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Composite for preservation of human saliva and preparation method thereof

A technology of composition and saliva, which is applied in the fields of medical inspection, genetic analysis and detection, can solve the problems of reliability reduction, DNA breakage damage, microbial contamination, etc., to prevent suspension breakage, increase system viscosity, and avoid nucleic acid enzymatic hydrolysis Effect

Inactive Publication Date: 2014-03-05
湖北维达健基因技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Patent application number 201010299215, the patent titled human saliva preservation fixative and its preparation and application discloses a saliva preservation solution, but the MgCl involved 2 The component belongs to the activator of nuclease, which is not conducive to the stable preservation of DNA
Although guanidine isothiocyanate is a denaturing lysis reagent that can inhibit the activity of nucleases, it will damage cells and leave DNA in a free state, which can easily cause DNA breakage and damage.
[0007] The DNA used for genetic testing should first ensure the purity and integrity. If the DNA is damaged such as breakage, it will inevitably directly affect subsequent tests such as PCR, thereby affecting the accuracy of the analysis results; in addition, saliva preservation fluid is used as the preservation of saliva DNA medium, also at risk of oxidation and microbial contamination
Microorganisms will use DNA as their nutrient material. What's more, these microorganisms may secrete enzymes that promote the accelerated degradation of DNA, destroying the integrity of DNA. At the same time, after saliva is contaminated by microorganisms, it is easy to make the DNA extracted from it impure. , and ultimately the integrity and purity of the DNA cannot be guaranteed, which in turn leads to a greatly reduced reliability of subsequent tests
In summary, the amount of DNA contained in saliva is small and easily damaged

Method used

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  • Composite for preservation of human saliva and preparation method thereof
  • Composite for preservation of human saliva and preparation method thereof
  • Composite for preservation of human saliva and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] The composition for human saliva preservation, its components and content are: EDTA 1mmol / L; Tris-HCl (pH 8) 10mmol / L; sucrose 0.3mol / L; NaOAc 3mol / L; Oxytryptamine 2mmol / L; Propyl p-hydroxybenzoate 3mg / mL; Diazolidinyl imidazolium 2mg / mL; Proteinase K 10μg / mL;

[0053] The preparation method is as follows:

[0054] (1) Prepare EDTA, Tris-HCl buffer solution (pH 8), sucrose storage solution and N-acetyl-5-methoxytryptamine storage solution in absolute ethanol to obtain 10mmol / L EDTA solution, 0.1 mol / L Tris-HCl buffer solution, 3mol / L sucrose aqueous solution, 20mmol / L N-acetyl-5-methoxytryptamine in absolute ethanol solution, sterilized at 121°C for 30min, and stored at room temperature;

[0055] (2) Under aseptic conditions, according to the final content of each component, mix the stock solution of each component prepared in step (1) with sterile water in proportion:

[0056] 10mmol / L EDTA aqueous solution 10ml

[0057] 0.1mol / L Tris-HCl buffer 10ml

[0058] 3mol...

Embodiment 2

[0096]The composition for human saliva preservation, its components and content are: EDTA 0.5mmol / L; Tris-HCl (pH 6) 5mmol / L; sucrose 0.2mol / L; NaOAc 2.5mol / L; N-acetyl-5 -Methoxytryptamine 3mmol / L; Propyl p-hydroxybenzoate 4mg / mL; Diazolidinyl imidazolium 1mg / mL; Proteinase K 10μg / mL, pH value of the system is 7.5.

[0097] The preparation method is as follows:

[0098] (1) Prepare EDTA, Tris-HCl buffer solution (pH 6), sucrose storage solution and N-acetyl-5-methoxytryptamine storage solution in absolute ethanol to obtain 10mmol / L EDTA solution, 0.1 mol / L Tris-HCl buffer solution, 3mol / L sucrose aqueous solution, 20mmol / L N-acetyl-5-methoxytryptamine in absolute ethanol solution, sterilized at 121°C for 30min, and stored at room temperature;

[0099] (2) Under aseptic conditions, according to the total volume of the pre-prepared human saliva preservation composition and the content of each component in the system, select the corresponding volume of EDTA, PH6 Tris-HCl buffer...

Embodiment 3

[0101] The composition for human saliva preservation, its components and contents are: EDTA 2mmol / L; Tris-HCl (pH 8.5) 20mmol / L; sucrose 0.4mol / L; NaOAc 3 mol / L; N-acetyl-5- Methoxytryptamine 1mmol / L; Propyl p-hydroxybenzoate 1mg / mL; Diazolidinyl imidazolium 3mg / mL; Proteinase K 10μg / mL, system pH 8.5.

[0102] Its preparation method is carried out with reference to Example 1.

[0103] Referring to Example 1, the saliva samples preserved by the human saliva preservation composition prepared in Example 2-3 were stored at room temperature for 7 days, and then extracted using the saliva genomic DNA extraction kit of "Tiangen" company to obtain Genomic DNA of each saliva sample after preservation.

[0104] Agarose gel electrophoresis and DNA concentration and purity detection are the same as above, the results are shown in Figure 5 and Table 3.

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Abstract

The invention relates to a composite for preservation of human saliva and a preparation method of the composite. The composite comprises the following components: Tris-HCl with pH being 6-8.5 (5-20mmol / L), EDTA (Ethylene Diamine Tetraacetic Acid) (0.5-2mmol / L), NaOAc (2.5-3.5mol / L), cane sugar (0.1-0.4mol / L), N-acetyl-5-methoxytryptamine (1-3mmol / L), propylparaben (1-4mg / mL), diazonium imidazolidinyl urea (1-3mg / mL), protease K(10mu g / mL) and a system with pH being 7.5-8.5. When used for preservation of human saliva, the composite is capable of inhibiting nuclease activity and preventing nuclease from being contaminated by external microbes or being oxidized by itself, thereby guaranteeing the integrality and the purity of genomic DNA (deoxyribonucleic acid). The composite is long in preservation period, wide in preservation temperature range and applicable to gene detection and related scientific researches.

Description

technical field [0001] The invention relates to a saliva sample preservation solution, in particular to a human saliva preservation composition and a preparation method thereof, which are applicable to the fields of genetic analysis and detection, medical examination and the like. Background technique [0002] Molecular techniques based on nucleic acid amplification have been increasingly used in forensic medicine, military affairs, human medicine, and scientific research. For example, in forensic identification, the identity of the deceased can be identified by extracting DNA from family members for nucleic acid amplification; Increase, after collecting relevant genetic information, analyze the various genetic conditions contained in it, so that people can understand their own genetic information, predict the risk of physical illness, and improve their living environment and develop good living habits. Avoid or delay the occurrence of disease. [0003] The body fluids typ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01N1/02
Inventor 徐谋胜王宁王珊袁雅燕
Owner 湖北维达健基因技术有限公司
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