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Method and biosensor for detecting target microbe

A technology for target microorganisms and biosensors, applied in a method for detecting target microorganisms and the field of biosensors, can solve the problems of detection of pathogenic microorganisms without nano-gold and aptamers, and achieve rapid detection, low-tech content, and avoidance of steps. Effect

Inactive Publication Date: 2012-08-22
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, there is no report on the combination of gold nanoparticles and aptamers for the detection of pathogenic microorganisms

Method used

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  • Method and biosensor for detecting target microbe
  • Method and biosensor for detecting target microbe
  • Method and biosensor for detecting target microbe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1. Preparation of Biosensor I

[0030] Material:

[0031] Gold nanoparticles: commercially purchased from Haide Biotechnology Co., Ltd., the product number is 25516.

[0032] Listeria monocytogenes aptamer A8:

[0033] 5’-ATC CAT GGG GCG GAG ATG AGG GGG AGG AGG GCG GGT ACC CGG TTG AT-3’

[0034] The 5' end was extended and thiol-modified, and Shanghai Bioengineering Co., Ltd. was commissioned to synthesize it to obtain an aptamer for the test. Its sequence is as follows:

[0035] 5'- TTTATGTTG ATC CAT GGG GCG GAG ATG AGG GGG AGG AGG GCG GGT ACC CGGTTG AT-3’

[0036] Adapter complementary chain, its sequence is as follows:

[0037] 5'-TAGGTACCCCGCCTCTACTCCCCCTCCTCCCGCCCATGGGCCAACTA-3'

[0038] method:

[0039] Step 1. Preparation of gold nanoparticles I and biosensor I

[0040] Prepare EP tube 1, add 400uL of gold nanoparticles and 100uL of 2.5uM aptamer complementary strand; adjust the pH value to 8.2, and place it at room temperature in the dark for...

Embodiment 2

[0043] Example 2. Preparation of Biosensor II

[0044] Step 1. Preparation of gold nanoparticles I and gold nanoparticles II

[0045] Material: gold nanoparticles: aptamer and its complementary chain are the same as in Example 1

[0046] Prepare EP tube 1, add 400uL of gold nanoparticles and 100uL 2.5uM aptamer complementary strand; prepare EP tube 2, add 400uL of gold nanoparticles and 100uL 2.5uM aptamer. Adjust the pH value to 8.2, and place it at room temperature to avoid light for 24 hours. Then put the two EP tubes in a high-speed centrifuge at 16000rp / min, centrifuge for 20 minutes, discard the supernatant (remove excess aptamer and its corresponding complementary chain), then wash with sodium citrate 4mM, and repeat the centrifugal washing Once, tube 1 and tube 2 contained gold nanoparticles I and II, respectively.

[0047] Step 2. Construction of Biosensor II

[0048] Materials: PBS solution: 10 mM Tris-HCl (pH 7.0), 4 mM MgCl2, 10 μM dithiothreitol.

[0049] Dis...

Embodiment 3

[0051] Example 3. Biosensor Sensitivity Detection

[0052] Materials: The standard strain of Listeria monocytogenes was purchased from the American Standard Biological Collection (ATCC), with the strain number 15313. It is also preserved in our laboratory and can be distributed to the public for verification tests.

[0053] Step 1: Suspend Listeria monocytogenes in PBS solution and perform the following doubling dilutions:

[0054] 8 samples: blank control, 1×10 2 CFU / ml, 1×10 3 CFU / ml, 1×10 4 CFU / ml, 1×10 5 CFU / ml, 1×10 6 CFU / ml, 1×10 7 CFU / ml. Put it in a refrigerator at 4 degrees Celsius for 2 minutes Take 8 EP reaction tubes, add 200 uL of the PBS solution of the biosensor II prepared in Example 2, adjust the pH value between 6.5-6.8, and put it in a refrigerator at 4 degrees Celsius for 2 minutes. Add 500 uL of 8 sample bacterial suspensions to 8 EP reaction tubes respectively, place them in a refrigerator at 4 degrees Celsius for 2 minutes, observe the color chang...

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Abstract

The invention relates to a method and a biosensor for detecting a target microbe, and belongs to the field of biological detection. The method is characterized in that a bacterial suspension prepared by a microbe to be detected is added into a colloidal gold particle solution containing aptamers and aptamer complementary strands of the target microbe for incubation; the aptamer complementary strands are connected to surfaces of the colloidal gold particle I; detection results are determined by the solution color change caused by the change of the aggregation or separation condition of the colloidal gold particles due to the recognition and combination of the target microbe with the aptamer. The invention also provides a biosensor based on the method. The invention can simply, rapidly, and accurately detect the presence of the target microbe, such as pathogenic bacteria, viruses, and the like.

Description

technical field [0001] The invention relates to a biological detection reagent, in particular to a method for detecting target microorganisms and a biological sensor. Background technique [0002] In 1990, two scientists screened the oligonucleotide sequences that specifically bind to organic dyes and phage DNA polymerases from artificially constructed oligonucleotide libraries by using a new technique combining in vitro screening and PCR. This new technique was later named systematic evolution of ligands by exponential enrichment (SELEX), and the screened oligonucleotides were named aptamers. [0003] Aptamers are artificially synthesized single-stranded oligonucleotides (DNA or RNA), which can bind to target substances with high specificity and affinity. Its function is similar to that of antibodies, but it has more advantages than antibodies, including the following Several aspects: (1) The range of target molecules is wide. This can range from small molecules such as m...

Claims

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Application Information

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IPC IPC(8): G01N21/78C12Q1/68C12Q1/04
Inventor 陈栋府伟灵王丰黄庆张晓莉黄君富苗杰刘星
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA