Free nucleic acid preservative and blood collection and preservation device

A free nucleic acid and preservative technology, applied in biochemical cleaning devices, enzymology/microbiology devices, biological material sampling methods, etc. and other problems, to prevent DNA dilution and contamination of target cfDNA, improve shelf life, and slow down the effect of rupture

Active Publication Date: 2019-04-09
NINGBO AJCORE BIOSCIENCES INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these existing protective agents/blood collection tubes still cannot fully meet the needs of clinical testing and research in terms of storage time and required storage conditions, especially large-scale disease screening a...

Method used

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  • Free nucleic acid preservative and blood collection and preservation device
  • Free nucleic acid preservative and blood collection and preservation device
  • Free nucleic acid preservative and blood collection and preservation device

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The preparation of embodiment 1 reagent

[0034] According to the different anticoagulants used, the formulations were divided into three groups. For the sake of simplification, the following experimental formulations all used glutamine 10mg / ml, sodium orthovanadate 800mg / ml, leupeptin 5mg / ml, dye Genistein 5mg / ml, Aluminum Fluoride 1mg / ml, Acetylcysteine ​​40mg / ml, Diazolidinyl Urea 100mg / ml. The table below shows only the representative ingredient ratios used in the final summary verification, and some wrong or unrepresentative ratios in the previous period are not shown.

[0035] During the preparation, dissolve the required amount of various reagents in RNase-free purified water.

[0036] Table 1 Formula based on EDTA-3K anticoagulant

[0037]

[0038]

[0039] Table 2 Formulas based on EDDHA–Na anticoagulant

[0040]

[0041]

[0042] Table 3 Formulas based on EDTA-3K and EDDHA–Na anticoagulants

[0043]

[0044]

[0045] Fill the above nuclei...

Embodiment 2

[0046] Each formula performance comparison of embodiment 2

[0047]Recruit 3 male volunteers aged 22-25 (after physical examination: normal blood routine indicators, no physical examination tumor indications, no inflammation indications), blood collection 170ml (formula 1-14 blood collection tubes, Kangjieheparin lithium blood collection tubes, Streck non-invasive vacuum blood collection tube, blank glass blood collection tube). Store in a thermostat at room temperature at 23°C until the experiment begins.

[0048] Perform the following tests on each sample on days 0 (within 2 hours after blood collection), 2, 5, 10, 14, and 21:

[0049] For hemolysis, check the 414nm OD value. Calculate the three sample average.

[0050] According to the instructions of the kit, use the QIAamp Circulating Nucleic Acid Kit cfDNA Extraction Kit to extract the cfDNA in the sample, and use the qPCR method to detect the total concentration of cfDNA in the sample. The detection object is GAPDH, ...

Embodiment 3

[0060] Embodiment 3 preferred formula actually detects application

[0061] The vacuum blood collection tubes made of formula 3 and formula 12 preservative of the present application are used together with Streck non-invasive vacuum blood collection tubes for the actual detection of EGFR T790M in tumor patients (QIAamp Circulating Nucleic Acid Kit kit extracts cfDNA, EGFR Mutation Test kit detection, operation according to the kit standard procedure), involving a total of 40 patients, each patient used three kinds of blood collection tubes for sampling, the first test was performed within 48 hours after blood collection, stored at room temperature for 14 days and 21 days and then two test and compare the test results.

[0062] Table 6 EGFR T790M detection results

[0063]

[0064]

[0065] Except that the test results of the three storage methods within 48 hours and on the 14th in the formula 3 group were consistent, there were 3 cases of inconsistency (false positive)...

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Abstract

The invention relates to a free nucleic acid preservative and a blood collection and preservation device. The free nucleic acid preservative is prepared from the following ingredients: 10-50mg/ml of glutamine, 200-1,000mg/ml of sodium orthovanadate, 1-5mg/ml of leupeptin, 1-5mg/ml of genistein, 1-5mg/ml of aluminum fluoride, 10-40mg/ml of acetylcysteine, 100-500mg/ml of diazolidinyl urea, 1-3mg/mlof an anticoagulant and 5-20mg/ml of a membrane protective agent. The free nucleic acid preservative has the advantages of effectively retarding the rupture of blood cells, preventing a target cfDNA(cell free deoxyribonucleic acid) from being diluted and polluted by DNAs in the blood cells, preventing the target cfDNA from being degraded by nucleases and prolonging the preservation time of a blood sample containing the target cfDNA.

Description

technical field [0001] The invention relates to the field of blood detection, in particular to a free nucleic acid preservation agent and a blood collection preservation device Background technique [0002] cfDNA (cell Free DNA), also known as circulating nucleic acid, refers to DNA molecules that are free outside cells. These DNA molecules are mainly derived from certain release processes of normal cells, cell apoptosis, cell necrosis, and the growth and infiltration of cancer cells. The size of the release is mainly concentrated in tens to hundreds of bp, and the half-life is about several hours. [0003] The level and type of cfDNA in the blood are associated with various diseases including tumors. In addition, fetal cfDNA can also be found in maternal blood. cfDNA detection in blood has been widely used in the screening of various cancers, certain autoimmune diseases such as lupus erythematosus, and the non-invasive detection of fetal genetic diseases such as Down syndr...

Claims

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Application Information

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IPC IPC(8): C12Q1/24C12M1/26C12M1/00
CPCC12Q1/24
Inventor 周杰锋王德明
Owner NINGBO AJCORE BIOSCIENCES INC
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