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Detection method formolecular typing of acinetobacter baumannii (Ab) Sv4-serotype O antigen

A technology for Acinetobacter baumannii, serotypes, applied in microorganism-based methods, biochemical equipment and methods, and microbial assay/inspection, etc. Highly repeatable and accurate results

Pending Publication Date: 2020-02-21
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at the serum level, identification using Bowman antiserum is limited by variability, availability, and specificity, so a more specific and feasible molecular typing scheme is needed

Method used

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  • Detection method formolecular typing of acinetobacter baumannii (Ab) Sv4-serotype O antigen
  • Detection method formolecular typing of acinetobacter baumannii (Ab) Sv4-serotype O antigen
  • Detection method formolecular typing of acinetobacter baumannii (Ab) Sv4-serotype O antigen

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Design of primers and probes

[0026] 1. Screening of specific genes

[0027]Acinetobacter baumannii Sv4 serotype O antigen synthesizes O antigen through two pathways, one is wzy The O antigen is synthesized through the ABC-transporter pathway, and the O antigen is synthesized through the ABC-transporter pathway. In the former, wxya / wzy Genes are very specific and can be directly used as candidate genes for designing probes, and in the latter, because there is no wxya / wzy Such a specific gene, so only relatively specific genes can be selected, usually some rare monosaccharide genes or glycosyltransferases. In the present invention, the all_vs_all_blast method is performed on all the genes in the gene cluster for comparison, and the matching number of the specific gene must be much smaller than that of the conservative gene. Combine the above methods to find specific genes and design probe primers for them.

[0028] 2. Design of primers and probes

[00...

Embodiment 2

[0037] Extraction of sample nucleic acid (separated from any environment suitable for the life of Acinetobacter baumannii, the crude extract of the pure culture of the sample)

[0038] 1. Sample processing: Take 1 mL of the overnight cultured bacterial solution and add it to a 1.5 mL centrifuge tube, centrifuge at 8000 rpm for 1 min at room temperature, discard the supernatant, and collect the bacteria. Add 400 µL Buffer Digestion, vortex to mix, and bathe in water at 65 °C for 1 h until the cells are completely lysed.

[0039] During the water bath process, invert and mix once every 10 minutes, which can promote the lysis of the sample, and the mixed solution becomes clear and transparent, indicating that the lysis is complete;

[0040] 2. Add 200 µL Buffer PB, mix thoroughly by inversion, and place in -20 ℃ refrigerator for 5 min;

[0041] 3. Centrifuge at 10,000 rpm for 5 minutes at room temperature, and transfer the supernatant (500-550 µL) to a new 1.5 mL centrifuge tube...

Embodiment 3

[0049] Sample PCR amplification

[0050] Use the extracted nucleic acid solution as a template for PCR reaction, PCR reaction system and reaction conditions:

[0051] PCR reaction system (25 µL):

[0052]

[0053]

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Abstract

The invention relates to analysis of an acinetobacter baumannii (Ab) Sv4-serotype O antigen by using an MGB probe-based real-time fluorescent TaZMan polymerase chain reaction rapid detection system. Aspecific gene, Wzy, in an acinetobacter baumannii O antigen gene cluster is adopted as a target gene so as to establish a primer and MGB probe for typing the acinetobacter baumannii O antigen, and areliable route is provided for typing of the O antigen of acinetobacter baumannii in blood, urine, pus and a respiratory tract. When the MGB probe is applied to detection of acinetobacter baumannii inthe blood, urine, pus and respiratory tract, and O-antigen typing of acinetobacter baumannii is performed, high sensitivity, high specificity, rapid detection and other advantages are achieved.

Description

technical field [0001] The invention relates to a Taqman-MGB technique for typing the O antigen of the Acinetobacter baumannii Sv4 serotype strain in a sample and a preparation method thereof. The present invention also designs a detection method using the MGB probe. Background technique [0002] Acinetobacter baumannii, a Gram-negative bacterium, is a strictly aerobic, non-lactose fermenting opportunistic pathogen. Acinetobacter baumannii is an important pathogen of nosocomial infection. It mainly causes respiratory tract infection, and can also cause bacteremia, urinary tract infection, secondary meningitis, surgical site infection, and ventilator-associated pneumonia. Its resistance rate to commonly used antibiotics has an increasing trend year by year, and has caused serious concern for clinicians and microbiologists. However, at the serum level, identification using Bowman antiserum is limited by variability, availability, and specificity, so a more specific and feasi...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6858C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689C12Q1/6858C12Q2600/156C12Q2531/113C12Q2561/101
Inventor 王磊刘倩宁可馨李樾华曹勃阳
Owner NANKAI UNIV
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