°C. After 12 hours of induction, E. coli was collected by centrifugation.
  • the supernatant was collected after crushing the Escherichia coli with an ultrasonic generator, and further purified with an anion exchange column (Q-Sepharose) and molecular sieve (Superdex 200 16/90).
  • the purified protein was analyzed by 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis).
  • the purified MspA nanoporin can be aliquoted and stored at -80°C. The aliquoted sample can remain stable for many years, and the nanopore remains structurally intact when it is thawed.
  • the nanopore electrophysiological signal detection experiment determines the best sample concentration and the best bacterial culture time
  • the nanopore electrophysiological signal detection experiment was performed on the two different concentrations of bacterial extract samples in Example 1.
  • the experimental method is:
  • the experiment was carried out in a room provided by Warner Instrument.
  • the nanopore electrophysiological signal detection experiment was carried out at a voltage of 150 mV.
  • the conductive buffer solution on the cis side and the trans side is a 400 mM KCl solution containing 10 mM HEPES, pH 7.0.
  • the bilayer lipid membrane (BLM) applied on both sides of the 150 ⁇ m pore is formed of 1,2-dihydroxyformyl-sn-glycero-3-phosphocholine (DPHPC).
  • DPHPC 1,2-dihydroxyformyl-sn-glycero-3-phosphocholine
  • Adding MspA to the solution in the cis chamber allows the MspA protein to be inserted and BLM is formed faster.
  • the insertion of a single MspA nanopore will increase the current, and the corresponding conductance is 1.2nS.
  • HEKA Heka EPC-10 patch clamp
  • 4MCF samples can be better detected in nanopore determination.
  • RNA extracted from carbapenem-resistant Klebsiella pneumoniae and carbapenem-sensitive Klebsiella pneumoniae was incubated with probe A and probe B, and the incubation was detected through MspA nanopores.
  • Solution B in Figure 4
  • the two parameters of the signal measured by the sample through the nanopore, the blocking rate and the residence time are plotted as a scatter diagram (C in Figure 4), and the significant difference in the residence time between different groups can be observed, especially in the blocking
  • the rate is 0.6 to 0.8, and the residence time is within the range of 100 milliseconds to 400 milliseconds. Therefore, the signal in this range is selected as the specific signal for diagnosis.
  • RNA Bacteria in blood samples from 20 Klebsiella pneumoniae infection patients provided by West China Hospital were cultured, and total RNA was extracted and used in double-blind experiments. Each sample was tested at least three times with MspA nanopores. After analysis, the number of 16S rRNA probe signals with a blocking rate of 0.6 to 0.8 and a residence time of 100 ms to 400 ms was collected and compared with the target signal transfer frequency threshold f threshold.
  • the nanopore measurement method of the present invention has the advantages of low cost and short time-consuming (Table 3). The results of 18 samples measured by the nanopore are correct (B in Figure 5), and two of them are false negative results.
  • sample ID is the patient ID in the hospital, and the sample number is the corresponding number in the study of the present invention.
  • sample ID is the patient ID in the hospital, and the sample number is the corresponding number in the study of the present invention.
  • the above embodiments use software Clampfit 10.6 and Origin Pro 8.0 for data analysis.
  • the blocking current is defined as ⁇ I/I 0 , where I 0 is the current of a fully opened pore, and ⁇ I is the amplitude of the blocking current caused by the transport molecule.
  • the retention time is collected by the single-channel search function of Clampfit 10.6. These two parameters were used to quantitatively analyze the target 16S rRNA from surviving carbapenem-resistant Klebsiella pneumoniae. All data are from 20-minute electrophysiological records, and the experimental group repeated 3 times independently.
  • the inventor team used the MspA nanopore to measure 20 clinical samples provided by West China Hospital.
  • 9 samples were correctly diagnosed, and 2 samples were tested as false negatives; in 9 carbapenem-sensitive pneumonias Among the Klebsiella samples, all 9 samples received correct diagnosis results.
  • the accuracy of the nanopore diagnosis method is 90%.
  • RNA degradation during sample storage or transfer is the main cause of 10% false negative diagnosis.
  • the transportation of clinical samples from the hospital to the laboratory and the time interval between sample processing and nanopore assays increase the possibility of RNA degradation, resulting in a decrease in the number of 16S rRNA and specific blocking signals.
  • the inventor’s team’s research confirmed that the nanopore single-molecule detection technology can be used for rapid clinical diagnosis of carbapenem-resistant Klebsiella pneumoniae.
  • the nanopore detection method Compared with the paper diffusion method or PCR method, the two most widely used methods in clinical diagnosis, the nanopore detection method has the advantages of low cost, high efficiency and easy operation.
  • the method can be used in clinical laboratory diagnosis as a supplement to the existing diagnosis method.
  • multiple clinical sample detection based on nanopore arrays will be further applied to clinical immediate diagnosis.

