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Digital loop-mediated isothermal amplification (LAMP) method based on track etching film and application

A loop-mediated isothermal and isothermal amplification technology, applied in the field of molecular biology, can solve the problems of limited scalability, difficult one-time use, high price, etc., and achieve the effect of flexible and concise operation, simple experimental steps, and simple operation.

Inactive Publication Date: 2020-02-28
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this non-digital detection method can only be used as a simple means of judgment, and cannot be used for accurate quantitative detection.
[0006] The existing dPCR technology 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 and expensive, making it difficult for one-time use

Method used

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  • Digital loop-mediated isothermal amplification (LAMP) method based on track etching film and application
  • Digital loop-mediated isothermal amplification (LAMP) method based on track etching film and application
  • Digital loop-mediated isothermal amplification (LAMP) method based on track etching film and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039]Example 1: Analysis of Escherichia coli (ATCC 10798) by a digital loop-mediated isothermal amplification detection method based on a track-etched membrane with a pore size of 25 μm

[0040] The primer sequences used are as follows:

[0041] F3: 5'-GCCATCTCCTGATGACGC-3'

[0042] B3: 5'-ATTTACCGCAGCCAGACG-3'

[0043] LF: 5'-CTTTGTAACAACCTGTCATCGACA-3'

[0044] LB: 5'-ATCAATCTCGATATCCATGAAGGTG-3'

[0045] FIP: 5'-CATTTTGCAGCTGTACGCTCGCAGCCCATCATGAATGTTGCT-3'

[0046] BIP: 5'-CTGGGGCGAGGTCGTGGTATTCCGACAAACACCACGAATT-3'

[0047] The actual original DNA concentrations of Escherichia coli in standard samples a to e are:

[0048] Standard sample a: The original concentration of Escherichia coli DNA is 11copy / μL;

[0049] Standard sample b: the original concentration of Escherichia coli DNA is 1.1×10 2 copy / μL;

[0050] Standard sample c: the original concentration of Escherichia coli DNA is 1.1×10 3 copy / μL;

[0051] Standard sample d: The original concentration of Esc...

Embodiment 2

[0060] Example 2: Analysis of Escherichia coli (ATCC 10798) based on a digital loop-mediated isothermal amplification detection method with a pore size of 14 μm track-etched membrane

[0061] The primer sequences used are the same as those in Example 1, but the actual original concentrations of Escherichia coli DNA in the standard samples a to d in this example are respectively:

[0062] Standard sample a: The original concentration of Escherichia coli DNA is 1×10 3 copy / μL;

[0063] Standard sample b: the original concentration of Escherichia coli DNA is 1×10 4 copy / μL;

[0064] Standard sample c: the original concentration of Escherichia coli DNA is 1×10 5 copy / μL;

[0065] Standard sample d: The original concentration of Escherichia coli DNA is 1×10 6 copy / μL;

[0066] Add 2.5μL standard samples a to e respectively with 1×LAMP buffer, 6mM MgSO 4 , 1.4mM dNTP, 640U / mL Bst DNA polymerase, 1.6μM FIP and BIP, 0.2μM F3 and B3, 0.8μM LF and LB, 1mg / mL BSA, 50μM calcein, 1m...

Embodiment 3

[0073] Example 3 Analysis of Salmonella (CVD 909) by a digital loop-mediated isothermal amplification detection method based on a track-etched membrane with a pore size of 25 μm

[0074] The primer sequences used are as follows:

[0075] F3: 5'-GACTTGCCTTTAAAAGATACCA-3'

[0076] B3: 5'-AGAGTGCGTTTGAACACTT-3'

[0077] LF: 5'-TCGGATGGCTTCGTTCCT-3'

[0078] LB: 5'-CAAGGGTTTCAAGACTAAGTGGTTC-3'

[0079] FIP: 5'-AACTTGCTGCTGAAGAGTTGGACCGAATGACTCGACCATC-3' BIP: 5'-CCTGGGGCCAAATGGCATTATGCACTAAGTAAGGCTGG-3'

[0080] The actual original DNA concentrations of Salmonella in standard samples a to e are:

[0081] Standard sample a: the original concentration of Salmonella DNA is 11copy / μL;

[0082] Standard sample b: the original concentration of Salmonella DNA is 1.1×10 2 copy / μL;

[0083] Standard sample c: the original concentration of Salmonella DNA is 1.1×10 3 copy / μL;

[0084] Standard sample d: the original concentration of Salmonella DNA is 1.1×10 4 copy / μL;

[0085] Stan...

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Abstract

The invention discloses a digital loop-mediated isothermal amplification (LAMP) method based on track etching film and application and relates to the technical field of molecular biology. The method is characterized in that pores in the track etching film are used as isolated nano reactors, a single bacterium is evenly dispersed into each hole of the film to perform LAMP to generate fluorescence,and ImageJ software is used to perform fluorescence photographing counting on the film system after the isothermal amplification to quantitatively calculate the original nucleic acid concentration ofto-be-measured objects in the film system so as to achieve absolute quantitative analysis of the bacteria. The method has the advantages that bacterium nucleic acid digital analysis can be completed without equipment such as complex micro-fluidic devices and chips which are high in cost, the method is simple in experiment steps, fast and efficient, simple to operate and low in cost, is not dependent on the target bacteria and can carry on the digital detection of the target bacteria only by designing matched primers, and a digital nucleic acid detection technology which is cheap and flexible and simple to operate is provided for gene research.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a digital ring-mediated isothermal amplification detection method based on a track-etched film and its application. Background technique [0002] Traditional pathogenic bacteria detection usually takes several days for bacterial / virus culture to achieve quantitative determination. In recent years, nucleic acid amplification techniques such as polymerase chain reaction (PCR) have been able to measure extremely small amounts of nucleic acids with high sensitivity. However, they require a real-time fluorescence quantitative thermal cycler for sample amplification and online fluorescence detection, which greatly limits their application in outdoor detection. [0003] More importantly, the traditional PCR method is difficult to carry out the absolute quantitative analysis of the number of bacteria. In recent years, digital nucleic acid detection technology has developed ra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844C12Q1/689
CPCC12Q1/6844C12Q1/689C12Q2531/119C12Q2563/159C12Q2545/114
Inventor 林星宇罗自生李莉蒋艳
Owner ZHEJIANG UNIV
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