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Kit and detection method for rapidly detecting enterobacteriaceae bacteria capable of producing A/B carbapenemase

A penemase Enterobacteriaceae and kit technology, which are applied in the field of high performance liquid chromatography tandem mass spectrometry, can solve the problems of high cost, slow discrimination and detection of A-type and B-type enzymes, complicated operation, etc. Simple operation and high detection accuracy

Active Publication Date: 2021-10-26
SHANGHAI EAST HOSPITAL EAST HOSPITAL TONGJI UNIV SCHOOL OF MEDICINE
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] In view of the existing problems in the prior art, the object of the present invention is to provide a test kit and detection method for rapidly detecting Enterobacteriaceae bacteria producing A / B class carbapenemases, and the described test kit and detection method What needs to be solved is that in the prior art, the technical problems of slow speed, high cost and complex operation of CRE and its produced Class A and Class B enzymes

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  • Kit and detection method for rapidly detecting enterobacteriaceae bacteria capable of producing A/B carbapenemase
  • Kit and detection method for rapidly detecting enterobacteriaceae bacteria capable of producing A/B carbapenemase
  • Kit and detection method for rapidly detecting enterobacteriaceae bacteria capable of producing A/B carbapenemase

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Embodiment 1

[0044] The present invention also provides a preparation method of the above-mentioned kit for detecting Enterobacteriaceae bacteria producing A / B class carbapenemase, comprising the following steps:

[0045] 1) A step of preparing a pharmaceutical reagent, weighing the meropenem drug, adding it to 0.85% normal saline, the concentration of the meropenem after adding is 50 mg / ml, completely dissolving it, and storing it for later use;

[0046] 2) A step of preparing bacterial culture medium, weighing MH broth dry powder, adding it to distilled water, the mass fraction of MH broth dry powder after adding is 21g / L, autoclaving at 121°C for 15 minutes, cooling and storing for later use;

[0047] 3) A step for preparing two kinds of enzyme inhibitors. Weigh APB and EDTA and add them to methanol and distilled water respectively. After adding, the concentration of APB is 300mg / ml, and the concentration of EDTA is 292mg / ml. After completely dissolving, save for future use ;

[0048] ...

Embodiment 2

[0051] Example 2 Establishment of HPLC-MS / MS detection method for rapid detection of Enterobacteriaceae bacteria producing A / B class carbapenemase 1. Instrument: high performance liquid chromatograph Shimadzu LC-20A (Shimadzu, JPN), in series Triple quadrupole mass spectrometer AB API3200 (AB sciex, USA), METTLER TOLEDO balance (Max = 220g, d = 0.1mg) (METTLER, Germany), Eclipse plus-C18 rapid separation high-throughput narrow-diameter column (3.0mm* 100mm*3.5μm) (Agilent, USA), Eppendorf 5424R low-temperature high-speed centrifuge (Eppendorf, Germany).

[0052]2. Commercially purchased reagents: Meropenem was purchased from Sigma Company of the United States with a purity of 98.0%; EDTA was purchased from Sinopharm Group Corporation of China; APB was purchased from West Asia Reagent Company of China; MH medium was purchased from Hangzhou Microbial Reagent Co., Ltd. of China; 0.85% Physiological saline was purchased from Kelun Pharmaceutical Co., Hunan, China; Methanol (LC / / MS...

Embodiment 3

[0066] Example 3 Preliminary Clinical Application of the Kit and Detection Method for Rapid Detection of Type A / B Carbapenemase-Producing Enterobacteriaceae Bacteria

[0067] 1. Research object

[0068] Enterobacteriaceae strains were collected from Shanghai Dongfang Hospital.

[0069] Selection criteria for strains: Fresh strains isolated and cultivated in clinical practice were determined to be Enterobacteriaceae after detection by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF) (identification confidence Degree ≥ 2.0)

[0070] Determination criteria for CRE: Enterobacteriaceae bacteria resistant to any carbapenems (such as meropenem inhibition zone diameter ≤ 19, imipenem inhibition zone diameter ≤ 19, etc.)

[0071] Determination criteria for the production of class A enzyme CRE: CRE strains are tested by enzyme inhibitor enhancement test, and the diameter of the inhibition zone of imipenem discs containing APB is 5mm or more larg...

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Abstract

A kit for rapidly detecting enterobacteriaceae bacteria capable of producing A / B carbapenemase comprises a medicine powder, a medicine dissolving agent, a bacteria culture medium, two enzyme inhibitors, a sample extract liquor and a mobile phase A / B, the medicine powder is 10 mg meropenem, the medicine dissolving agent is 0.85% normal saline, the bacteria culture medium is an MH culture medium, the two enzyme inhibitors are 300 mg / ml of 3-aminophenylboronic acid and 292 mg / ml of ethylenediamine tetraacetic acid, the sample extract liquor is pure methanol, the mobile phase A is a formic acid aqueous solution with the volume ratio of 0.01%-0.1%, and the mobile phase B is a formic acid methanol solution with the volume ratio of 0.01%-0.1%. The invention also provides a detection method adopting the kit. The kit is high in accuracy and short in time consumption, and has important significance on rapid detection of enterobacter capable of producing A / B carbapenemase, guidance of clinical medication, reduction of antibiotic abuse and the like.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and relates to a detection technology based on high-performance liquid chromatography tandem mass spectrometry. A kit and method for class A and class B enzyme type detection. Background technique [0002] β-lactam antibiotics refer to a large class of antibiotics with a β-lactam ring in their chemical structure (see appendix figure 1 ), including penicillins, cephalosporins, monobactams (aztreonam), and carbapenems and other antibiotics. Carbapenems are currently a type of β-lactam antibiotics with the strongest antibacterial activity and the broadest antibacterial spectrum. infection, representative drugs include imipenem, meropenem, ertapenem, etc. Enterobacteriaceae bacteria mainly include Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae and Proteus mirabilis, which are common pathogens causing nosocomial infections. In recent years, with the irrational use of an...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06G01N30/30G01N30/32G01N30/34G01N30/72G01N30/86
CPCG01N30/02G01N30/06G01N30/30G01N30/32G01N30/34G01N30/72G01N30/8675G01N2030/324Y02A50/30
Inventor 范列英李根叶致含陈念贞
Owner SHANGHAI EAST HOSPITAL EAST HOSPITAL TONGJI UNIV SCHOOL OF MEDICINE
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