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Abstract

一种检测细菌耐药性的方法、装置及其应用，其特征在于，通过利用纳米孔检测探针与细菌生物标识结合后产生的复合物的特异性信号，并利用对所述细菌生物标识的定量检测来检测细菌生长。与现有技术相比，检测灵敏度高、速度快，在临床微生物的快速耐药性检测方面具有潜在的应用价值。

Description

一种利用纳米孔进行细菌耐药性的快速检测方法、装置和系统
该申请要求申请号为201910660192.1，申请日为2019年7月22日，名称为“一种利用纳米孔进行细菌耐药性的快速检测方法、装置和系统”的中国专利的优先权。
技术领域
本发明属于生物工程技术领域，具体涉及一种检测细菌耐药性的方法及其应用、一种16S rRNA-探针复合物及其应用、一种检测耐碳青霉烯类肺炎克雷伯菌的装置和试剂盒。
背景技术
肺炎克雷伯氏菌是临床感染中最严重的机会性病原体之一，通常存在于人和动物的肠中，可引起严重的临床后果，包括中枢神经系统感染或腹腔感染等。使用抗菌药物是肺炎克雷伯菌感染的主要治疗方法；早期使用和正确使用抗菌药物是肺炎克雷伯菌感染治愈的关键。然而，广谱抗菌药物的广泛使用导致肺炎克雷伯菌的强耐药性，这导致治疗的延长和失败。肺炎克雷伯菌HS11286(PMID：26169555)的碳青霉烯抗性可能由生物膜形成、活性抗微生物外排和β-内酰胺酶生成引起。
准确、快速地诊断感染患者肺炎克雷伯菌的抗菌药物耐药性对于治疗非常重要，因为它可以帮助医生选择适当种类的抗菌药物，缩短治疗周期，改善预后。细菌耐药性表型检测，β-内酰胺酶检测和耐药性基因检测是目前用于耐药性检测的主要方法。但是，细菌耐药 表型的检测需要足够的时间来培养肺炎克雷伯菌，通常是耗时的；β-内酰胺酶检测速度快，但检测范围相对较小，仅能检测很窄浓度的区间；耐药性的基因检测具有高精度，但是它也是昂贵且耗时的。
因此需要研发一种更优化、高效的细菌耐药性检测方法和装置。
发明内容
为满足临床需求，本发明提出了一种基于纳米孔传感技术的细菌耐药性检测方法，此方法具有时间成本低、准确度高、无需昂贵一期设备等优点。
具体地，本发明提供了一种检测细菌耐药性的方法，其特征在于，所述方法通过利用纳米孔检测探针与细菌生物标识结合后产生复合物的特异性信号，并利用对所述细菌生物标识的定量检测来检测细菌的生长。
进一步地，所述细菌生物标识为16S rRNA。
进一步地，所述细菌为耐碳青霉烯类肺炎克雷伯菌。
进一步地，所述细菌为肠埃希菌、肺炎克雷伯菌、产酸克雷伯菌、粪肠球菌和屎肠球菌的一种或多种。
进一步地，所述探针为探针A和B，所述探针A和B的核苷酸序列分别如SEQ ID NO.1和SEQ ID NO.2所示。
进一步地，所述检测细菌耐药性方法包括以下步骤：1)提取目标细菌总RNA；2)设计探针，并制备16SrRNA-探针复合物；3)检测纳米孔电生理信号。
进一步地，步骤1)培养细菌浓度大约至2MCF～10MCF时，提取总RNA。
进一步地，步骤1)所述培养细菌浓度大约为4MCF。
进一步地，步骤1)培养细菌时间长度大约为1小时～8小时，提取总RNA。
进一步地，步骤1)培养细菌时间长度大约为4小时。
进一步地，步骤1)所述细菌为肺炎克雷伯菌，培养时加入终浓度为16mg/L的亚胺培南。
进一步地，步骤2)所述探针为探针A和B，所述探针A和B的核苷酸序列分别如SEQ ID NO.1和SEQ ID NO.2所示。
进一步地，步骤2)所述16S rRNA-探针复合物由探针A和B和肺炎克雷伯菌的16S rRNA通过退火形成。
进一步地，步骤3)所述纳米孔电生理信号检测在50～200毫伏的电压下进行。
进一步地，步骤3)所述纳米孔电生理信号检测在150毫伏的电压下进行。
进一步地，所述纳米孔为MspA、alpha溶血素、氮化硅或石墨烯纳米孔。
具体地，本发明还提供了一种16S rRNA-探针复合物，其特征在于，所述复合物由探针A和B和肺炎克雷伯菌的16S rRNA通过退火形成，所述探针A和B的核苷酸序列分别如SEQ ID NO.1和SEQ ID NO.2所示。
进一步地，本发明还提供了所述16S rRNA-探针复合物在检测耐碳青霉烯类肺炎克雷伯菌中的应用。
具体地，本发明还提供了一种检测耐碳青霉烯类肺炎克雷伯菌的装置，其特征在于，所述装置包括纳米孔、探针、肺炎克雷伯菌RNA提取试剂单元、纳米孔电生理信号检测单元。
