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Detection kit for detecting bladder cancer cells, preparation method of detection kit and detection method of bladder cancer cells

A bladder cancer cell and detection kit technology, which is applied in the detection kit for bladder cancer cells and the detection field of bladder cancer cells, can solve problems such as false positive results, and achieve the effect of improving accuracy and detection sensitivity

Pending Publication Date: 2022-04-01
上海国奥源华安生物科技有限公司
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the use of a single target often results in false positives

Method used

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  • Detection kit for detecting bladder cancer cells, preparation method of detection kit and detection method of bladder cancer cells
  • Detection kit for detecting bladder cancer cells, preparation method of detection kit and detection method of bladder cancer cells
  • Detection kit for detecting bladder cancer cells, preparation method of detection kit and detection method of bladder cancer cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] The hybridization chain reaction (HCR) triggered by CRISPR / Cas13a provided by the present invention is used for the method for the analysis of bladder cancer cells, comprising the following steps:

[0042] a. Combine a certain number of cells with the trigger probe linked to the CD47 antibody and the activate probe linked to the EpCAM antibody through antigen-antibody reaction, incubate at 37°C for 1 hour, wash and discard the unbound antibody-linked DNA after the reaction.

[0043] b. Add 20 μL of T7 RNA polymerase, 20 μL of buffer, 4 μL of NTPs, 148 μL of PBS, 2 μL of 100 μM T7 sequence to the reaction system in step a, react at 37°C for 1 hour, add 4 μL of Cas13a, 2 μL of CrRNA, continue the reaction for 30 minutes, and centrifuge after the reaction Discard the supernatant;

[0044] c. Add 20 μL of annealed H1 (20 μM) and H2 (20 μM) 20 μL, then add 160 μL of PBS solution, and react at 37 ° C for 1 h;

[0045] see figure 1 , a schematic diagram of the principle of t...

Embodiment 2

[0048] Adjust the base numbers of the stem-loop complementary duplexes in the trigger probe to 10, 11, 12, and 13, respectively. All the other steps are the same as in Example 1.

[0049] Fluorescence value detection: put the reacted solution into a fluorescence cuvette, and measure the spectrum within the wavelength range of 500nm to 600nm.

[0050] Please refer to figure 2 , is a histogram of fluorescence intensity at 520nm measured under different complementary base numbers. Depend on figure 2 It can be seen that when the number of complementary bases is 12, the corresponding fluorescence intensity difference reaches the maximum.

[0051] In summary, the hybridization chain reaction (HCR) triggered by CRISPR / Cas13a provided by the present invention is used in the analysis and detection method of bladder cancer cells, and the optimal number of stem-loop complementary double-stranded bases in the trigger probe is 12.

Embodiment 3

[0052] Example 3 Cell Qualitative Chart

[0053] The number of stem-loop double-strand complementary bases in the trigger probe was determined to be 12, and the difference from Example 1 was that three sets of control reactions were added, namely only adding the trigger probe, only adding the activate probe, and neither added to the cells. All the other steps are the same as in Example 1.

[0054] Please refer to image 3 , is the qualitative result of the cell system. Depend on image 3 It can be seen that the reaction can only occur when there are two proteins on the cell surface at the same time, and a higher fluorescence intensity will appear. In other cases, the reaction cannot occur, and the observed results are all background values.

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Abstract

The invention provides a detection kit for detecting bladder cancer cells, a preparation method of the detection kit and a detection method of the bladder cancer cells, and belongs to the technical field of biological analysis, the detection kit comprises DNA, crRNA, Cas13a, T7 RNA polymerase, NTPs, an antibody and a PBS buffer system. According to the method, T7 in-vitro transcription, CRISPR / Cas13a and HCR triple signal amplification technologies are adopted, so that the detection sensitivity is remarkably improved; the application of the'and 'logic gate which can output the signal only when the two proteins exist at the same time improves the analysis accuracy.

Description

technical field [0001] The invention relates to the technical field of biological analysis, in particular to a detection kit for detecting bladder cancer cells, a preparation method thereof, and a detection method for bladder cancer cells. Background technique [0002] Bladder cancer is the tenth most common malignant tumor of the urinary system worldwide, with a high recurrence rate and poor clinical prognosis. The tumor mutation rate of bladder cancer ranks third among all tumors, is more common in men, and is the sixth most common cancer in men, and the morbidity and mortality of men are about 4 times that of women. Currently, the gold standard for the diagnosis of bladder cancer is cystoscopy and urine cytology. However, cystoscopy is costly, invasive, and has suboptimal sensitivity. Although urine cytology is a non-invasive test, its sensitivity is low and it is related to the pathological staging and grading of bladder cancer. For the treatment of bladder cancer, th...

Claims

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Application Information

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IPC IPC(8): C12Q1/682
CPCY02A50/30
Inventor 张娟王沛彭爽赵春山魏兴华
Owner 上海国奥源华安生物科技有限公司
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