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Stomach cancer serological detection and identification kit and method

A technology of serology and kits, which is applied in gastric cancer serology detection and identification kits and fields, can solve the problems of high false positive rate, patient mental stress and property loss, low detection sensitivity and specificity, etc.

Active Publication Date: 2017-05-31
ZHEJIANG PROVINCIAL HOSPITAL OF TRADITIONAL CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

CA19-9, CA24-2 and CEA are currently the main serological indicators for the diagnosis of gastric cancer, but the sensitivity and specificity of these tests are still not high, and the false positive rate is high, because people's fear of cancer, false positive detection As a result, it is easy to cause huge mental pressure and property loss to the patient

Method used

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  • Stomach cancer serological detection and identification kit and method
  • Stomach cancer serological detection and identification kit and method
  • Stomach cancer serological detection and identification kit and method

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0049] Experimental example 1 Screening of exosomal miRNAs highly correlated with gastric cancer

[0050] 1. Serum exosome isolation and RNA extraction

[0051] 1) Take 2ml of fresh blood tested positive for gastric cancer by routine blood testing and put it into a vacuum blood collection tube-separating gel / coagulant tube (purchased from Greiner Bio-One Company), and let stand at room temperature for 15 minutes.

[0052] 2) Thermo ST16R refrigerated centrifuge at 1000×g, centrifuge at 4°C for 10 minutes.

[0053] 3) Carefully transfer the serum to a 15ml centrifuge tube and centrifuge at 2000×g for 30 minutes. Transfer the supernatant to a new 15ml centrifuge tube.

[0054] 4) Centrifuge at 10,000×g in a Thermo ST16R refrigerated centrifuge at 4°C for 40 minutes. The supernatant was transferred to a new ultracentrifuge tube (Beckman Company, Cat No. / ID: 355654). Beckman Optima L-100XP Ultracentrifuge Ultracentrifuge at 100,000×g, 4°C for 70 minutes.

[0055] 5) Discard t...

experiment example 2

[0079] Experimental example 2 Preparation and use of gastric cancer serum detection and identification kit

[0080] 1. Kit

[0081] The kit provided in this example consists of cel-miR-39-5p fragment, primers, vacuum blood collection tube-separation gel / coagulant tube and vacuum blood collection needle assembly, exosome isolation kit, and exosome miRNA extraction It consists of kits, reverse transcription reagents and real-time fluorescent quantitative PCR reagents.

[0082] Primers (see Table 1, Table 2) are as follows: cel-miR-39-5p, hsa-miR-375, hsa-miR-590-5p, hsa-miR-19b-3p, hsa-miR-100-5p and hsa - miR-16.

[0083] hsa-miR-122-3p, hsa-miR-122-5p, hsa-miR-125a-3p, hsa-miR-125a-5p, hsa-miR-193b-5p, hsa-miR-205-5p, hsa- miR-214-3p, hsa-miR-1307-3p, hsa-miR-19a-3p, hsa-miR-21-3p, hsa-miR-215-5p, hsa-miR-33a-5p, hsa-miR- 494-3p, hsa-miR-501-3p and hsa-miR-598-3p. Primers for real-time quantitative PCR were synthesized by Shanghai Shenggong Company or Suzhou Jinweizhi Bio...

experiment example 3

[0151] Experimental Example 3 Preparation and use of gastric cancer serum detection and identification kit

[0152] Table 7 clinical serological test results:

[0153]

[0154] Steps (1)-(4) are the same as in Example 2.

[0155] (5) Analysis of fluorescence quantitative detection results

[0156] Taking the expression level of cel-miR-39-5p as a reference, when calculating the expression level of miRNA, use ΔCt=Ct(miRNA)-Ct(cel-miR-39-5p), ΔΔCt=ΔCt(patient)-ΔCt(normal individual ), the relative expression of miRNA is 2-ΔΔCt.

[0157] For patients whose serum test results of gastric cancer CA19-9, CA24-2 and CEA are positive or all negative, by analyzing the expression of miRNA in the serum exosomes of the patients and the normal human serum exosomes , and the judging criteria of 20 miRNAs combinations can also be used.

[0158] Table 8: 20 miRNA qPCR results, C T value

[0159]

[0160] Table 9: Result of relative expression of 20 miRNAs

[0161]

[0162]

...

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Abstract

The invention discloses a stomach cancer serological detection and identification kit and a method. The stomach cancer serological detection and identification kit is prepared from a cel-miR-39-5p fragment, a primer, an exosome isolating reagent, an exosome miRNA (Micro Ribonucleic Acid) extracting reagent, a reverse transcription reagent and a real-time fluorescent quantitative PCR (Polymerase Chain Reaction) reagent, wherein the primer comprises a cel-miR-39-5p internal reference primer, an hsa-miR-375 primer, an hsa-miR-590-5p primer, an hsa-miR-19b-3p primer, an hsa-miR-100-5p primer and an hsa-miR-16 primer. When any results of detecting stomach cancer CA (Carbohydrate Antigen)19-9, CA24-2 and CEA (Carcino Embryonie Antigen) of a patient by using serum are positive, and the stomach cancer serological detection and identification kit provided by the invention is then used for carrying out aided detection, false positive results of serological detection can be effectively removed, so that huge mental stress and property loss brought to the patient can be avoided; meanwhile, aiming at the situation that when the results of detecting the stomach cancer CA19-9, CA24-2 and CEA of the patient by using the sera are negative, and the stomach cancer serological detection and identification kit provided by the invention is then used for carrying out the aided detection,, the false negative results of the serological detection can be effectively found, so that the life of the patient can be rescued in time.

Description

[0001] (1) Technical field [0002] The invention relates to a gastric cancer serum detection and identification kit and a detection method. [0003] (2) Background technology [0004] MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs with regulatory functions found in eukaryotes, with a length of about 20-25 nucleotides. Mature miRNAs are produced by the cleavage and processing of long primary transcripts pri-miRNA by a series of nucleases, and then assembled into the RNA-induced silencing complex, which recognizes the target mRNA by base pairing, and according to The difference in the degree of complementarity directs the silencing complex to degrade the target mRNA or repress the translation of the target mRNA. Recent studies have shown that miRNAs are involved in a variety of regulatory pathways, including development, virus defense, hematopoietic process, organ formation, cell proliferation and apoptosis, fat metabolism and so on. Animal miRNAs are usually tra...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6851C12Q1/6886C12Q2600/158C12Q2600/178C12Q2531/113C12Q2545/101C12Q2563/107
Inventor 程向东徐志远吕航郑国淀
Owner ZHEJIANG PROVINCIAL HOSPITAL OF TRADITIONAL CHINESE MEDICINE
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