Detection method for molecular typing of O antigens of serotypes such as providencia O19, O20, O28, O30 and like

A Providencia, O antigen technology, applied in the field of Taqman-MGB technology and its preparation, can solve the problems of variation availability and specificity, and achieve the effects of high accuracy, strong repeatability and easy operation

Inactive Publication Date: 2020-10-09
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the widespread use of antibiotics and immunosuppressants, drug-resistant bacteria are on the rise, but at the serum level, identification using wound antisera 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 for molecular typing of O antigens of serotypes such as providencia O19, O20, O28, O30 and like
  • Detection method for molecular typing of O antigens of serotypes such as providencia O19, O20, O28, O30 and like
  • Detection method for molecular typing of O antigens of serotypes such as providencia O19, O20, O28, O30 and like

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Design of primers and probes

[0035] 1. Screening of specific genes

[0036] Providencia O19, O20, O28, O30, O31, O36 serotype O antigens synthesize O antigens through two ways, 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.

[0037] 2. Design of primers and pr...

Embodiment 2

[0046] Extraction of sample nucleic acid (separated from any environment suitable for the life of Providencia bacteria, the crude extract of the pure culture of the sample)

[0047] 1. Dilute the sample to be tested with sterile water, usually the dilution factor is 1:10 (for example, 10g solid sample or 10ml liquid sample is dissolved in 90ml sterile water) to make a bacterial liquid mother solution (only the liquid is taken).

[0048] Dilute the bacterial solution mother solution with sterile water for 5 gradients: 1×10 -1 , 1×10 -2 , 1×10 -3 , 1×10 -4 , 1×10 -5 , each gradient bacterial solution was evenly spread on LB solid medium and cultured at 37°C. When a single colony grows on the plate, pick up the colony and inoculate it in LB liquid medium, and cultivate overnight at 37°C and 180rpm (this step of inoculation is performed in a super-clean bench).

[0049] 2. Sample processing: Take 1 mL of overnight cultured bacterial liquid and add it to a 1.5 mL centrifuge tu...

Embodiment 3

[0060] positive test

[0061] 1. Real-Time PCR system (25 µL, take O19 as an example)

[0062]

[0063] 2. Real-Time PCR reaction conditions:

[0064]

[0065] 3. Test results

[0066] figure 1 It is the detection results of the probes for each serotype of Providencia bacteria. The results show that each reaction system has a good amplification curve, indicating that the detection system of each serotype can achieve accurate detection of the corresponding serotype.

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Abstract

For O antigens of Providencia O19, O20, O28, O30, O31 and O36 serotypes, a real-time fluorescent TaqMan polymerase chain reaction rapid detection system based on an MGB probe is used for analysis. According to the invention, the specific gene of the O antigen gene cluster of the Providencia is used as a target gene; a primer and an MGB probe for O antigen typing of the Providencia are designed andscreened, a corresponding real-time fluorescent PCR (RT-PCR) detection method is established, and a credible way is provided for O antigen typing of the aeromonas hydrophila by utilizing a molecularbiological means. The MGB probe disclosed by the invention is used for detecting the prowedenia in excrement, urethral infection, wounds, burns and bacteremia specimens, and O antigen typing is carried out on the Providencia, so that the method has the advantages of high sensitivity, high specificity, rapid detection and the like.

Description

technical field [0001] The invention relates to a Taqman-MGB technique for typing O antigens of Providencia bacteria O19, O20, O28, O30, O31 and O36 serotype strains in samples and a preparation method thereof. The present invention also designs a detection method using the MGB probe. Background technique [0002] Providencia, 0.6-0.8×1.5-2.5μm, conforms to the general definition of Enterobacteriaceae, facultatively anaerobic, belongs to Gram-negative straight bacteria, moves with perinatal flagella, and does not appear to cluster. Oxidizes desaminophenylalanine and tryptophan. Isolated from diarrheal stools, urinary tract infections, wounds, burns and bacteremia specimens. Providencia is an opportunistic pathogen that can cause diarrhea, extraintestinal infections, and urinary tract infections. Common Providencia bacteria include P.alcalifaciens, P.rettgeri, P.rustigianii, Wittenella (P. stuartii). With the widespread use of antibiotics and immunosuppressants, drug-res...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11
CPCC12Q1/689C12Q1/686C12Q2600/16
Inventor 王磊鲁阁阁夏香红刘斌郭玺
Owner NANKAI UNIV
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