Method for rapidly and quantitatively detecting enterobacter sakazakii based on cascade amplification

A technology for quantitative detection of Enterobacter sakazakii, applied in biochemical equipment and methods, measurement/inspection of microorganisms, resistance to vector-borne diseases, etc., to achieve the effect of ensuring specificity, high biological activity, and easy operation

Active Publication Date: 2022-02-08
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The visual detection method of Enterobacter sakazakii in dairy products based on antibody-aptamer immune sandwich-assisted EXPAR cascade amplification has not been reported yet

Method used

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  • Method for rapidly and quantitatively detecting enterobacter sakazakii based on cascade amplification
  • Method for rapidly and quantitatively detecting enterobacter sakazakii based on cascade amplification
  • Method for rapidly and quantitatively detecting enterobacter sakazakii based on cascade amplification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Principle of rapid detection method for Enterobacter sakazakii

[0047] The principle of rapid quantitative and visual detection of Enterobacter sakazakii figure 1 As shown, first, the classic EDC / NHS method is used to construct immunomagnetic beads, that is, to couple antibodies to the magnetic beads; when the target exists, the antibody can be used as a capture recognition element, and then the fusion nucleic acid aptamer (IAPT) is added to form Highly specific sandwich structure. In addition, since the fusion nucleic acid aptamer contains the amplification template of EXPAR, Nt.BstNBI Dicer recognition site and G-rich antisense sequence, so in Bst. Under the action of DNA polymerase, EXPAR can be triggered, and the product contains a large number of G-rich sequences; this sequence combines with Hemin to form G4 / HeminDNAzyme, which catalyzes H 2 o 2 The colorless TMB is oxidized to blue, and finally the experimental results can be evaluated by the di...

Embodiment 2

[0051] Example 2 Construction of immunomagnetic beads and related characterization

[0052] Carboxyl magnetic beads were washed with 500 μL of reaction buffer (0.05 M 2(N-morpholine)ethanesulfonic acid, 0.5 M NaCl, pH=6.0) for three times and then magnetically separated and activated by classical EDC / NHS method. Then add 200 μL of coupling solution (0.1M sodium phosphate, 0.15 M NaCl, pH=7.5) to wash the activated magnetic beads three times, and remove the supernatant after separation. Dissolve 0.5 mg antibody in 0.5 mL coupling solution as stock solution, add antibody to magnetic beads according to the ratio of magnetic beads:antibody = 10:1, shake and couple at room temperature for 2 h, perform magnetic separation to remove supernatant, and then add 200 μL of blocking solution (add 1 M glycine to the coupling solution, pH=8.5), shake at room temperature for 30 min, block unreacted activating groups, and finally dilute to working concentration with preservation solution and s...

Embodiment 3

[0054] Example 3 Feasibility of detection of Enterobacter sakazakii

[0055] After the immune sandwich structure was constructed, the EXPAR reaction was carried out, and the specific EXPAR reaction system is shown in Table 2. In order to verify the feasibility of using cascade amplification to detect Enterobacter sakazakii, the occurrence of EXPAR amplification was first verified by gel electrophoresis.

[0056] Table 2 EXPAR reaction system

[0057]

[0058] The result is as image 3 As shown in A, the clear band in lane 1 represents IAPT while the cDNA (lane 2) has no obvious band due to its short single strand. Lane 3 represents the hybridization result of IAPT and cDNA, compared with lane 1, its mobility is reduced, indicating that the two hybridize successfully. Only when IAPT, cDNA, Bst . DNA polymerase, Nt.BstNBI The characteristic band of lane 8 will appear only when cleavage enzyme and dNTP co-exist, which indicates the generation of short DNA fragments (i.e...

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Abstract

The invention constructs a biosensor for rapidly and quantitatively detecting enterobacter sakazakii based on cascade amplification. According to the sensor, quadruple cascade amplification of enterobacter sakazakii is realized through magnetic enrichment, antibody-nucleic acid aptamer recognition, EXPAR and DNAzyme signal accumulation. A method comprises steps: firstly, immunomagnetic beads are prepared for magnetic enrichment; secondly, a target is directly captured from a complex matrix through magnetic separation, then a nucleic acid aptamer is added, an immune sandwich structure of an antibody-target-fusion nucleic acid aptamer is formed, and high specificity of the sensor is guaranteed; thirdly, exponential amplification of the signal is realized through EXPAR; and finally, a quadruplex with enzyme-like activity is formed through a G-rich sequence in the product, and color development signal output is catalyzed. The method has the advantages of high sensitivity, high specificity, rapidness, simplicity and convenience, the quantitative detection of enterobacter sakazakii can be realized within 2 hours, the whole process is extraction-free, the result is visual, and the problems of complex sensor preparation and long determination time are effectively solved.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a method for rapidly and quantitatively detecting Enterobacter sakazakii by using cascade amplification. Background technique [0002] Enterobacter sakazakii ( Cronobacter spp.) is an opportunistic pathogen mainly present in milk powder, which can cause infantile meningitis, necrotizing enterocolitis, sepsis and other diseases, which is very detrimental to the healthy growth of infants. In addition, people with low immunity, especially the elderly, may also be infected. In some cases, the mortality rate of diseases caused by E. sakazakii can reach 40% to 80%. Therefore, it is necessary to develop a method for rapid detection of Enterobacter sakazakii. [0003] At present, the detection of Enterobacter sakazakii mainly includes traditional microbial culture methods, immunological detection methods, and modern molecular biotechnology detection methods. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6804C12Q1/10
CPCC12Q1/6804C12Q2525/205C12Q2563/143C12Q2563/149C12Q2531/119C12Q2537/1376C12Q2521/301C12Q2565/607C12Q2521/345Y02A50/30
Inventor 朱龙佼许文涛许秀媛田洪涛田晶晶
Owner CHINA AGRI UNIV
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