进一步地，所述探针为探针A和B，所述探针A和B的核苷酸序列分别如SEQ ID NO.1和SEQ ID NO.2所示。
进一步地，所述肺炎克雷伯菌RNA提取试剂单元含有TRIZOL、乙醇、DEPC水/无RNase水,和RNase抑制剂。
进一步地，所述纳米孔为MspA、alpha溶血素、氮化硅或石墨烯纳米孔。
进一步地，，所述纳米孔电生理信号检测单元含有HEPES、KCl、膜和DPHPC。
进一步地，所述膜为双层脂质膜或高分子膜。
具体地，本发明还提供了一种检测耐碳青霉烯类肺炎克雷伯菌的试剂盒，其特征在于，所述试剂盒由纳米孔、探针和RNA提取试剂组成，所述探针为探针A和B，所述探针A和B的核苷酸序列分别如SEQ ID NO.1和SEQ ID NO.2所示。
进一步地，所述RNA提取试剂包括TRIZOL、乙醇、DEPC水/无RNase水,和RNase抑制剂。
进一步地，所述纳米孔为MspA、alpha溶血素、氮化硅或石墨烯纳米孔。
具体地，本发明还提供了利用所述装置检测耐碳青霉烯类肺炎克雷伯菌的方法，其特征在于，包括以下步骤：
(1)在肺炎克雷伯菌RNA提取试剂单元中，用TRIZOL提取肺炎克雷伯菌的总RNA，再用乙醇洗涤，加入DEPC水/无RNase水溶解后再加入RNase抑制剂进行储存；
(2)将探针A和B与步骤(1)储存的样品通过退火形成16S rRNA-探针复合物；
(3)将所述纳米孔和步骤(2)的16S rRNA-探针复合物置于纳米孔电生理信号检测单元进行检测；
(4)对检测到的电生理信号进行数据分析，对肺炎克雷伯菌进行定量检测。
具体地，本发明还提供了利用本发明所述方法在检测微生物耐药性方面的应用。
进一步地，所述微生物为细菌。
进一步地，所述细菌为肺炎克雷伯菌。
具体地，在本发明的方法中，我们使用特异性探针与肺炎克雷伯菌中的16S rRNA结合，并通过纳米孔测定记录核酸读取过程。目标核酸通过纳米孔转运的特定信号的频率反映了活肺炎克雷伯菌的数量。因此，可以通过此方法对碳青霉烯的剩余活肺炎克雷伯菌进行定量分析。根据特异性阻塞信号的阻塞率和滞留时间，我们可以检测出具有碳青霉烯抗性的肺炎克雷伯菌样品中的16S rRNA，整个检测方法的过程和需要的时间如图6所示。
具体地，本发明提供了一种基于纳米孔的新颖，高效和快速的检测方法，在单一分子水平上区分耐碳青霉烯类肺炎克雷伯菌和碳青霉烯敏感类肺炎克雷伯菌。通过MALDI-TOF MS鉴定的这些菌株分别与亚胺培南一起培养数小时。16S rRNA具有高度保守性和特异性，可以作为基因检测技术中病原体检测和鉴定的有力工具，因此选择它作为在抗生素下培养后测量活肺炎克雷伯菌量的参数。换句话说，由于基于16S rRNA的种属鉴定是微生物组研究中最常用的方法，因此本发明所提供的检测方案适用于多数细菌的鉴定，再结合含抗生素环境/无抗生素环境的对照培养实验，即可实现对细菌耐药性的鉴定。
对本领域技术人员而言，很显然地，本发明的应用不局限于肺炎克雷伯菌和16S rRNA，根据实际需求，其它具有高度保守性和特异性的生物标记同样适用于本发明所提供的检测方法和装置，例如：rpoB,SodA,gyrB,groEL,recN等同样是可以被用于本发明的细菌生物标识；本发明也同样可以被用来检测肠埃希菌、产酸克雷伯菌、粪肠球菌和屎肠球菌等细菌的生长情况，以及基于此的耐药性检测
(例如，利用含抗生素环境/无抗生素环境的对照培养)。另外，常见的纳米孔均可以适用于本发明：例如，MspA、alpha溶血素、氮化硅和石墨烯纳米孔。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作一简单地介绍。显而易见地，下面描述中的附图是本发明的一些实施例、实验数据和 结果，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图内容衍生出其它实施例、进行其它实验，获得其它实验结果。
图1为纳米孔的结构和纳米孔测定的单通道记录设置。
图2为16S rRNA-探针复合物及其纳米孔信号。
图3为探针组的易位信号。
图4为区分耐碳青霉烯类肺炎克雷伯菌和碳青霉烯敏感类肺炎克雷伯菌及单通道记录信号。
图5为临床样品的双盲测试和测定准确度的评估。
图6为检测流程图和总时间成本。
图7为纳米孔检测碳青霉烯抗性肺炎克雷伯氏菌的方案程序。
本发明有益效果：
