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Kit for detecting pancreatic cancer cells in peripheral blood

A pancreatic cancer cell and kit technology, applied in the biological field, can solve the problems of no pancreatic cancer, difficult to identify, poor patient prognosis, etc., and achieve the effects of small sample size, high detection sensitivity, and small damage.

Active Publication Date: 2018-03-27
北京牛牛基因技术有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the diagnosis and treatment methods of tumors have been continuously improved and improved in recent years, the prognosis of many tumors has been improved, but according to the statistics of the American Cancer Society, the improvement of the prognosis of pancreatic cancer patients is the least obvious
The reasons for the high mortality rate of pancreatic cancer are as follows: 1) Although imaging methods play an important role in the detection and treatment of pancreatic cancer, pancreatitis, pancreatic intraepithelial neoplasia and other benign lesions are still closely related to pancreatic cancer for small lesions. Difficult to identify, only 7% of pancreatic cancers are diagnosed at the initial stage
2) There is no effective treatment for advanced pancreatic cancer, leading to poor prognosis for most patients. The 5-year relative survival rate of patients with surgically resectable local lesions is 22%, while the survival rate of unresectable advanced metastatic lesions is only 1-2%.
However, these factors have their own defects and cannot be used alone as diagnostic markers for pancreatic cancer.

Method used

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  • Kit for detecting pancreatic cancer cells in peripheral blood
  • Kit for detecting pancreatic cancer cells in peripheral blood
  • Kit for detecting pancreatic cancer cells in peripheral blood

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1: Preparation of magnetic beads bound to pancreatic cancer antibody:

[0069] The antibodies used were anti-EpCAM monoclonal antibody and anti-CK20 monoclonal antibody. After the above two antibodies were labeled respectively, the above two labeled magnetic beads were mixed and used.

[0070] Labeling method: magnetic beads produced by Invitrogen, USA M-450Tosylactivated. The marking method is strictly in accordance with the product instructions.

[0071] The labeling ratio is: label 500 μL magnetic beads per 100 μg antibody, and mix in equal amounts after labeling.

Embodiment 2

[0072] Example 2: Isolation of pancreatic cancer cells:

[0073] 2.1 Sample handling:

[0074] Take 5 mL of peripheral blood from patients with pancreatic cancer, anticoagulate with EDTA, store at 4 degrees, and use within 48 hours.

[0075] 2.2 Magnetic bead treatment:

[0076] Wash the magnetic beads bound with pancreatic cancer antibody (antibody-labeled magnetic beads, which are mixed magnetic beads labeled with two antibodies, and prepare antibody-labeled magnetic beads according to the ratio of 1 mL sample to 25 μL magnetic beads) with PBS repeatedly for three times, and separate the magnetic beads. Beads, kept on ice;

[0077] The specific operation is: draw 125μL of marked magnetic beads into a 1.5mL centrifuge tube, gently blow and mix with a pipette (note that a vortexer cannot be used), and then place it on the magnetic bead enricher for 1min. , to attach the magnetic beads to the wall of the centrifuge tube, discard the supernatant; then add 1mL PBS, place it on...

Embodiment 3

[0084] Example 3: Detection of pancreatic cancer cell markers:

[0085] 3.1 mRNA purification

[0086] Mix 40 μL magnetic beads with oligonucleotide Oligo(dT) into a 1.5 mL centrifuge tube, add 500 μL lysis / binding buffer to wash twice, separate magnetic beads, add supernatant, mix well, and incubate at room temperature 10min, let the mRNA bind to the magnetic beads; then separate the magnetic beads, use 500 μL buffer A to wash the magnetic beads twice, separate the magnetic beads, then wash the magnetic beads twice with 500 μL buffer B, separate the magnetic beads, and then use 100 μL RNase- Wash once with free water, separate the magnetic beads, resuspend the magnetic beads with 29.5 μL RNase-free water, incubate in a water bath (or metal bath) at 55°C for 5 min, and place on ice for 2 min to obtain the mRNA / magnetic bead mixture.

[0087] The mRNA / bead mix cannot be stored and needs to be reverse transcribed immediately.

[0088] 3.2 Reverse transcription:

[0089] The r...

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Abstract

The invention provides a kit for detecting pancreatic cancer cells in peripheral blood. The kit comprises magnetic beads combined with pancreatic cancer antibodies and a corresponding buffer reagent.According the kit, through magnetic bead sorting, RT-PCR and other molecular biological methods, biological activity of tumor cells in the blood circulation of pancreatic cancer patients is detected and a drug treatment targeting gene is determined, and through detection of the expression level of EpCAM, KRT19 and MUC16, the development and prognosis of pancreatic cancer are determined. The kit provides the theoretical basis and experimental evidence for individualized treatment on pancreatic cancer. The kit needs a small sample size, has small damage to the patient and has high detection sensitivity and a detection rate of 80% or more.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a pancreatic cancer detection kit, in particular to a high-accuracy detection kit for pancreatic cancer cell markers in peripheral blood. Background technique [0002] 1.1 Overview of Pancreatic Cancer [0003] Pancreatic carcinoma (pancreatic carcinoma) is a common malignant tumor of the digestive tract, about 90% of which are ductal adenocarcinoma (pancreatic ductal adenocarcinoma, PDAC) originating from the ductal epithelium, its morbidity and mortality have increased significantly in recent years, 90% of The survival period of pancreatic cancer patients is less than one year, and less than 5% of patients survive more than five years. It is one of the malignant tumors with the worst prognosis. The factors affecting the unsatisfactory treatment effect of pancreatic cancer include atypical early symptoms, complex pathophysiological mechanisms, early diagnosis and lack of prognostic ma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574
CPCG01N33/57438
Inventor 谭焕然牛刚郝纯毅徐巍杨晓伟
Owner 北京牛牛基因技术有限公司
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