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MGB probe multiple fluorescence quantitative PCR method for detecting colibacillus 0157

A multiple fluorescence quantitative, Escherichia coli technology, applied in the direction of fluorescence/phosphorescence, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problems of single determination of bacteria and inability to detect virulence factors, etc., and achieve high sensitivity and specific effects

Inactive Publication Date: 2007-04-25
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This article failed to use the fluorescent quantitative PCR method to quantify the sample. In addition, the above method can only determine the serotype of the bacteria alone, and cannot detect whether it contains important virulence factors

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] 1 Design, synthesis of rfbE, vt2 primers and probes

[0061] The primer sequence is

[0062] rfbE-F cta gga ccg cag agg aaa gag a

[0063] rfbE-R ttc cga gta cat tgg cat cgt

[0064] vt2-F acc aca tcg gtg tct gtt att aac c

[0065] vt2-R aac gaa ccc ggc cac ata ta

[0066] The Taqman-MGB probe sequence is

[0067] rfbE-probe att aag gaa tca cct tgc aga t

[0068] vt2-probe ttt gct caa taa tca gac gaa

[0069] 2 Cloning of rfbE and vt2 genes

[0070] According to the designed rfbE and vt2 primers, the target genes of rfbE and vt2 were amplified respectively, and the PCR products were electrophoresed on 2% agarose gel, and then analyzed by the agarose gel imaging analysis system, and the target bands were visible at about 100bp and 150bp . The gene of interest was recovered from the PCR product.

[0071] 3 Preparation of standard samples

[0072] Preparation of DH5α Competent Escherichia coli Cells

[0073] Pick a single colony from the Escherichia coli DH5α pl...

Embodiment 2

[0077] 1 Pre-enrichment of clinical samples

[0078] Mince 1 gram of meat sample, put into 9 ml of modified broth containing novobiocin (20 mg / ml), shake at 37°C, 200 rpm for 8 h.

[0079] 2 Genomic DNA extraction

[0080] Take 3ml of the bacterial solution cultured by shaking, 12000rpm, 5min; resuspend the pellet with 567ul of TE (pH 8.0), add 30ul of 10% SDS and 3ul of 20mg / ml proteinase K, mix well, and put in a water bath at 60°C for 20 min; add 100ul of 5mol / ml proteinase K LNacl solution, mix well, then add 80ul CTAB / Nacl solution, mix well, 60°C water bath for 20min; add 780ul phenol:chloroform, mix well, 12000rpm, 10min; transfer 600ul supernatant into a new tube. Repeat the previous step; take 400ul supernatant to a new tube; add 400ul chloroform, mix well, 12000rpm, 10min, absorb 300ul supernatant; add twice the volume of ice-cold ethanol, mix well, place at -20°C for 30min, 12000rpm, 20min , the precipitate was washed three times with 70% ethanol, dried in a 37°C ...

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Abstract

A method of testing colibacillus O157 with MGB probe multiline fluorescence gauging PCR, is disclosed, the recombinant plasmid is constructed, the synthesized primer and the Taqman-MGB probe are designed, diploid fluorescence gauges PCR, the segment of saving sequences of rfbE, vt2 of the colibacillus O157 are augmented by PCR to obtain the goal segment, the goal segment is connected with PMD 18-T Vector to obtain the recombinant plasmid, then the recombinant plasmid is converted to DH5a competence bacillus coli cell, enlarged the culture to obtain the recombinant plasmid with high concentration, then the recombinant plasmid with high concentration is diluted by 10 times ratio to be the standard sample, the standard sample and the sample to be measured are combined together to gauge the PCR by diploid fluorescence, the calibration curve is formed by the standard sample, the content of the colibacillus O157 can be calculated in the sample to be measured based on the calibration curve. The MGB probe is used in the invention that has higher sensibility and specificity, and detects the content of the colibacillus O157 in the sample quickly and precisely; two specificity virogenes of the colibacillus O157 can be detected at the same time, so it is a quick and precise method of testing colibacillus O157.

Description

technical field [0001] The invention relates to a detection method in the field of biotechnology, in particular to a method for detecting Escherichia coli O157 by multiple fluorescent quantitative PCR with MGB probes. Background technique [0002] Escherichia coli O157 is a highly pathogenic intestinal pathogen, mainly causing hemorrhagic enteritis and hemolytic uremic syndrome in humans and animals, resulting in relatively high mortality. The infection dose of Escherichia coli O157 is relatively low, and more than 10 bacteria per gram of infection carrier may cause infection and even cause death. Therefore, it is urgent to establish a rapid and sensitive detection method. The current industry standard for detecting O157 in food in my country is to adopt the classic selective isolation culture, biochemical test and serological methods. The whole process takes 4-6 days, which is time-consuming and cumbersome to operate, and cannot meet the timely, rapid and correct evaluatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64G01N21/76C12Q1/10
Inventor 严亚贤朱向玲孙建和陆承平
Owner SHANGHAI JIAO TONG UNIV
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