Immunochromatography multiple gene detection method based on Cas9 nucleic acid isothermal amplification

A technology of isothermal amplification and immunochromatography, which is applied in the fields of biotechnology and medical testing, can solve the problems of high cost, single detection object, and low universality, and achieve the advantages of simple operation, short detection time, and instant readable results Effect

Active Publication Date: 2020-08-07
ZHEJIANG UNIV OF TECH
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Problems solved by technology

[0005] In order to overcome the problems of high cost, single detection object, and low universality of the traditional nucleic acid detection method, the present invention provides a low-cost, no special requirements for instruments, can simultaneously detect multiple nucleic acid products, fast and efficient, and visualized detection results The immunochromatographic multiple gene detection method based on isothermal amplification of Cas9 nucleic acid can be widely used in grassroots detection and family detection

Method used

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  • Immunochromatography multiple gene detection method based on Cas9 nucleic acid isothermal amplification
  • Immunochromatography multiple gene detection method based on Cas9 nucleic acid isothermal amplification
  • Immunochromatography multiple gene detection method based on Cas9 nucleic acid isothermal amplification

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Embodiment 1 Multiple immunochromatographic diagnostic test strips detect Escherichia coli and Salmonella

[0038] In this example, the DNA amplification template comes from the genome or plasmid extracted from the kit, the primers are commissioned to be synthesized by a biotechnology company, the sgRNA is obtained by in vitro transcription, and the 20 bases at the 5' end of the sgRNA are the seed sequence complementary to the prespacer.

[0039] refer to figure 1 The detection principle of the immunochromatographic multiplex gene Escherichia coli and Salmonella is shown:

[0040] (1) Extract the genomic DNA to be detected from the sample to be detected: Escherichia coli ATCC35128 and Salmonella ASL1174 are cultured on a shaker in LB liquid medium until the exponential growth phase (OD=0.4-0.6), and 1 ml of the bacterial liquid is taken to extract the bacteria DNA (target DNA),

[0041] (2) Using the target DNA as a template, perform multiple isothermal amplification:...

Embodiment 2

[0056] The difference between Example 2 and Example 1 is that the immunochromatographic test paper is different, the sample pad and the latex microsphere pad use polyester cellulose membrane, the temperature of the water bath in the multiple isothermal amplification process is 42 ° C, and the rest of the process is completely the same. The detection result of this embodiment is equivalent to that of Embodiment 1, and will not be repeated here.

Embodiment 3

[0058] Escherichia coli and Salmonella were mixed into milk, stored overnight at room temperature, and then used as samples for experimental testing, and the testing method was the same as in Example 1. like Figure 5 As shown, the method still has good specificity for bacteria in real food samples. like Image 6 As shown, the method has a detection limit of 100 colonies / mL for bacteria in real food samples.

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Abstract

The invention relates to the technical field of biotechnology and medical examination and relates to an immunochromatography multiple gene detection method based on Cas9 nucleic acid isothermal amplification. The method comprises the following steps of: (1) extracting a to-be-detected genome DNA from a to-be-detected sample; (2) carrying out multiple isothermal amplification with the to-be-detected genome DNA adopted as a template to obtain a multiple amplified nucleic acid product; and (3) dropwise adding the multiplex amplification nucleic acid product onto immunochromatography test paper which is provided with a plurality of detection lines and a quality control line, observing the colors of the detection lines and the quality control line, and determining a detection result. The detection method disclosed by the invention can be used for simultaneously detecting various nucleic acid products, is simple and convenient to operate, short in detection time, enables instantaneity in reading results, can be widely applied to primary detection and household detection, and is not limited by instruments.

Description

technical field [0001] The invention relates to the technical fields of biotechnology and medical testing, in particular to an immunochromatographic multiple gene detection method based on Cas9 nucleic acid isothermal amplification. Background technique [0002] Nucleic acid detection has been widely used in many fields, including criminal investigation, detection of pathogenic microorganisms, and disease diagnosis. In general, the target nucleic acid content in environmental samples or physiological samples is very small, and in order to make the results more accurate, it is often necessary to amplify the target nucleic acid fragments. Among them, the polymerase chain reaction (PCR) is the most widely used nucleic acid amplification technique. However, this technique relies on thermal cycling, requires expensive professional equipment, and is not suitable for detection in ordinary homes or remote areas. [0003] With the requirements of biotechnology in clinical and on-si...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/558G01N33/50C12Q1/6806
CPCG01N33/558G01N33/5008C12Q1/6806C12Q2537/143C12Q2521/327Y02A50/30
Inventor 叶邦策王露莹尹斌成沈幸盈
Owner ZHEJIANG UNIV OF TECH
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