本发明所述的快速检测细菌耐药性的方法可定量检测细菌中16S rRNA的量，并通过对细菌中16S rRNA的量判断细菌是否耐药；
本发明所述的快速检测细菌耐药性的方法与常规的纸片扩散方法和PCR法相比，具有高灵敏度，实时操作、低成本和耗时少的优势，经验证细菌培养时间仅需4小时，准确度为90％；
本发明所述的快速检测细菌耐药性的方法若清除因样品储存或转移过程中的RNA降解的原因，其准确度会更高，因此在临床快速耐药性检测方面具有巨大潜在的应用价值。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将 结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述。显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
除非另外指定，否则术语“包含有”和“包含”及其在语法上的变化是用来表示“开放式”或者“包括”的语言，从而它们包括列举的技术特征但也允许包括另外的没有列举的技术特征。
如在本说明书中使用的，术语“大约”，典型地表示为所述值的+/-5％，更典型的是所述值的+/-4％，更典型的是所述值的+/-3％，更典型的是所述值的+/-2％，甚至更典型的是所述值的+/-1％，甚至更典型的是所述值的+/-0.5％。
在本说明书中，某些实施方式可能以一种处于某个范围的格式公开。应该理解，这种“处于某个范围”的描述仅仅是为了方便和简洁，且不应该被解释为对所公开范围的僵化限制。因此，范围的描述应该被认为是已经具体地公开了所有可能的子范围以及在此范围内的独立数字值。例如，范围
Figure PCTCN2020102381-appb-000001
的描述应该被看作已经具体地公开了子范围如从1到3，从1到4，从1到5，从2到4，从2到6，从3到6等，以及此范围内的单独数字，例如1，2，3，4，5和6。无论该范围的广度如何，均适用以上规则。
耐碳青霉烯类肺炎克雷伯菌近几十年来在全球范围内迅速流行，对当今的临床实践造成了巨大挑战。快速检测耐碳青霉烯类肺炎 克雷伯菌可以减少不恰当的抗菌治疗并挽救生命。传统的耐碳青霉烯类肺炎克雷伯菌检测方法非常耗时，PCR和其他测序方法过于昂贵且技术要求较高，难以满足临床需求。纳米孔检测具有高灵敏度，实时操作和低成本的优势，已经应用于疾病生物标志物的筛选。在这项研究中，我们通过检测细菌与抗生素亚胺培南短期培养后的核酸提取物中16S rRNA的量来反映细菌的生长情况，以此区分碳青霉烯敏感类肺炎克雷伯菌和耐碳青霉烯类肺炎克雷伯菌。利用MspA纳米孔可以记录到探针与16S rRNA结合后产生的特异性信号，以此完成对16S rRNA的超灵敏和快速定量检测。我们证明纳米孔检测方法仅需要4小时的培养时间即可区分耐碳青霉烯类肺炎克雷伯菌和对碳青霉烯敏感类肺炎克雷伯菌。该法的时间成本为纸片扩散方法的5％左右，同时达到了与纸片扩散法相近的准确度。这个新方法在临床微生物的快速耐药性检测方面具有潜在的应用价值。
具体地，纳米孔传感技术有助于其在第三代DNA单分子测序中广泛应用。纳米尺寸的蛋白质孔嵌入磷脂膜中，磷脂膜将蛋白质孔腔室分成两部分(顺式和反式)。当在包含一定浓度的离子溶液的腔室上施加电压时，系统中的带电检测物质通过孔隙被驱动到另一个腔室。膜片钳传感器检测纳米孔的电流变化信号。通过纳米孔传输的不同分子可以引起相应的电流阻塞信号，使用特定的转运信号和转运频率，可以实现对检测到的分子的定性和定量分析。这种纳米孔传感技术具有无标记，快速，实时操作和高灵敏度的优点，仅需少量样品。因此，这些特征适用于疾病的快速诊断和生物标志物的检测。
具体地，耻垢分枝杆菌(Mycobacterium smegmatis)孔蛋白A( MspA)纳米孔蛋白是分枝杆菌的外膜蛋白之一，长度为9.6nm，如图1所示，直径为1.3nm。纳米孔高效地结合到双层脂质膜中，并且允许单链核酸转运通过孔，由于其短而窄的通道，MspA纳米孔是非常适合于纳米孔测序的。当然，除了MspA纳米孔以外，其它常见纳米孔，例如alpha溶血素、氮化硅和石墨烯纳米孔均可适用于纳米孔测序。另外，除了双层脂质膜，高分子膜也可以适用于本发明。
