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Method for screening mcr-3 positive bacteria in intestinal sample through alkaline peptone water bacteria proliferation

A peptone and positive technology, applied in the fields of molecular biology and medical microbiology, can solve difficult problems such as infection treatment, and achieve the effect of reducing the workload of PCR

Inactive Publication Date: 2017-11-24
张嵘
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, carrying the carbapenem resistance gene (bla NDM and bla KPC ) and mcr‐1 Enterobacteriaceae, making the treatment of infections caused by multidrug-resistant bacteria more difficult

Method used

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  • Method for screening mcr-3 positive bacteria in intestinal sample through alkaline peptone water bacteria proliferation
  • Method for screening mcr-3 positive bacteria in intestinal sample through alkaline peptone water bacteria proliferation
  • Method for screening mcr-3 positive bacteria in intestinal sample through alkaline peptone water bacteria proliferation

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Experimental program
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Effect test

Embodiment 1

[0026] 1. Sample pretreatment

[0027] Inoculate the sample into 5ml of nutrient broth, and incubate the inoculated broth sample in a 37°C incubator for 24‐48 hours, ready for testing. The composition of the nutrient broth is shown in Table 1.

[0028] Table 1: List of Nutrient Broth Components

[0029] ingredient name

content

Peptone (tryptone)

10.0g / L

Yeast extract

5.0g / L

NaCl

10.0g / L

[0030] 2. Direct PCR detection of polymyxin resistance gene mcr‐3 in broth samples after enrichment:

[0031] Take 1ml of the above enrichment solution into a 1.5ml EP tube, centrifuge at 8000r / min for 3min, remove the supernatant; add 1ml of normal saline to suspend the sediment, wash thoroughly, centrifuge at 8000r / min for 3min, remove the supernatant; Add 70 μl of ultrapure water to suspend the precipitate, put the suspension in boiling water for 8‐10 minutes to free the DNA; centrifuge the suspension at 8000 r / min for 3 minutes, and th...

Embodiment 2

[0043] 1. Sample pretreatment

[0044] Inoculate the sample into 5ml of alkaline peptone water, and incubate the inoculated broth sample in a 37°C incubator for 24‐48 hours for testing. The composition of alkaline peptone water is shown in Table 4, and the pH is 8.8±0.4.

[0045] Table 4: Alkaline Peptone Water Table

[0046] ingredient name

content

Peptone (tryptone)

15.0g / L

Beef Extract

4.0g / L

NaCl

10.0g / L

[0047] 2. Direct PCR detection of polymyxin resistance gene mcr‐3 in broth samples after enrichment:

[0048] Take 1ml of the above enrichment solution into a 1.5ml EP tube, centrifuge at 8000r / min for 3min, remove the supernatant; add 1ml of normal saline to suspend the sediment, wash thoroughly, centrifuge at 8000r / min for 3min, remove the supernatant; Add 70 μl of ultrapure water to suspend the precipitate, put the suspension in boiling water for 8-10 minutes to free the DNA; centrifuge the suspension at 8000r / min ...

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Abstract

The invention discloses a method for screening plasmid or chromosome-encoded polymyxin drug resistance gene mcr-3 bacteria in an intestinal sample through alkaline peptone water bacteria proliferation. The method comprises the following steps: after inoculating the intestinal sample to alkaline peptone water for bacterial proliferation, carrying out direct PCR (Polymerase Chain Reaction) detection on broth; inoculating an mcr-3 broth sample obtained through the direct PCR detection to an SS (Salmonella Shigellaagar) flat plate for screening, separating and purifying; carrying out secondary PCR detection on the separated and purified bacteria, thus screening out mcr-3 positive bacteria. According to the method disclosed by the invention, firstly, through alkaline peptone water bacteria proliferation enrichment, separation of aeromonas, vibrio and the like is increased; secondly, through obtained mcr-3 positive alkaline peptone water, the PCR workload of separation and purification and separated and purified bacteria in the later period can be reduced, and the method can be applied to the intestinal sample for screening drug resistance gene mcr-3 positive bacteria.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and medical microbiology, and in particular relates to a method for screening out plasmid or chromosome-mediated polymyxin-resistant gene mcr-3 bacteria from human or animal intestinal samples. Background technique [0002] Antibiotic resistance is one of the biggest challenges in global public health in the 21st century. The extensive use of many antibiotics has led to the continuous growth of drug-resistant strains. Due to the emergence of plasmid-mediated drug-resistant genes for β-lactam antibiotics, which are widely used clinically, the drug-resistant strains are increasing at an alarming rate. Extended-spectrum beta-lactamases (ESBLs) have attracted widespread attention due to their strong hydrolytic activity against antibiotics such as third- and fourth-generation cephalosporins and aztreonam. Since the beginning of this century, The European Antimicrobial Resistance Surveillan...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/10C12N1/20
CPCC12N1/20C12Q1/686C12Q2531/113Y02A50/30
Inventor 张嵘王汉宇黄子衔孙巧玲李佳萍
Owner 张嵘
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