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Biological sample pretreatment method, method for detecting rna, and pretreatment kit

一种生物样本、检测方法的技术，应用在生物化学设备和方法、微生物的测定/检验、DNA制备等方向

Inactive Publication Date: 2013-05-08
DNAFORM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, the detection of influenza virus needs to be carried out in the clinical field

Method used

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  • Biological sample pretreatment method, method for detecting rna, and pretreatment kit
  • Biological sample pretreatment method, method for detecting rna, and pretreatment kit
  • Biological sample pretreatment method, method for detecting rna, and pretreatment kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] (Nucleic acid amplification of template RNA mixed in human nasal mucosa)

[0088] In this example, it was shown whether the pretreatment kit of the present invention can suppress the nucleic acid amplification inhibitory activity of template RNA derived from human nasal mucosa. The pretreatment kit was prepared to consist of 10 mM MES buffer (pH 5.8), 2.5 mM ferric sulfate, 20 mM sodium bicarbonate, 0.1% SDS, and 20 mg / ml activated alumina (manufactured by Wako Pure Chemical Industries, Ltd., molecular weight 101.96) Kit (pretreatment reagent). In this kit, the human nasal mucosa of a healthy person is suspended, and then, the template RNA having the same partial sequence as the virus (influenza A(H1N1)) prepared as follows is subjected to 10 7 Mixed replicates were used to make samples of this example. This sample was submitted to the RT-SmartAmp method to detect the presence of RNA. In addition, in the comparative example, except that iron sulfate and sodium bicarb...

reference example 1

[0110] (presence of lactoferrin and lysozyme C in human nasal mucosa)

[0111] Such as figure 2 As shown in the electrophoresis photograph of , the presence of lactoferrin and lysozyme C was confirmed in human nasal mucosa. In addition, the presence of lactoferrin and lysozyme C in human nasal mucosa was confirmed as follows.

[0112] Nasal mucosa samples (mucosa of the upper throat) of healthy individuals were collected with a swab (MEN-TIP, manufactured by Nippon Cotton Swab Co., Ltd.), and each swab was suspended in 300 μL of phosphate buffered solution (PBS). The obtained suspension was subjected to gel filtration chromatography (phosphate buffer mobile phase, column HiLoad 16 / 60 Superdex 200 pg, manufactured by GE Healthcare), and size-selected into four fractions (F1 to F4). In addition, this suspension was applied to a protein G affinity spin column (manufactured by Dojin Chemical Co., Ltd.) to obtain a column-bound fraction. The gel filtration screening preparation...

Embodiment 2

[0114] (Inhibitory effect of iron ions and carbonate ions on the RNA decomposition activity of human lactoferrin)

[0115] Such as image 3 As shown, the inhibitory effect of iron ions and carbonate ions on the RNA degradation activity of human lactoferrin was confirmed.

[0116] In this example, the same RNA as the synthesized template RNA was used as a substrate for RNA degradation activity (RNase activity). image 3 In the left panel, a nasal mucosa sample (mucosal membrane of the upper throat) of a healthy person was collected by a swab (MEN-TIP, manufactured by Japanese cotton swabs) and suspended in 250 μL of 40 mM Tris-HCl (pH 7.5). Next, 1.5 mM ferric chloride was added to 9 μL of the suspension, followed by the addition of 5 mM, 10 mM or 15 mM NaHCO 3 solution, the final volume was 18 μL. Then, the suspension was incubated at 37°C for 10 minutes. To its 2.5 μL suspension, 10 μL of 50 ng / μL RNA was added and incubated at 25° C. for 10 minutes. After the reaction, ...

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Abstract

The present invention is to provide a pretreatment method that allows RNA to be detected promptly and simply. RNA degradation activity due to lactoferrin present in the human rhinal mucosa is inhibited, for example, by adding iron ion and carbonate ion to a biological sample that contains the human rhinal mucosa. With the pretreated biological sample, an RNA virus gene can be amplified by a reverse transcriptase. Iron ion and carbonate ion can also inhibit reverse transcriptase inhibition due to lysozyme C contained in the human rhinal mucosa. Further, it is preferable to remove the envelope of the RNA virus by adding SDS to the biological sample that contains the human rhinal mucosa.

Description

technical field [0001] The invention relates to a pretreatment method of biological samples containing RNA, a detection method of RNA and a pretreatment kit. Background technique [0002] As a method of detecting a virus in a biological sample such as blood, there is a method of amplifying and detecting a virus gene. However, most biological samples contain inhibitory substances for gene amplification. Therefore, prior to gene amplification, the biological samples are pretreated to remove or inactivate the inhibitory substances. Pretreatment methods include, for example, the method of treating biological samples with surfactants, solubilizers, heating, etc., and then further purifying them with solid phase carriers or ion exchange resins (Patent Documents 1 to 3). However, these methods require special equipment, and require many operational steps, and thus are cumbersome. [0003] Especially in the detection of viruses with RNA as the gene, in order to perform the amplifi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C07K14/79C12N9/36C12N9/99
CPCC12N15/09G01N1/34C12N9/99C12N15/1003C12Q1/6806
Inventor 林崎良英臼井健悟合田砂织野村和仁川井雄辉
Owner DNAFORM
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