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Fluorescence detection kit for intestinal flora

A technology for detecting kits and intestinal flora, which is applied to the determination/testing of microorganisms, methods based on microorganisms, biochemical equipment and methods, etc., to achieve the effect of streamlining operation steps

Active Publication Date: 2022-07-15
李莉
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods require multiple steps, including DNA extraction, PCR, electrophoresis or hybridization, imaging detection, etc., usually need 6-7 hours; and these methods require more specialized instruments, such as PCR instrument, electrophoresis instrument, Gene chips, fluorescence imaging devices, etc.

Method used

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  • Fluorescence detection kit for intestinal flora
  • Fluorescence detection kit for intestinal flora
  • Fluorescence detection kit for intestinal flora

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1. Preparation of DNA fluorescent probes:

[0085] According to the nucleic acid sequences of SEQ ID No. 1 to SEQ ID No. 10, the isolated nucleic acids of SEQ ID No. 1 to SEQ ID No. 10 were prepared using nucleic acid sequence synthesis methods known in the art.

[0086] The Alexa488 fluorescent dye was coupled to the 5' end of each isolated nucleic acid of SEQ ID No. 1 to SEQ ID No. 10 according to coupling methods known in the art.

[0087] For ease of description, the DNA fluorescent probes synthesized in this example are named as follows in the following examples:

[0088]

Embodiment 2

[0089] Embodiment 2, detect stool sample

[0090] In order to confirm the feasibility of the probe and detection method, an appropriate amount of 7 kinds of bacteria were mixed into the feces collected clinically to make experimental samples.

[0091] 1. Pick 100 mg of fecal sample with a cotton swab and put it into a sterile test tube, add 500 ul of lysis solution, and shake well. The cleavage product was incubated at 97°C for 5 minutes and then placed in an ice-water bath. Centrifuge at 15,000g for 5min. Carefully aspirate the upper brown solution and transfer to a sterile tube. The composition of the lysate was: 500 mM Tris, 16 mM EDTA, 10 mM NaCl, pH 9.0.

[0092] 2. Add well-mixed 400ul dynabeads DNA direct (Invitrogen) to the sample, let it stand at room temperature for 5 minutes, place it on a magnetic separation rack for 2 minutes, and carefully aspirate the supernatant. Remove the sample from the separation rack, add 400ul of cleaning solution, and place it on the...

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PUM

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Abstract

The invention is a method for detecting intestinal flora by utilizing nanometer magnetic beads to purify stool samples combined with fluorescence flow cytometry. In the present invention, specific DNA probe sequences for Clostridium difficile, Salmonella, Shigella, Campylobacter jejuni, Enterohemorrhagic Escherichia coli, Vibrio enteritidis and Yersinia pestis are provided, using the sequence coupled to the probe Fluorescent substances on the microfluidics were used to detect intestinal flora. The detection method of the invention has the advantages of rapidity, accuracy and high sensitivity.

Description

technical field [0001] The invention belongs to the field of biological diagnosis and detection, and relates to a molecular biology detection method of intestinal flora, in particular to a detection technology using DNA fluorescent probes, a kit for detecting bacterial flora DNA and its application. Background technique [0002] There are a large number of bacteria in the human intestinal tract, that is, "gut flora". These intestinal flora are considered to play an important role in the human body and can greatly affect human health. Many chronic diseases, such as obesity, an important cause of diabetes. The latest research shows that changes in gut flora are closely related to neurodegenerative diseases such as Parkinson's, and even depression. Adjusting gut flora may be a new direction for the treatment of such diseases. Therefore, it is of great significance to establish an accurate and rapid detection system and method of intestinal flora. [0003] In the existing inte...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/6816C12Q1/10C12Q1/04C12N15/11C12R1/145C12R1/42C12R1/19C12R1/63C12R1/01
CPCC12Q1/6816C12Q1/689C12Q2600/166C12Q2563/107C12Q2565/626C12Q2545/113
Inventor 李莉
Owner 李莉