Method for determining nucleotide metabolites in filter paper dried blood spots

A technology for filter paper dried blood slices and metabolites, which is applied in the field of kits for determining nucleotide metabolites in filter paper dried blood slices to achieve the effect of improving accuracy

Inactive Publication Date: 2020-06-23
SHENZHEN HUADA GENE INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, methods for the detection of nucleoside metabolites still need to be further developed and improved

Method used

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  • Method for determining nucleotide metabolites in filter paper dried blood spots
  • Method for determining nucleotide metabolites in filter paper dried blood spots
  • Method for determining nucleotide metabolites in filter paper dried blood spots

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] The kit for this example includes the following components individually packaged:

[0059] (A) Isotope internal standard, 1 bottle; it is composed of three kinds of ribose 2-13C-adenosine, 15N5-guanosine and 15N4-inosine in equal amounts; the quality of each of the above internal standards is 0.1 mg;

[0060] (B) Quality control products, 3 sets; quality control products are bovine serum filter paper dried blood spots containing 6 kinds of nucleoside metabolites, 3 sets of quality control products include 6 bovine serum filter paper dried blood spots in total, of which low concentration quality 2 sheets of control products, 2 sheets of medium-concentration quality control products, and 2 sheets of high-concentration quality control products;

[0061] (C) V-bottom 96-well plate, 10 pieces;

[0062] (D) hydrophilic PVDF filter plate, 5 pieces;

[0063] (E) Aluminum foil sealing film, 10 sheets;

[0064] (F) Instruction Manual, 1 copy

[0065] The kit should be stored ...

Embodiment 2

[0095] The optimization of embodiment 2 liquid phase methods

[0096] In the comparison case, the sample injection detection method using the liquid flow method (that is, the sample directly enters the mass spectrometry through the liquid phase without connecting to the chromatographic column) can only realize the detection of one of ADA or PNP diseases. In this example, the six nucleosides related to ADA and PNP were combined and detected by liquid flow method tandem mass spectrometry, and it was found that there was mutual interference between the detection targets, so simultaneous detection could not be achieved (ie, the detection did not meet the specificity). That is, a single standard solution and an internal standard solution (both 1ppm) of the six nucleoside metabolites were prepared respectively, and injected for detection. The detection signal intensity (peak height) of each target analyte is shown in Table 4 for details.

[0097] Table 4: Detection signal of single...

Embodiment 3

[0108] The optimization of embodiment 3 extract

[0109] Take the same sample of dried blood film and extract with different extract components, the extract components include pure water, 25% methanol, 50% methanol, 75% methanol and 100% methanol (the extract contains 3 internal standards , the concentration is 10ppb). After sample pretreatment, on-machine testing and evaluation (see Example 1 for the pre-processing steps and on-machine testing methods).

[0110] Under different extraction conditions, the detection and response results of each target analyte are shown in the attached Figure 1 ~ Figure 3 , it can be seen from the data that when the proportion of methanol is less than 50%, the internal standard will be degraded to varying degrees, and when the concentration of methanol is greater than 75%, the response of each target analyte meets the detection requirements (the comprehensive response of 75% methanol is the best) . However, in order to reduce the nitrogen bl...

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Abstract

The invention provides a method for determining nucleotide metabolites in a filter paper dried blood spot. The nucleotide metabolites comprise adenosine, 2'-deoxyadenosine, guanosine, 2'-deoxyguanosine, inosine and 2'-deoxyinosine. The method comprises the following steps: 1) performing extraction treatment on a to-be-detected filter paper dried blood slice in an extraction working solution to obtain an extraction solution containing the nucleotide metabolite; 2) carrying out liquid chromatography-mass spectrometry detection on the extract liquor; and 3) determining the yield of the nucleotidemetabolites in the filter paper dried blood spot based on a liquid chromatography separation-mass spectrometry detection result. According to the detection method provided by the embodiment of the invention, six nucleotide metabolites in the filter paper dried blood spot can be simultaneously detected, and the detection method is used for scientific research or synchronous screening and diagnosisof ADA and PNP diseases, so that the detection cost is reduced, and the efficiency is improved. The method has the advantages of high sensitivity, strong specificity and high accuracy.

Description

technical field [0001] The invention relates to the field of biological detection, in particular, the invention relates to a method for determining nucleotide metabolites in filter paper dried blood slices and a kit for determining nucleotide metabolites in filter paper dried blood slices. Background technique [0002] Nucleosides are a very important class of biological macromolecules, which play a key role in the regulation of cell structure, metabolism, energy and function. As the basic building block of nucleic acid, nucleosides participate in the molecular mechanisms of the retention, replication and transcription of genetic information in organisms. In addition, nucleosides have various biological functions such as anti-virus, anti-tumor, immune regulation, anti-inflammation, anti-myocardial ischemia, and improvement of cardiovascular and cerebrovascular circulation. By measuring the content of nucleoside metabolites in human blood, it can be used to evaluate the nucl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06
Inventor 王远高新彦陈欣徐子晨刘勤程丹任艳刘斯奇
Owner SHENZHEN HUADA GENE INST
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