具体地，存在于所有细菌中的16S rRNA是原核生物核糖体中30S亚基的组分，其功能不随时间而变化。16S rRNA可用于鉴定细菌物种，因为它含有所有细菌共有的高度保守区域和不同细菌差异的高变区。它已被证明是一种可靠的遗传标记，常用于细菌分类且有文献证明它可用于鉴定临床病原体。当然，除了采用16S rRNA作为细菌生物标识，其它细菌生物标识，例如rpoB,SodA,gyrB,groEL,recN，同样适用于本发明，来检测细菌的生长情况，以及基于此的耐药性检测(例如，利用含抗生素环境/无抗生素环境的对照培养)。
材料
试剂4-(2-羟乙基)哌嗪-1-乙磺酸(HEPES，纯度>99.5％，CAS#7365-45-9)，氯化钾(KCl，纯度>99.0％，CAS#7447-40-7)，琼脂糖(纯度>99.0％，CAS#：9012-36-6)，氯仿(纯度>99.0％，CAS：67-66-3)，异丙醇(纯度>99.0％，CAS#：67-63-0)和乙醇(纯度>99.0％，CAS#：64-17-5)购自Sigma-Aldrich。RNase抑制剂(5KU)，pET-28b质粒和所有DNA均由Sangon Biotech 提供，1,2-二乙酰基-sn-甘油-3-磷酸胆碱(DPHPC)购自Avanti，PrimeSTAR HS DNA聚合酶购自TaKaRa，亚胺培南(CAS#：64221-86-9)购自MSD。
临床标本：
2例肺炎克雷伯菌感染患者的血液样本由四川大学华西医院检验科提供。本发明研究是根据中国国家生物医学研究涉及人类伦理审查和赫尔辛基WMA宣言的建议进行的。该方案经四川大学华西医院生物医学伦理委员会批准。该发明研究使用了剩余的标本，即用于常规临床护理或分析的标本残留物，这些标本将被丢弃，并符合放弃知情同意的标准。四川大学华西医院生物医学伦理委员会给予了知情同意权的豁免。
实施例一 检测16S rRNA-探针复合物
1.制备细菌提取物
两组来自临床患者的肺炎克雷伯氏菌样本由四川大学华西医院提供。将肺炎克雷伯菌样品培养至两种不同浓度，第一组的浓度为0.5MCF，第二组的浓度为4MCF。在培养开始时，两组中使用的亚胺培南的最终浓度为16mg/L，通过TRIZOL方法提取肺炎克雷伯菌的总RNA。首先，收集100μL细菌溶液。离心后取出上清液(8000g，4℃，2分钟)。用溶菌酶沉淀并在37℃下孵育10分钟。裂解肺炎克雷伯菌，提取总RNA并用乙醇洗涤。取下离心管帽，在室温下干燥5-10min，加入DEPC水或溶解于无rnas的水中。将RNase抑制剂加入到溶解的溶液中至终浓度为20U/μL用于储存。
2.设计探针并与样本孵育
设计探针与16S rRNA的特定片段结合，以便发明人团队可以通过纳米孔识别关于目标核酸的特定信号。由于目标16S rRNA长932bp，因此难以在没有探针或单个探针的情况下区分16S rRNA-探针复合物，因此发明人团队设计了两个探针以结合肺炎克雷伯菌的特异性表达的16S rRNA。所述两个探针为探针A和B的核苷酸序列如SEQ ID NO.1和SEQ ID NO.2所示。将所述探针A和B与储存的样品进行退火，并用使用琼脂糖凝胶电泳来验证探针16S rRNA-探针复合物的形成(图2中的A)。
3.结果
琼脂糖凝胶电泳的结果表明成功获得了16S rRNA-探针复合物(图2中的B)。在耐碳青霉烯类肺炎克雷伯菌的样品中16S rRNA-探针复合物的转运信号的滞留时间在100-400ms范围内，峰值为196.98ms，单链DNA转运的滞留时间在0-100ms的范围内，峰值为12.03ms(图2中的C和D)。探针A和探针B的滞留时间在0-70ms的范围内(图3)。这些结果表明长滞留时间的信号是由16S rRNA-探针复合物引起的。
实施例二 细菌浓度的优化和标准样品测试
1.MspA纳米孔的表达和纯化
将MspA纳米孔的基因克隆到pET-28b质粒中，将携带MspA基因的pET-28b质粒转移到工程菌BL21大肠杆菌感受态细胞中。在37℃温度下，成功转移的大肠杆菌用LB培养基培养，卡那霉素加至50微 克/毫升。当光密度(600nm)接近0.8时，将0.8mM IPTG加入LB(溶原性发酵液)培养基中，诱导温度为15℃。经过12个小时的诱导后，通过离心收集大肠杆菌。用超声波发生器破碎大肠杆菌后收集上清液，用阴离子交换柱(Q-Sepharose)和分子筛(Superdex 200 16/90)进一步纯化。通过10％SDS-PAGE(十二烷基硫酸钠聚丙烯酰胺凝胶电泳)分析纯化的蛋白质。可以将纯化的MspA纳米孔蛋白分装并在-80℃下储存。分装的试样可以保持稳定多年，并且纳米孔在解冻时保持结构完整。
