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Assay kit and method for simultaneously extracting and purifying ribose nucleic acid (RNA) and deoxyribose nucleic acid (DNA)

A kit and co-extraction technology, applied in the field of kits for simultaneous extraction and purification of RNA and DNA, can solve problems such as the influence of glass beads

Inactive Publication Date: 2013-02-06
PEOPLES REPUBLIC OF CHINA BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the residue of glass beads and drying problems have affected the application of this technology

Method used

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  • Assay kit and method for simultaneously extracting and purifying ribose nucleic acid (RNA) and deoxyribose nucleic acid (DNA)
  • Assay kit and method for simultaneously extracting and purifying ribose nucleic acid (RNA) and deoxyribose nucleic acid (DNA)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Preparation of Mixed Samples

[0039] In order to verify the extraction effect of the kit of the present invention, the inactivated foot-and-mouth disease virus and Streptococcus suis preserved in our laboratory were selected as test materials, and a mixed sample containing animal bacteria and viruses was prepared.

[0040] Asian type 1 foot-and-mouth disease virus (strain code: As-1 / PK-1 / 2005) cell culture. The concentration of virus before inactivation is: 10 9 TCID 50 / mL.

[0041] Inactivated Streptococcus suis type 2 (gifted by the China Veterinary Drug Administration) bacterial suspension, the concentration of the bacterial suspension before inactivation is 10 8 CFU / mL.

[0042] Concrete preparation process is as follows:

[0043] (1) In a secondary biological safety cabinet, dilute the concentrated suspensions of the above two test bacteria / strains to 10 with sterilized PBS (pH 7.2) 5 TCID 50 / mL and 10 5 CFU / mL.

[0044] (2) Take 5mL of the t...

Embodiment 2

[0045] Embodiment 2: Trizol method separately extracts the RNA of mixed sample

[0046] Extract the RNA of mixed sample in example 1 with Trizol method, concrete steps are as follows:

[0047] (1) Take 5 sterilized 1.5mL sterilized centrifuge tubes, numbered 1-5;

[0048](2) Add 600 μL of lysate to each tube, add 100 μL each of the tested sample, negative control, and positive control, and use a tip for each sample, then add 200 μL of chloroform, shake and mix on the mixer for 5 seconds (not too strong, In order to avoid an emulsified layer, it can also be mixed by hand upside down). Centrifuge at 4°C, 12000r / min for 15min;

[0049] (3) Take five sterilized 1.5mL sterilized centrifuge tubes, add 500μL isopropanol (pre-cooled at -20°C), and mark them. Pipette the supernatant from each tube in 2) and transfer to the corresponding tube. The supernatant should be pipetted at least 500 μL, the middle layer cannot be sucked out, and the mixture should be mixed by inversion;

[0...

Embodiment 3

[0054] Embodiment 3: Boiling method extracts mixed sample DNA separately

[0055] Extract the DNA in the mixed sample with DNA extraction reagent, the specific operation is as follows:

[0056] (1) Take n 1.5mL sterilized centrifuge tubes, where n is the sum of the number of samples to be tested, one tube of positive control and one tube of negative control, and number each tube.

[0057] (2) Add 200 μL of DNA extraction solution I to each tube, then add 100 μL each of the sample to be tested, the negative control and the positive control, and use a tip for each sample; shake and mix for 5 seconds on the mixer. Centrifuge at 13000r / min for 10min at 4°C~25°C.

[0058] (3) Aspirate and discard the supernatant as much as possible without touching the precipitate, then add 10 μL of DNA Extraction Solution II, shake and mix for 5 seconds on a mixer. Centrifuge at 2000r / min for 10s at 4°C~25°C.

[0059] (4) Dry bath or boiling water bath at 100°C for 10 minutes; centrifuge at 500...

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Abstract

The invention discloses an assay kit and a method for simultaneously extracting and purifying ribose nucleic acid (RNA) and deoxyribose nucleic acid (DNA). The assay kit comprises extraction lysis buffer, 2M sodium acetate, phenol water, chloroform / isoamyl alcohol mixed liquor, isopropyl alcohol, 70% or 75% diethylpyrocarbonate (DEPC) alcohol, sterilized DEPC water, back extract and a nucleic acid purification column. The assay kit and the method have the advantages that the method for simultaneously extracting RNA and DNA is built and the assay kit is assembled. Compared with the classical separate extraction method which separately extracts RNA or DNA, the method has the comparative extraction efficiency, and moreover, the method also provides a method for further purifying RNA and DNA. The method has the advantages of simultaneously extracting and purifying RNA and DNA, and users can select different methods to extract nucleic acids according to the practical detection requirements. In addition, the assay kit and the method also provide a good basic platform for a detection technology for detecting two different types of nucleic acids.

Description

technical field [0001] The invention relates to a kit and method for simultaneously extracting and purifying RNA and DNA, belonging to the field of inspection and quarantine. Background technique [0002] In animal quarantine, according to quarantine regulations, one sample often needs to be tested for multiple diseases. With the wide application of high-throughput detection methods such as multiplex PCR and gene chips, it is possible to achieve multiple quarantine epidemic diseases in one test. The extraction and purification of nucleic acid (DNA and RNA) is the basis and premise of the application of these technologies. [0003] Nucleic acid extraction and purification are mainly divided into the following methods: [0004] 1. Phenol chloroform extraction method. This method is one of the most classic methods in nucleic acid separation and purification technology. It was first extracted by researchers in Cold Spring Harbor Laboratory, and has been improved and used by a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 张伟蒲静高志强凌凤俊汪琳谷强乔彩霞尹义张利峰张鹤晓
Owner PEOPLES REPUBLIC OF CHINA BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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