Kit for endometriosis diagnosis and detection method thereof

A technology for endometriosis and endometriosis, which is applied in the determination/testing of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of uneven detection efficiency and reliability, and facilitate early treatment , damage reduction, and high repetition stability

Inactive Publication Date: 2017-07-21
SHANGHAI RUNDARONGJIA BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the detection reagents and amplification primers used in the detection process of this method are very random, resulting in uneven detection efficiency and reliability.

Method used

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  • Kit for endometriosis diagnosis and detection method thereof
  • Kit for endometriosis diagnosis and detection method thereof
  • Kit for endometriosis diagnosis and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] A method for detecting miRNA-141 gene in human blood:

[0047] 1. Collection and processing of blood samples

[0048] First, draw 2 mL of peripheral venous blood into EDTA-K2 anticoagulant tubes, dispense 200 μL into Trizol-prepacked cryopreservation tubes within 30 minutes, mix well and store at -80 degrees.

[0049] 2. Total RNA Extraction

[0050] 1) Add 1mL Trizol to fully lyse the cells.

[0051] 2) Add 200 μL of chloroform, shake vigorously for 15 s, and after standing at room temperature for 3 min, centrifuge at 12000 r / min at 4 °C for 15 min.

[0052] 3) Pipette the upper aqueous phase into another centrifuge tube, add 1.5 times the volume of isopropanol and mix well, let stand at room temperature for 10 minutes, then centrifuge at 12000 r / min at 4°C for 10 minutes.

[0053]4) Discard the supernatant, add 600 μL of 75% ethanol, and mix well. Centrifuge at 12000r / min at 4°C for 5 minutes.

[0054] 5) Discard the supernatant, add 800 μL absolute ethanol, and ...

Embodiment 2

[0080] Kit for the diagnosis of endometriosis, comprising:

[0081] (1) Collection and preservation of peripheral blood kits, including: EDTA anticoagulant tubes, cryopreservation tubes, and Trizol reagents;

[0082] (2) total RNA extraction kit in blood, including: chloroform, isopropanol, 75% ethanol, absolute ethanol, DEPC water;

[0083] (3) A kit for detecting the expression of miRNA-141 and U6 genes, including a reverse transcription reaction solution and a fluorescent PCR reaction solution. Wherein the reverse transcription reaction solution includes 10× reverse transcription Buffer, reverse transcriptase, dNTP, primers and DEPC water as shown in SEQ ID NO:1,5; the fluorescent PCR reaction solution includes Premix Ex TaqTM II (2×), primers and DEPC water as shown in SEQ ID NO:2-5;

[0084] (4) Calculation formula of blood miRNA-141 fraction in the sample: miRNA-141 fraction = miRNA-141Ct value - U6Ct value.

[0085] When the miRNA-141 score is higher than or equal t...

Embodiment 3

[0087] Utilize the test kit of embodiment 2 to detect clinical samples:

[0088] Peripheral blood samples were collected from gynecological female patients, a total of 65 patients with endometriosis, aged 22-49 years, with an average age of 33.7 years; 18 patients with blocked fallopian tubes without endometriosis were used as normal controls , aged 30-43 years old, with an average of 36.3 years old.

[0089] Blood total RNA was extracted according to the method described in Example 1, and the blood miRNA-141 score was detected by fluorescent PCR method. Each sample was repeated 3 times, and positive, negative, and blank controls were made at the same time. Make judgments based on the scoring results.

[0090] The miRNA-141 score in the endometriosis group was 10.5±0.93, and in the normal control group was 4.89±1.25. The score of miRNA-141 was significantly different between the two groups, P<0.01.

[0091] This kit uses miRNA-141 score 8.3 as the cut-off value for the dia...

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Abstract

The invention provides a kit for endometriosis diagnosis. The kit comprises 1) an EDTA-K2 anticoagulative tube for collecting peripheral blood, 2) a cryopreservation tube for preloading Trizol, 3) a reagent for extracting total RNA in peripheral blood, 4) a reagent for detecting miRNA-141 and U6 gene expression, and 5) a formula of calculating miRNA-141 scores in blood: miRNA-141 score = miRNA-141Ct value - U6Ct value. The kit has high repeatability and stability and no repeated experiment is required. MiRNA-141 and U6 are utilized to amplify a nucleic acid sequence, the diagnosis kit for determining miRNA score in the blood is constructed, the goal of noninvasive diagnosis of endometriosis can be achieved, the damage to patients caused by laparoscope detection is greatly reduced, and the kit is favorable for early treatment of the endometriosis.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a kit for diagnosing endometriosis and a detection method thereof. Background technique [0002] Endometriosis (Endometriosis, EMs), referred to as endometriosis, is a common gynecological disease. The incidence rate of this disease is about 10% in women of childbearing age, and the incidence rate is as high as 30-60% in infertile women. Its basic feature is the emergence of endometrial tissue (glands and stroma) with growth function in other parts of the uterus covering the endometrium and myometrium, and the main clinical manifestations are chronic pelvic pain, dyspareunia and infertility 1 . Although endometriosis is a benign gynecological disease, it has malignant behaviors such as hyperplasia, invasion, and metastasis, and the malignant transformation rate is 0.7-1%. Ectopic endometrial tissue is mainly located in the pelvic cavity (such as uterosacral ligament, ut...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6883C12Q2600/118C12Q2600/178C12Q2600/158
Inventor 钱学庆胡雄敏秦炜黄欣
Owner SHANGHAI RUNDARONGJIA BIOLOGICAL TECH CO LTD
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