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Method for rapidly detecting bacteria in complex sample

A technology for complex samples and detection methods, applied in the field of molecular biology, can solve the problems of limited scalability, high price, difficult one-time use, etc., and achieve the effect of simple experimental steps, flexible and simple operation, and low price

Active Publication Date: 2021-02-26
ZHEJIANG UNIV
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AI Technical Summary

Problems solved by technology

[0006] The existing digital loop-mediated isothermal amplification method (digitalLAMP) based on the microfluidic chip array reaction chamber has relatively high design and use costs, limited scalability, and more importantly, the processing process of the microfluidic device is too cumbersome. Moreover, it is expensive and difficult to use at one time, and this method also has disadvantages such as low light-to-dark ratio and easy to be affected by inhibitors.

Method used

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  • Method for rapidly detecting bacteria in complex sample
  • Method for rapidly detecting bacteria in complex sample
  • Method for rapidly detecting bacteria in complex sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Analysis of Pathogenic Escherichia coli O157:H7 (CICC 10907) in Human Whole Blood Samples Using a Nanoporous Hydrogel-Based Loop-Mediated Isothermal Amplification Method

[0044] 1. The primer sequence is as follows:

[0045] F3: 5'-GCCATCTCCTGATGACGC-3';

[0046] B3: 5'-ATTTACCGCAGCCAGACG-3';

[0047] LF: 5'-CTTTGTAACAACCTGTCATCGACA-3';

[0048] LB: 5'-ATCAATCTCGATATCCATGAAGGTG-3';

[0049] FIP: 5'-CATTTTGCAGCTGTACGCTCGCAGCCCATCATGAATGTTGCT-3';

[0050] BIP: 5'-CTGGGGCGAGGTCGTGGTATTCCGACAAACACCACGAATT-3';

[0051] 2. The actual original DNA concentrations of Escherichia coli in human whole blood samples a to e are:

[0052] Human whole blood sample a: The original concentration of E. coli DNA is 1 CFU / μL;

[0053] Human whole blood sample b: the original concentration of E. coli DNA is 10 CFU / μL

[0054] Human whole blood sample c: the original concentration of Escherichia coli DNA is 20 CFU / μL;

[0055] Human whole blood sample d: the original concen...

Embodiment 2

[0062] Example 2 Analysis of Listeria monocytogenes (CICC 21633) in milk tea samples by a ring-mediated isothermal amplification method based on nanoporous hydrogel

[0063] 1. The primer sequence is as follows:

[0064] F3: 5'-TTGCGCAACAAACTGAAGC-3';

[0065] B3: 5'-GCTTTTACGAGAGCACCTGG-3';

[0066] LF: 5'-TAGGACTTGCAGGCGGAGATG-3';

[0067] LB: 5'-GCCAAGAAAAGGTTACAAAGATGG-3';

[0068] FIP: 5'-CGTGTTTCTTTTCGATTGGCGTCTTTTTTTCATCCATGGCACCACC-3';

[0069] BIP: 5'-CCACGGAGATGCAGTGACAAATGTTTTGGATTTCTTCTTTTTCTCCAC AAC-3';

[0070] 2. Add 1×LAMP buffer, 6mM MgSO to 0.83μL, 1.67μL and 2.50μL milk tea samples respectively 4 , 1.4mM dNTP, 640U / mL Bst 2.0 DNA polymerase, 1.6μM FIP and BIP, 0.2μM F3 and B3, 0.8μM LF and LB, 1mg / mL BSA, 0.1mg / mL lysozyme, 1X EvaGreen, 2.0μL bacteria Liquid or DNA solution, 1.6mg Four-arm PEGAcrylate and 1.1mg SH-PEG-SH were used to prepare the LAMP reaction hydrogel system mixture (25μL) with 3 different milk tea sample concentrations.

[0071] 3. T...

Embodiment 3

[0073] Example 3 Analysis of Salmonella typhi (CICC 10871) in bovine whole blood samples by a loop-mediated isothermal amplification method based on nanoporous hydrogel

[0074] 1. The primer sequence is as follows:

[0075] F3: 5'-GGCGATATTGGTGTTTATGGGG-3';

[0076] B3: 5'-AACGATAAACTGGACCACGG-3';

[0077] LF: 5'-GACGAAAGAGCGTGGTAATTAAC-3';

[0078] LB: 5'-GGGCAATTCGTTATTGGCGATAG-3';

[0079] FIP: 5'-GACGACTGGTACTGATCGATAGTTTTTCAACGTTTCCTGCGG-3';

[0080] BIP: 5'-CCGGTGAAATTATCGCCACACAAAACCCACCGCCAGG-3'.

[0081] 2. Add 1×LAMP buffer, 6mM MgSO to 0.5μL, 1.0μL and 2.0μL bovine whole blood samples respectively 4 , 1.4mM dNTP, 640U / mL Bst 2.0 DNA polymerase, 1.6μM FIP and BIP, 0.2μM F3 and B3, 0.8μM LF and LB, 1mg / mL BSA, 0.1mg / mL lysozyme, 1X EvaGreen, 2.0μL bacteria Liquid or DNA solution, 1.6mg Four-arm PEGAcrylate and 1.1mg SH-PEG-SH prepared three LAMP-reactive hydrogel system mixtures (25μL) with different bovine whole blood sample concentrations.

[0082] 3. The sa...

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Abstract

The invention discloses a method for rapidly detecting bacteria in a complex sample, and belongs to the technical field of molecular biology. The method comprises the following steps of: (1) adding asample to be detected into a loop-mediated isothermal amplification reaction system solution containing lysozyme, adding a hydrogel monomer into the reaction system, and forming hydrogel at room temperature; (2) performing isothermal amplification reaction on the hydrogel system under a constant temperature condition; and (3) analyzing a fluorescence signal in the hydrogel system after the isothermal amplification reaction by utilizing a fluorescence imaging technology, and calculating the concentration of target bacteria in the sample to be detected. According to the method disclosed by the invention, the LAMP reaction system is innovatively converted into a hydrogel state from the previous solution state, LAMP in-situ amplification is performed, and multiple nano holes in the hydrogel can achieve an effect of isolating inhibitors such as organic matters, heavy metals and the like, so that absolute quantitative analysis of the bacteria is realized, and a digital nucleic acid detectiontechnology which is low in price and flexible and simple in operation is provided for detecting bacteria in a real complex sample.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a ring-mediated isothermal amplification method based on a nanoporous hydrogel system to quickly detect bacteria in complex samples. Background technique [0002] Traditional pathogen detection uses bacterial culture to achieve quantitative determination, but they are too time-consuming to perform and have limitations in identifying certain species. Nucleic acid amplification reaction can shorten the detection time to several hours, such as loop-mediated isothermal amplification reaction (LAMP), which is a new type of isothermal nucleic acid amplification method, which is characterized by the design of 6-8 regions of the target gene 4-6 kinds of specific primers, under the action of strand-displacing DNA polymerase, can be amplified at a constant temperature of 65°C and can achieve 10 in about 60 minutes 9 This method has the characteristics of simple operation, strong...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844C12Q1/689C12Q1/06C12Q1/10C12R1/01C12R1/19C12R1/42
CPCC12Q1/6844C12Q1/689C12Q2531/119C12Q2545/114C12Q2563/155C12Q2563/107
Inventor 林星宇罗自生李莉
Owner ZHEJIANG UNIV
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