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A screening method and mcr-1 technology, applied in the fields of molecular biology and medical microbiology, can solve the problems of poor plate screening method and low positive rate, and achieve the effect of reducing the workload of PCR
Inactive Publication Date: 2017-07-21
张嵘
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Problems solved by technology
However, the strains carrying the mcr-1 gene have a minimum inhibitory concentration (Minimum Inhibitory Concentration, MIC) of about 2-16ug / ml to polymyxin, and the two-component regulatory system mediated by PmrA / PmrB and PhoP / PhoQ The MIC value of the bacteria is much higher than that of the bacteria carrying the mcr-1 gene, and the effect of the plate screening method is poor
In the early stage, our research team used the plate screening method to screen children's intestinal samples, and found that the positive rate of the test was as low as 2.14%.
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Embodiment 1
[0029] 1. Sample Inoculation
[0030] Inoculate a matchhead-sized sample onto an SS plate containing 2ug / ml, and incubate in a 37°C incubator for 18-24 hours. The composition of the SS plate is shown in Table 1.
[0031] Table 1: SS Tablet Composition List
[0032] ingredient name content Peptone (tryptone) 5.0g / L Beef Extract 5.0g / L lactose 10.0g / L Agar 20.0g / L Neutral red 0.025g / L Sodium Thiosulfate 8.5g / L No.3 Bile Salt 3.5g / L Sodium citrate 8.5g / L Ferric citrate 1.0g / L Brilliant Green 0.00033g / L
[0033] 2. Isolation of mcr-1 positive bacteria
[0034] Pick a single colony on the plate and transfer it to a new plate for purification, and incubate in a 37°C incubator for 18-24 hours; pick the above-mentioned single colony for PCR detection of mcr-1. PCR electrophoresis picture as figure 1 As shown, those with the same band as the positive control are positive for mcr-1; further screen ...
Embodiment 2
[0036] 1. Sample pretreatment
[0037] Inoculate the sample into 5ml of nutrient broth, and incubate the inoculated broth sample in a 37°C incubator for 24-48 hours for testing. The composition of the nutrient broth is shown in Table 2.
[0038] Table 2: Nutrient Broth Composition List
[0039] ingredient name content Peptone (tryptone) 10.0 / L Yeast extract 5g.0 / L NaCl 10.0g / L
[0040] 2. Direct PCR detection of polymyxin resistance gene mcr-1 in broth samples after enrichment:
[0041] 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 Suspend the sediment in 70ul ultrapure water, boil the suspension in boiling water for 8-10min to free the DNA; centrifuge the suspension at 8000r / min for 3min, and the supernatant is the DNA template for the PC...
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Abstract
The invention discloses a method for screening out bacteria of a plasmid or chromosome mediated polymyxin drug-resistant gene mcr-1 from an intestinal / environmental sample. After bacterium increase of the sample, direct PCR detection is performed, a mcr-1 positive broth sample is further subjected to bacterium increase, screening and purification, mcr-1 of purified bacteria is detected through PCR, and a mcr-1 positive bacterium is screened out. According to the method, the mcr-1 positive broth sample is firstly obtained, the interference in a drug-containing flat plate screening method due to mediated polymyxin drug-resistant bacteria of PmrA / PmrB and PhoP / PhoQ bi-component regulation and control systems is avoided, then the mcr-1 positive broth sample is only inoculated to a drug-containing enterobacterium increase broth and a flat plate, the PCR working amount of purification and later purified bacteria is decreased through rescreening of the drug-containing enterobacterium increase broth and the flat plate, and the method can be applied to screening of the positive bacteria of the drug-resistant gene mcr-1 from the intestinal / environmental sample.
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 colistin-resistant gene mcr-1 bacteria from human or animal intestines and environmental 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. β-lactam antibiotics, which are widely used clinically, have grown at an alarming rate due to the emergence of plasmid-mediated drug-resistant genes. 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 Surveillance Network (The Eu...
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