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Cervical cell Warburg effect simple detection reagent as well as preparation method and application thereof

A technology for detecting reagents and cervical cells, applied in the field of medical testing, can solve the problems of high operating cost of PET-CT, human radiation hazards, no simple screening and detection, etc., to achieve high specificity, good stability and high sensitivity. Effect

Active Publication Date: 2017-09-05
山东天倪生物技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the problems of high operating cost of PET-CT, serious radiation hazards to the human body and lack of simple screening and detection reagents for cervical cancer, the present invention provides a reagent that can quickly and accurately detect the Warburg effect of cervical cells

Method used

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  • Cervical cell Warburg effect simple detection reagent as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Reagent Preparation

[0047] (1) Preparation of Reagent A:

[0048] Add 5.00 g of subtilisin to 100 mL of 0.9 % NaCl isotonic solution and dissolve to obtain reagent A;

[0049] (2) Preparation of reagent B:

[0050] a) Dissolve 82g sodium acetate in 1000mL water to obtain 2 mol / L sodium acetate solution; dissolve 15g glacial acetic acid in 200mL water to obtain 2 mol / L acetic acid solution;

[0051] b) Mix the above solution at a ratio of 5:1 to obtain reagent B.

[0052] (3) Preparation of Reagent C:

[0053] a) Dissolve 0.15 g of 3,3’,5,5’-tetramethylbenzidine in 30 mL of absolute ethanol;

[0054] b) Add 0.25 mL of cumene peroxide and 0.125 mL of 6-methoxyquinoline to the above solution in a) in order to dissolve;

[0055] c) Dilute the purified water to 100mL to obtain reagent C;

[0056] (4) Preparation of reagent D:

[0057] a) Dissolve 0.05 g of toluidine blue O in 100 mL of purified water;

[0058] b) Add 0.5 mL of Triton X-100 to the above so...

Embodiment 2

[0059] The usage method of embodiment 2 reagent

[0060] (1) Gently brush the exfoliated cells of the cervical epithelium with a cell brush, then place the cell brush in 2mL of reagent A and gently stir to make a sample solution;

[0061] (2) Centrifuge the above sample solution at a high speed of 3000 rpm for 3 minutes;

[0062] (3) Take the supernatant of step (2), add 1mL of reagent B to mix well, then add 1mL of reagent C, observe the color of the reaction solution in 3 minutes, which shows the determination of the free ferrous protoporphyrin content of cells: If it does not change color, it is negative, light blue is weak positive, blue is positive, and dark blue is strong positive;

[0063] (4) Add 1mL of reagent D to the precipitate in step (2), and observe the color of the reaction solution at 3 minutes, which shows the content of reactive oxygen species in the cells: if it does not change color (blue), it is negative, and the blue color becomes Light blue is positive,...

Embodiment 3

[0065] Example 3 Clinical verification

[0066] 1. Sensitivity

[0067] (1) Specimen source: 100 cervical exfoliated cell samples from cervical cancer patients received by the gynecology department of a medical unit, and all cervical cancers were confirmed by pathological examination.

[0068] (2) Operation method: operate according to Example 2.

[0069] (3) Verification results: see Table 1.

[0070] Table 1 Sensitivity of simple detection reagents for Warburg effect in cervical cells

[0071] Test items positive feminine suspicious positive Sensitivity (%) Cell Free Ferrous Protoporphyrin Detection 87 13 -- 87 Cellular reactive oxygen species detection 64 36 -- 64 Test results 64 13 23 87

[0072] 2. Specificity

[0073] (1) Specimen source: 100 cervical exfoliated cell samples from non-cervical cancer patients received by the general gynecology department of a medical unit. All non-cervical cancers were ruled out by path...

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Abstract

The invention relates to a cervical cell Warburg effect simple detection reagent as well as a preparation method and application thereof. The reagent is composed of a reagent A, a reagent B, a reagent C and a reagent D, wherein the reagent A is 4-6% of a subtilisin isotonic solution; the reagent B is an acetate buffer solution of which the pH value is 3.5-6.0; the reagent C is composed of a 3,3'5,5' tetramethyl benzidine solution with the concentration of 0.1-0.25g / L, a 6-methoxy quinoline solution with the concentration of 1.0-2.5mL / L and a cumene hydroperoxide solution with the concentration of 2-5mL / L; and the reagent D is composed of 0.03-0.07% of a toluidine blue O solution and a triton x-100 solution with the concentration of 2.5-7.5mL / L. The reagent can be used for qualitatively detecting the contents of cellular free ferroprotoporphyrin and active oxygen of cervical exfoliated cells so as to judge the strength of the Warburg effect of cervical cells.

Description

technical field [0001] The invention relates to a simple detection reagent for Warburg effect of cervical cells and its preparation method and application, which belong to the field of medical examination. Background technique [0002] Cervical cancer is the second "killer" of women's health. There are nearly 500,000 new cases of cervical cancer every year in the world, and it kills 300,000 women worldwide every year. The number of new cases of cervical cancer in China exceeds 150,000 every year, accounting for 73-93% of the incidence of malignant tumors of the female reproductive system. The mortality rate is about 50%. Cervical cancer has seriously threatened the health and life of young and middle-aged women in my country. The key to improving the cure rate of cancer lies in the "three early" (early detection, early diagnosis, early treatment). Evidence shows that women's chances of catching cervical cancer early and nipping it in the bud are much higher if they are pr...

Claims

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Application Information

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IPC IPC(8): G01N21/78
CPCG01N21/78G01N2021/775
Inventor 郑德春
Owner 山东天倪生物技术有限公司
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