2.纳米孔电生理信号检测实验确定较佳的样品浓度和最佳的细菌培养时间
2.1确定较佳的样品浓度
对实施例一中的两种不同浓度的细菌提取物样品进行纳米孔电生理信号检测实验。实验方法为：
所述实验在Warner Instrument提供的室中进行。纳米孔电生理信号检测实验在150毫伏的电压下进行。顺式侧和反式侧的导电缓冲溶液是含有10mM HEPES，pH7.0的400mM KCl溶液。在150μm孔的两侧上涂抹的双层脂质膜(BLM)由1,2-二羟甲酰基-sn-甘油-3-磷酸胆碱(DPHPC)形成。将MspA添加到顺式腔室中的溶液中，允许MspA蛋白质插入，BLM更快的形成。单个MspA纳米孔嵌入将导致电流增加，对应的电导为1.2nS。在通过Heka EPC-10膜片钳(HEKA)时记录插入单个MspA纳米孔电流信号后，将样品添加到顺式侧。
使用两种浓度的肺炎克雷伯菌优化检测效率。在0.5MCF的样品中，对照组的目标RNA转运信号频率为0.02±0.02/分钟(n＝3)，耐碳青霉烯类肺炎克雷伯菌组的目标RNA转运信号频率为0.13±0.05/min(n＝3)。而在4MCF的样品中，对照组的目标RNA转运信号频率为每分钟0(n＝3)，耐碳青霉烯类肺炎克雷伯菌组的易位频率为每分钟0.33±0.07(n＝3)(图4中的A)。与0.5MCF样品相比，4MCF样品在纳米孔测定中能被较好的检测。
2.2.确定最佳的细菌培养时间
将提取自耐碳青霉烯类肺炎克雷伯菌和碳青霉烯敏感类肺炎克雷伯菌的总RNA与探针A和探针B一起孵育，并分别通过MspA纳米孔检测孵育后的溶液(图4中的B)。将样品通过纳米孔测定得到的信号的两个参数，阻塞率和滞留时间绘制成散点图(图4中的C)，可以观察到不同组之间的滞留时间的明显差异，尤其是在阻塞率0.6至0.8，滞留时间100毫秒至400毫秒范围内。因此，选择该范围内的信号作为诊断的特异信号。在比较来自空白，对照，耐碳青霉烯类肺炎克雷伯菌和碳青霉烯敏感类的肺炎克雷伯菌样品的给定范围内的16S rRNA-探针信号的数量后，使用f＝0.1·min -1作为目标信号转运频率阈值来区分肺炎克雷伯氏菌的碳青霉烯抗性。为了确定区分耐碳青霉烯类肺炎克雷伯菌和碳青霉烯敏感类的肺炎克雷伯菌所需的最小细菌培养时间，通过MspA纳米孔检测具有不同细菌培养时间的样品，包括2小时，4小时和8小时，实验结果表明，4小时是兼顾灵敏度和效率的最佳细菌培养时间。
实施例三 MspA纳米孔检测临床样品的双盲试验
对来自华西医院提供的20名肺炎克雷伯菌感染患者的血液样品中的细菌进行培养，提取总RNA并用于双盲实验。用MspA纳米孔检测每个样品至少三次。分析后，收集阻塞率为0.6至0.8，滞留时间为100ms至400ms的16S rRNA探针信号数目，并与目标信号转运频率阈值f阈值进行比较。
在20个样本中，如表1所示，其中9个高于阈值(0.1·min -1)并且被判定为耐碳青霉烯类肺炎克雷伯菌。如表2所示，其他11个样本低于阈值0.1·min -1，这些临床样本被判定为碳青霉烯敏感类肺炎克雷伯菌样品(图5中的A)。与从标准临床方法(纸盘扩散法或PCR)获得的测定结果相比，本发明的纳米孔测定方法具有成本低、耗时短的优点(表3)。通过纳米孔测定的18个样品的结果是正确的(图5中的B)，有两个为假阴性结果。
表1.碳青霉烯敏感类肺炎克雷伯菌的临床样本信息
Figure PCTCN2020102381-appb-000002
Figure PCTCN2020102381-appb-000003
注：样本ID是医院中的患者ID，样本编号是本发明研究中的相应编号。
表2.耐碳青霉烯类肺炎克雷伯菌的临床样本信息
样品ID 样品# SCIM(mm) 耐药基因 耐药基因
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 负 -
17008404-1 11 6 KPC 没有
17012837-3 12 6 KPC KPC-2
17020362-3 20 6 KPC KPC-2
注：样本ID是医院中的患者ID，样本编号是本发明研究中的相应编号。
表3.不同耐碳青霉烯类肺炎克雷伯菌检测方法的比较
Figure PCTCN2020102381-appb-000004
