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Circulating tumor cell detection kit and application thereof

A detection kit and tumor cell technology, which is applied in the field of circulating tumor cell detection kits, can solve the problems of high detection cost and unsuitability for high-throughput detection, and achieve the effect of simple operation

Active Publication Date: 2014-06-18
上海顿慧医疗科技发展有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a new chip technology, CTC-chip has the advantages of high sensitivity and good specificity, but it requires special detection equipment, and the detection cost is high, so it is not suitable for high-throughput detection

Method used

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  • Circulating tumor cell detection kit and application thereof
  • Circulating tumor cell detection kit and application thereof
  • Circulating tumor cell detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] A circulating tumor cell detection kit, consisting of the following reagents:

[0033] 1. RosetteSep rare cell enrichment reagent: RosetteSep Human CD45 Depletion Cocktail (Stemcell, Canada), 5*2ml, Cat.No.15162.

[0034] 2. Mini Kit (50) (Qiagen, Germany), Cat. No. 74104.

[0035] 3. Reverse Transcription Kit (Qiagen, Germany), Cat. No. 205313.

[0036] 4. qPCR SuperMix-UDG (Invitrogen, USA), Cat. No. C11730-025.

[0037] 5. Taq-Man probe (self-made, 10μM):

[0038] EpCAM probe: FAM-ACGGCGACTTTTGCCGCAGCT-TAM;

[0039] β-actin probe: FAM-ATCACTGCCCTGGCACCCAGCA-TAM.

[0040] 6. Specific primers (self-made, 10μM):

[0041] EpCAM-F: 5-CTCGCGTTCGGGCTTCT-3;

[0042] EpCAM-R: 5'-TGTAGTTTTCACAGACACATTCTTCCT-3';

[0043] β-actin-F: 5'-TGGCATTGCCGACAGGAT-3';

[0044] β-actin-R: 5'-CTCAGGAGGAGCAATGATCTTGAT-3'.

[0045] Use the above-mentioned circulating tumor cell detection kit to detect circulating tumor cells, mix different numbers of Hep3B cells into the periph...

Embodiment 2

[0066] A circulating tumor cell detection kit, the composition of which is the same as in Example 1.

[0067] Use the above-mentioned circulating tumor cell detection kit to detect circulating tumor cells. The blood samples come from the peripheral blood of healthy people who have undergone physical examination. The specific steps are as follows:

[0068] 1, adopt the method for embodiment 1 step 1.1.-1.2 to prepare 10,10 2 、10 3 、10 4 Hep3B cells / 5ml peripheral blood sample. RosetteSep (Stemcell, Canada) and MACS microbeads (Mitenly, Germany) were used to enrich cells containing 10 and 10, respectively, according to the instructions of the kit. 2 、10 3 、10 4 CTCs in Hep3B cells / 5ml peripheral blood samples.

[0069] 2. Using the method in Step 1.4 of Example 1, extract total RNA from the CTCs enriched in Step 1 and reverse transcribe to synthesize cDNA.

[0070] 3. Using the method in Step 1.5 of Example 1, using fluorescent-labeled Taqman probe technology, using EpCAM...

Embodiment 3

[0073] A circulating tumor cell detection kit, the composition of which is the same as in Example 1.

[0074] Take the peripheral blood of healthy people for physical examination, and evaluate the imprecision within the batch: 1, 10 Hep3B cells / 5ml healthy human peripheral blood samples are prepared by using the steps 1.1.-1.2 of Example 1, and the circulating tumor cells of the present invention are used The detection kit was repeated 5 times (method is the same as in Example 1 step 1.3.-1.6.) to assess the imprecision within the batch of the detection system ( Figure 3A , 3B ).

[0075] like Figure 3A and Figure 3B As shown, β-actin was used as an internal reference marker, EpCAM was used as a CTC surface marker, and fluorescently labeled Taqman probe qRT-PCR was used as a detection technology to verify the precision of the new platform for detecting CTCs. The results show that the method established in the present invention detects CTC with accurate results and good re...

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Abstract

The invention provides a circulating tumor cell detection kit and an application thereof. The circulating tumor cell detection kit is characterized by comprising a RosetteSep rare cell enriching reagent, an RNA (Ribonucleic Acid) extraction reagent, an RNA reverse transcription reagent, a fluorescence labeling Taqman probe, a specific primer and a qPCR (Quantitative Polymerase Chain Reaction) reaction system reagent. The kit can be applied to tumor patient minimally invasive liquid biopsy, early diagnosis and early warning on tumor metastasis and reappearance are achieved, the curative effect in antineoplaston and the tumor development situation are monitored, molecular subtyping based on circulating tumor cells is achieved, and sensitive medicines which specifically aim at the circulating tumor cells are selected, understanding about tumor metastasis, replase and relevant medicine resistance mechanisms is improved, and individualized treatment on tumor patients is guided, so that the total survival of tumor patients is remarkably improved, and great clinical significance is achieved.

Description

technical field [0001] The invention relates to a circulating tumor cell detection kit and application thereof. Background technique [0002] In recent years, the morbidity and mortality of hepatocellular carcinoma have risen rapidly, ranking fifth among malignant tumors. Although treatment techniques have been greatly improved, the 5-year survival rate has not improved significantly, and about 50% of patients have tumor recurrence or distant metastasis 5 years after surgery. Therefore, predicting the postoperative recurrence of liver cancer patients for early necessary intervention is the key to further improve the prognosis of liver cancer. There is an urgent need for an effective and reliable detection technology to solve the problems faced in the treatment of HCC such as early diagnosis of disease, screening of high-risk recurrence, evaluation of anti-tumor treatment effect, prevention and control of distant metastasis. [0003] Circulating tumor cells (CTCs) refer to ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/04C12Q1/686C12Q2561/101
Inventor 樊嘉杨欣荣郭伟孙云帆周俭
Owner 上海顿慧医疗科技发展有限公司
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