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Method, device and system for rapidly detecting bacterial drug resistance by utilizing nanopores

A nanopore, drug-resistant technology, applied in the field of bioengineering, can solve problems such as time-consuming, expensive, and small detection range

Pending Publication Date: 2021-01-29
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the detection of bacterial drug-resistant phenotypes requires sufficient time to culture Klebsiella pneumoniae, which is usually time-consuming; the detection of β-lactamase is fast, but the detection range is relatively small, and only a narrow concentration can be detected. interval; genetic testing for drug resistance is highly accurate, but it is also expensive and time-consuming

Method used

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  • Method, device and system for rapidly detecting bacterial drug resistance by utilizing nanopores
  • Method, device and system for rapidly detecting bacterial drug resistance by utilizing nanopores
  • Method, device and system for rapidly detecting bacterial drug resistance by utilizing nanopores

Examples

Experimental program
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Effect test

Embodiment 1

[0070] Example 1 Detection of 16S rRNA-probe complex

[0071] 1. Preparation of Bacterial Extracts

[0072] Two groups of Klebsiella pneumoniae samples from clinical patients were provided by West China Hospital of Sichuan University. Klebsiella pneumoniae samples were cultured to two different concentrations, the first group had a concentration of 0.5 MCF and the second group had a concentration of 4 MCF. At the beginning of the culture, 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. First, collect 100 µL of the bacterial solution. The supernatant was removed after centrifugation (8000g, 4°C, 2 minutes). Precipitate with lysozyme and incubate at 37 °C for 10 min. Klebsiella pneumoniae were lysed, total RNA was extracted and washed with ethanol. Remove the centrifuge tube cap, dry at room temperature for 5-10min, add DEPC water or dissolve in rnas-free water. Add the R...

Embodiment 2

[0077] The optimization of embodiment two bacterial concentration and standard sample test

[0078] 1. Expression and purification of MspA nanopore

[0079] The gene of the MspA nanopore was cloned into the pET-28b plasmid, and the pET-28b plasmid carrying the MspA gene was transferred into the competent cells of the engineering bacteria BL21 Escherichia coli. At a temperature of 37°C, the successfully transferred Escherichia coli was cultured with LB medium, and kanamycin was added to 50 μg / ml. When the optical density (600nm) was close to 0.8, 0.8mM IPTG was added into the LB (lysogenic fermentation broth) medium, and the induction temperature was 15°C. After 12 hours of induction, E. coli were collected by centrifugation. The supernatant was collected after crushing E. coli with an ultrasonic generator, and further purified with an anion exchange column (Q-Sepharose) and molecular sieves (Superdex200 16 / 90). Purified proteins were analyzed by 10% SDS-PAGE (sodium dodecyl...

Embodiment 3

[0087] Example 3 Double-blind test of MspA nanopore detection of clinical samples

[0088] Bacteria in blood samples from 20 patients with Klebsiella pneumoniae infection provided by West China Hospital were cultured, total RNA was extracted and used for double-blind experiments. Each sample was assayed at least three times with the MspA nanopore. After analysis, the number of 16S rRNA probe signals with a blocking rate of 0.6 to 0.8 and a residence time of 100 ms to 400 ms was collected and compared with the target signal translocation frequency threshold fthreshold.

[0089] Among the 20 samples, as shown in Table 1, 9 of them are above the threshold (0.1 min -1 ) and was judged to be carbapenem-resistant Klebsiella pneumoniae. As shown in Table 2, the other 11 samples are below the threshold of 0.1 min -1 , these clinical samples were determined to be carbapenem-sensitive Klebsiella pneumoniae samples ( Figure 5 in A). Compared with assay results obtained from standar...

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Abstract

The invention relates to a method and a device for detecting bacterial drug resistance and application of the method and the device. The method and the device are characterized in that bacterial growth is detected by utilizing a specific signal of a compound generated after a nanopore detection probe is combined with a bacterial biomarker and conducting quantitative detection on the bacterial biomarker. Compared with the prior art, the method and the device are high in detection sensitivity and high in speed, and have potential application value in the aspect of rapid drug resistance detectionof clinical microorganisms.

Description

[0001] The application claims the priority of the Chinese patent with the application number 201910660192.1 and the application date of July 22, 2019, entitled "A Rapid Detection Method, Device and System for Bacterial Drug Resistance Using Nanopores". technical field [0002] 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. Background technique [0003] Klebsiella pneumoniae is one of the most serious opportunistic pathogens in clinical infection. It usually exists in the intestines of humans and animals and can cause serious clinical consequences, including central nervous system infection or intra-abdominal infection. The use of antibacterial drugs is the main treatment for Klebsiella pneumoniae infection; early and correct use o...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6816C12Q1/10C12R1/22
CPCC12Q1/689C12Q1/6816C12R2001/22C12Q2565/631C12Q1/10G01N15/00G01N33/487C12N1/205Y02A50/30
Inventor 耿佳魏于全
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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