以上实施例采用软件Clampfit 10.6和Origin Pro 8.0进行 数据分析。阻塞电流被定义为ΔI/I 0，其中I 0是一个完全打开的孔的电流，并且ΔI是转运分子引起的阻塞电流的振幅。由Clampfit10.6的单信道搜索功能收集滞留时间。这两个参数用于定量分析来自存活的耐碳青霉烯肺炎克雷伯菌的目标16S rRNA。所有数据均来自20分钟的电生理记录，实验组独立重复3次。
本发明技术问题与解决方案总结
快速准确的肺炎克雷伯菌对碳青霉烯类耐药性的检测在临床治疗过程中非常重要。然而现在的检测技术并不能完全满足临床需求。我们的目标是开发一种基于纳米孔传感技术的肺炎克雷伯菌对碳青霉烯抗性的新型检测方法以解决临床中面临的问题。
在过去的20年中，蛋白质纳米孔表达纯化和电生理学检测技术得到了快速发展，基于多种类型蛋白质纳米孔的核酸检测方法已经非常成熟。在本发明中，发明人团队设计了两个DNA探针来特异性结合具有碳青霉烯抗性肺炎克雷伯菌的16S rRNA，16S rRNA-探针复合物易位通过MspA纳米孔将造成在100ms至400ms之间的滞留时间。根据特异性阻塞信号的阻塞率和滞留时间，可以检测出具有碳青霉烯抗性的肺炎克雷伯菌样品中的16S rRNA(图6)。通过对具有碳青霉烯类抗性肺炎克雷伯菌标准样品和对碳青霉烯敏感的肺炎克雷伯菌标准样品的检测，证实了该方法可用于区分对碳青霉烯类耐药性和对碳青霉烯敏感的肺炎克雷伯菌样品且细菌样品的培养时间仅需4小时。
此外，发明人团队利用MspA纳米孔测定了华西医院提供的20 个临床样本。在11例碳青霉烯类耐药的肺炎克雷伯菌临床样本中，9例样本得到了正确的诊断结果，2例样本被检测为假阴性；在9个对碳青霉烯敏感的肺炎克雷伯菌样本中，9个样本全部得到了正确的诊断结果。纳米孔诊断方法的准确率为90％。样品储存或转移过程中的RNA降解是导致10％假阴性诊断的主要原因。临床样品从医院到实验室的运输过程以及样品处理和纳米孔测定之间的时间间隔增加了RNA降解的可能性，导致16S rRNA和特异性阻塞信号的数量减少。
上述结果是基于新型纳米孔单分子诊断技术的试验性研究，旨在提高灵敏度的进一步验证研究可以围绕以下几个方面展开：优化细菌培养条件和RNA提取技术；用于16S rRNA-探针复合物检测的蛋白质纳米孔的修饰和改造；用大量临床样本进行检验以及改进数据的处理统计方法。在-80℃下将分装的MspA蛋白稳定储存使得能够批量生产纳米孔用于后续的多次测试。
总之，发明人团队的研究证实了纳米孔单分子检测技术可用于对碳青霉烯类耐药性肺炎克雷伯菌的快速临床诊断。与纸片扩散法或PCR法这两种临床诊断中使用最广泛的方法相比，纳米孔检测方法具有成本低，效率高，易于操作的优势。该方法可以作为现有诊断方法的补充应用于临床实验室诊断。随着纳米孔芯片技术的发展，基于纳米孔阵列的多重临床样本检测将进一步应用于临床即时诊断。
需要说明的是，在本文中，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者装置不仅包括那些要素，而且还包括没有明确列 出的其他要素，或者是还包括为这种过程、方法、物品或者装置所固有的要素。在没有更多限制的情况下，由语句“包括一个……”限定的要素，并不排除在包括该要素的过程、方法、物品或者装置中还存在另外的相同要素。
上面结合附图对本发明的实施例进行了描述，但是本发明并不局限于上述的具体实施方式，上述的具体实施方式仅仅是示意性的，而不是限制性的，本领域的普通技术人员在本发明的启示下，在不脱离本发明宗旨和权利要求所保护的范围情况下，还可做出很多形式，这些均属于本发明的保护之内。

Claims (31)

  1. 一种检测细菌耐药性的方法，其特征在于，所述方法通过利用纳米孔检测探针与细菌生物标识结合后产生的复合物的特异性信号，并利用对所述细菌生物标识的定量检测来检测细菌生长。
  2. 根据权利要求1所述的方法，其特征在于，所述细菌生物标识为16S rRNA。
  3. 根据权利要求2所述的方法，其特征在于，所述细菌为耐碳青霉烯类肺炎克雷伯菌。
  4. 根据权利要求2所述的方法，其特征在于，所述细菌为肠埃希菌、肺炎克雷伯菌、产酸克雷伯菌、粪肠球菌和屎肠球菌的一种或多种。
  5. 根据权利要求2所述的方法，其特征在于，所述探针为探针A和B，所述探针A和B的核苷酸序列分别如SEQ ID NO.1和SEQ ID NO.2所示。
  6. 根据权利要求2所述的方法，其特征在于，包括以下步骤：
    1)提取目标细菌总RNA；
