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RNA type sample preservation and dilution liquid and preparation thereof

A diluent and sample technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of short storage time at room temperature, inability to use the same one, poor quality of effect, etc., and achieve easy preservation and inhibition of RNase activity. Effect

Active Publication Date: 2016-04-20
GUANGZHOU BDS BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages are: 1. Different types of RNA samples (serum, plasma, tissue, urine, secretions, culture fluid, etc.) cannot use the same buffer or diluent; 2. Various buffers or diluents preserved The quality of the effect is poor, and the RNA is easy to degrade; 3. The stored samples should be quickly stored in liquid nitrogen or low temperature storage, and the storage time at room temperature is too short

Method used

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  • RNA type sample preservation and dilution liquid and preparation thereof
  • RNA type sample preservation and dilution liquid and preparation thereof
  • RNA type sample preservation and dilution liquid and preparation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: the preparation (100ml) of RNA preservation diluent

[0032] 1) Measure 40ml DEPC·H with a graduated cylinder 2 O is added in the sterilized beaker;

[0033] 2) Add 0.1ml of β-mercaptoethanol into the beaker, and stir evenly;

[0034] 3) Add 2.5ml of 8-hydroxyquinoline (2mol / L) into the beaker, and stir evenly;

[0035] 4) Weigh 16.4g of sodium acetate into the beaker, and stir to dissolve;

[0036] 5) Weigh 10g of sodium lauryl sarcosinate into the beaker, and stir to dissolve;

[0037] 6) Weigh 15g of guanidine isothiocyanate into the beaker, and stir to dissolve;

[0038] 7) Weigh 0.12g of Tris-HCl into the beaker, and stir to dissolve;

[0039] 8) Add 10ml ENDA (0.5mol / L) into the beaker and stir evenly;

[0040] 9) Add 0.5ml Triton-100 into the beaker and stir evenly;

[0041] 10) Draw 5ml of glycerol into the beaker, and stir evenly;

[0042] 11) Weigh 0.1g of sodium azide into the beaker, and stir to dissolve;

[0043] 12) Adjust the pH val...

Embodiment 2

[0046] Example 2: Plasma Sample Dilution Test

[0047] 1. Instruments, reagents and samples

[0048] 1) Main instruments: ABI7500 PCR detector, TGL-18R high-speed refrigerated centrifuge, TDL-5-A desktop centrifuge, K10CD dry constant temperature metal bath, BSC-1500IIA2-X biological safety cabinet, pipette, etc.

[0049] 2) Main reagents: Hepatitis C virus nucleic acid quantitative detection kit (PCR-fluorescent probe method) from Sun Yat-Sen University Daan Gene Co., Ltd.

[0050] 3) Sample: fresh HCV RNA clinically positive plasma sample (1.0E+07IU / ml).

[0051] 2. Experimental plan

[0052] The HCV RNA-positive plasma samples were diluted 10-fold with common diluent and RNA sample preservation diluent. Two sets of dilutions in the control group and the experimental group were designed, and the samples diluted in the two sets of different dilutions were extracted with the hepatitis C virus nucleic acid detection kit (PCR-fluorescent probe method) of Sun Yat-Sen Universit...

Embodiment 3

[0061] Embodiment 3: Plasma sample preservation test

[0062] 1. Instruments, reagents and samples

[0063] 1) Main instruments: ABI7500 PCR detector, TGL-18R high-speed refrigerated centrifuge, TDL-5-A desktop centrifuge, K10CD dry constant temperature metal bath, BSC-1500IIA2-X biological safety cabinet, pipette, etc.

[0064] 2) Main reagents: Hepatitis C virus nucleic acid quantitative detection kit (PCR-fluorescent probe method) from Sun Yat-Sen University Daan Gene Co., Ltd.

[0065] 3) Sample: fresh HCV RNA clinically positive plasma sample (1.0E+07IU / ml).

[0066] 2. Experimental plan

[0067] Dilute HCV RNA-positive plasma samples 10 times and 10,000 times with RNA sample storage diluent, and mark them as high and low concentration test samples as P1 and P2, respectively. Place them in -20±5°C environment, 2-8°C environment, room temperature (20-25°C) environment and 37°C environment respectively. The design of this experiment is as follows: mark the diluted sample...

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Abstract

The invention aims at providing RNA type sample preservation and dilution liquid and a preparation method thereof and belongs to the scopes of dilution and preservation of ribonucleic acid (RNA) type samples in the technical field of polymerase chain reactions (PCR). The preparation method comprises the steps of adding a certain amount of RNA preservative and chemical reagent to deionized purified water; adjusting pH value to 4.8-5.2, and then maintaining the constant volume; conducting sealing and autoclaved sterilization, and leaving the mixture for standby application at the room temperature. The RNA type sample preservation and dilution liquid is a white transparent solution, has the advantages of being stable, easy to preserve, convenient and the like and is applicable to preservation and / or dilution of RNA type samples such as serum, plasma, tissues, urine and secreta. After the RNA samples are immersed in the RNA type sample dilution and preservation liquid, RNA in fresh histocyte can be preserved in a good condition for one day at the temperature of 37 DEG C, a week at the temperature of 25 DEG C, one month at the temperature of 4 DEG C and a long time at the temperature of minus 20 DEG C or minus 80 DEG C.

Description

[0001] Field [0002] The invention belongs to the technical field of biological products, belongs to the dilution and preservation category of ribonucleic acid (RNA) samples in the technical field of polymerase chain reaction (PCR), and relates to a preparation method and application of RNA sample preservation diluents. Background of the invention [0003] Polymerase chain reaction (PCR) technology is an important molecular biology technique, which plays a very important role in the development of modern molecular biology. PCR technology is characterized by high sensitivity, high specificity, time-saving and rapidity. Samples for direct detection of target genes are divided into DNA samples or RNA samples, but the requirements for sample dilution and storage are very high. Ribonucleic acid (abbreviated as RNA, Ribonucleic Acid), the genetic information carrier present in biological cells and some viruses and viroids. RNA is a long chain molecule formed by condensation of rib...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6806C12Q2527/125
Inventor 李尔华高旭年钟凤然吴淑贤
Owner GUANGZHOU BDS BIOLOGICAL TECH CO LTD
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