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Detection method for O antigen molecular typing of citrobacter O4 and O12 serotype

A technology of citric acid bacteria and detection method, applied in the direction of microorganism-based method, biochemical equipment and method, microorganism measurement/inspection, etc., to achieve the effect of simple operation, strong repeatability and high accuracy

Inactive Publication Date: 2020-10-30
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, serotyping schemes based on O antigen changes are still the mainstream method for the detection and identification of Gram-negative pathogens in clinical specimens and environmental samples.

Method used

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  • Detection method for O antigen molecular typing of citrobacter O4 and O12 serotype
  • Detection method for O antigen molecular typing of citrobacter O4 and O12 serotype
  • Detection method for O antigen molecular typing of citrobacter O4 and O12 serotype

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Primer design

[0028] 1. Screening of specific genes

[0029] The O-antigen processing genes wzy, wzx, wzm, and wzt are highly serotype-determining and thus have been widely used as target genes for molecular serotyping of many Gram-negative bacteria. Citrobacter O12, select wxya as a specific gene. But in Citrobacter O4, because there are no specific genes like wzx / wzy, only relatively specific genes can be selected, usually some rare monosaccharide genes or glycosyltransferase genes, O4 serotype is the selection Glycosyltransferase gene GT was used as a specific gene.

[0030] 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 primers for them.

[0031] 2. Design of primers

[0032]The primers for LAMP were designed using the selecte...

Embodiment 2

[0038] Extraction of sample nucleic acid (crude extract of a pure culture of a sample isolated from any environment suitable for the life of Citrobacter)

[0039] 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).

[0040] Dilute the stock solution with sterile water for another 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).

[0041] 2. Sample processing: Take 1 mL of overnight cultured bacterial liquid and add it to a 1.5 mL centrifuge tube, centrifuge at 8000 rpm fo...

Embodiment 3

[0052] Positive and Specific Detection

[0053] The extracted nucleic acid solution is used as the template for the LAMP reaction, the LAMP reaction system and reaction conditions:

[0054] 1. Example of LAMP reaction system (25 µL):

[0055]

[0056] 2. LAMP reaction conditions:

[0057]

[0058] 3. Test results:

[0059] See figure 1 As shown, the genomes of Citrobacter O12, O15, O20, O22, O24, O29, O32, O39 and O41 were added to the LAMP system of Citrobacter genome sample O4, and the LAMP primers of Citrobacter O4 had good specificity.

[0060] See figure 2 As shown, the genomes of Citrobacter O4, O15, O20, O22, O24, O29, O32, O39 and O41 were added to the LAMP system of Citrobacter genome sample O12, and the LAMP primers of Citrobacter O4 had good specificity.

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Abstract

The invention relates to a loop-mediated isothermal amplification (LAMP) detection method for O antigen typing of Citrobacter youngae (CY) O4 serotype and Citrobacter gillenill (CG) O12 serotype. According to the invention, specific genes in O antigen gene clusters of Citrobacter youngae O4 and O12 are used, GT and wzx are taken as target genes, four primers for O antigen typing of Citrobacter youngae O4 and O12 are designed and screened, and a credible way is provided for O antigen typing of Citrobacter youngae in food, water, soil and intestinal tracts. The LAMP primers are used for detecting Citrobacter youngae in food water, soil and intestinal tracts, O antigen typing is carried out on Citrobacter youngae, and the LAMP primers have the advantages of being easy to operate, rapid, efficient, high in sensitivity and the like.

Description

technical field [0001] The invention relates to a LAMP technology and a preparation method for typing O antigens of Citrobacter O4 and O12 serotype strains in samples. The present invention also designs a detection method using the LAMP primers. Background technique [0002] Citrobacter is a facultative anaerobic Gram-negative bacilli belonging to the family Enterobacteriaceae. The cell size in liquid medium is 6.0*1-2μm. Usually uncapsulated and moves with peripsiform hairs. Metabolism includes respiration and fermentation. Citrobacter strains are usually isolated from animal and human water, soil, food and gut. May cause bacteremia, meningitis, sepsis, peritonitis, urinary tract infection, respiratory tract infection and postoperative infection, especially in infants, minor children under 6 years old and immunocompromised people, the infection rate of this bacteria is higher high. In particular, invasive Citrobacter infections are associated with high mortality, with...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/10C12N15/11C12R1/01
CPCC12Q1/689C12Q1/6844
Inventor 王磊朱文轩郭玺刘斌鲁阁阁
Owner NANKAI UNIV
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