    2)设计探针，并制备16SrRNA-探针复合物；
    3)检测纳米孔电生理信号。
  7. 根据权利要求6所述的方法，其特征在于，步骤1)培养细菌浓度大约至2MCF～10MCF时，提取总RNA。
  8. 根据权利要求7所述的方法，其特征在于，步骤1)所述培养细菌浓度大约为4MCF。
  9. 根据权利要求6所述的方法，其特征在于，步骤1)培养细菌时间 长度大约为1小时～8小时，提取总RNA。
  10. 根据权利要求9所述的方法，其特征在于，步骤1)培养细菌时间长度大约为4小时。
  11. 根据权利要求6所述的方法，其特征在于，步骤1)所述细菌为肺炎克雷伯菌，培养时加入终浓度为16mg/L的亚胺培南。
  12. 根据权利要求6所述的方法，其特征在于，步骤2)所述探针为探针A和B，所述探针A和B的核苷酸序列分别如SEQ ID NO.1和SEQ ID NO.2所示。
  13. 根据权利要求6所述的方法，其特征在于，步骤2)所述16SrRNA-探针复合物由探针A和B和肺炎克雷伯菌的16S rRNA通过退火形成。
  14. 根据权利要求6所述的方法，其特征在于，步骤3)所述纳米孔电生理信号检测在50～200毫伏的电压下进行。
  15. 根据权利要求6所述的方法，其特征在于，步骤3)所述纳米孔电生理信号检测在150毫伏的电压下进行。
  16. 根据权利要求2所述的方法，其特征在于，所述纳米孔为MspA、alpha溶血素、氮化硅或石墨烯纳米孔。
  17. 一种16S rRNA-探针复合物，其特征在于，所述复合物由探针A和B和肺炎克雷伯菌的16S rRNA通过退火形成，所述探针A和B的核苷酸序列分别如SEQ ID NO.1和SEQ ID NO.2所示。
  18. 权利要求17所述的16S rRNA-探针复合物在检测耐碳青霉烯类肺炎克雷伯菌中的应用。
  19. 一种检测耐碳青霉烯类肺炎克雷伯菌的装置，其特征在于，所述装置包括纳米孔、探针、肺炎克雷伯菌RNA提取试剂单元、纳米孔电生理信号检测单元。
  20. 根据权利要求19所述的装置，其特征在于，所述探针为探针A和B，所述探针A和B的核苷酸序列分别如SEQ ID NO.1和SEQ ID NO.2所示。
  21. 根据权利要求19所述的装置，其特征在于，所述肺炎克雷伯菌RNA提取试剂单元含有TRIZOL、乙醇、DEPC水/无RNase水,和RNase抑制剂。
  22. 根据权利要求19所述的装置，其特征在于，所述纳米孔为MspA、alpha溶血素、氮化硅或石墨烯纳米孔。
  23. 根据权利要求19所述的装置，其特征在于，所述纳米孔电生理信号检测单元含有HEPES、KCl、膜和DPHPC。
  24. 根据权利要求23所述的装置，其特征在于，所述膜为双层脂质膜或高分子膜。
  25. 一种检测耐碳青霉烯类肺炎克雷伯菌的试剂盒，其特征在于，所述试剂盒由纳米孔、探针和RNA提取试剂组成，所述探针为探针A和B，所述探针A和B的核苷酸序列分别如SEQ ID NO.1和SEQ ID NO.2所示。
  26. 根据权利要求25所述的试剂盒，其特征在于，所述RNA提取试剂包括TRIZOL、乙醇、DEPC水/无RNase水,和RNase抑制剂。
  27. 根据权利要求25所述的试剂盒，其特征在于，所述纳米孔为 MspA、alpha溶血素、氮化硅或石墨烯纳米孔。
  28. 利用权利要求19-27任一项所述装置检测耐碳青霉烯类肺炎克雷伯菌的方法，其特征在于，包括以下步骤：
    (1)在肺炎克雷伯菌RNA提取试剂单元中，用TRIZOL提取肺炎克雷伯菌的总RNA，再用乙醇洗涤，加入DEPC水/无RNase水溶解后再加入RNase抑制剂进行储存；
    (2)将探针A和B与步骤(1)储存的样品通过退火形成16S rRNA-探针复合物；
    (3)将所述纳米孔和步骤(2)的16S rRNA-探针复合物置于纳米孔电生理信号检测单元进行检测；
    (4)对检测到的电生理信号进行数据分析，对肺炎克雷伯菌进行定量检测。
  29. 权利要求1-16任一项所述的方法在检测微生物耐药性方面的应用。
  30. 根据权利要求29所述的应用，其特征在于，所述微生物为细菌。
  31. 根据权利要求29所述的应用，其特征在于，所述细菌为肺炎克雷伯菌。
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