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Molecular internal standard quality control method and kit for biological sample nucleic acid detection

An internal molecular standard, biological sample technology, applied in the field of biochemical detection technology and clinical molecular diagnosis, to achieve the effect of improving quality control reliability, high accuracy and low cost

Active Publication Date: 2013-03-20
GENESKY DIAGNOSTICS SUZHOU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the existing field of clinical diagnostic testing, there is no quality control method that uses molecular internal standards to mark samples, and detects internal standards through follow-up experiments to distinguish samples and sample contamination.

Method used

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  • Molecular internal standard quality control method and kit for biological sample nucleic acid detection
  • Molecular internal standard quality control method and kit for biological sample nucleic acid detection
  • Molecular internal standard quality control method and kit for biological sample nucleic acid detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1. Preparation of molecular internal standard

[0057] 1.1 Design of the original sequence

[0058] Using Random DNA Sequence online biology software (http: / / www.bioinformatics.org / sms2 / random_dna.html) to design a 1380bp DNA sequence, as shown in Seq No.1, the GC content of this sequence is between 0.4 and 0.6 Among them, after comparison of genome homology in the NCBI database, it was confirmed that there is no homologous sequence above 40 bp in a row, which means that the designed sequence does not exist in any organism.

[0059] 1.2 Site-directed mutation of Seq No.1 sequence

[0060] The method of artificially synthesizing DNA synthesizes this sequence as a template, and uses site-directed mutagenesis technology to change the bases at specific positions in the DNA sequence to achieve the effect of sequence marking, that is, use site-directed mutagenesis technology to design 6 kinds of missing bases at specific positions ( Deletion of 2bp, 4bp, 6b...

Embodiment 2

[0063] Example 2. Molecular internal standard labeling method

[0064] The sample is human whole blood as an example:

[0065] 2.1 Source or preparation of whole blood samples

[0066] The hospital can draw blood in the blood collection tube, without any follow-up processing after collection;

[0067] 2.2 Preparation of deletion-type internal standard sequences I, II, and III

[0068] 2.3 Molecular internal standard labeling method

[0069] (1) Proportionally add 0.05ng of missing molecular internal standards I, II, and III to the 200ul whole blood samples in the original tubes of the three samples, make a record of the correspondence between the samples and the internal standards, and then perform the following DNA extraction steps (using QIAamp DNA Mini Kit from QIAGEN);

[0070] (2) Add 20ul QIAGEN protease or proteinase K to the bottom of a 1.5ml centrifuge tube;

[0071] (3) Add 200ul samples (whole blood, plasma, serum, buffy coat, or 5×106 lymphoc...

Embodiment 3

[0082] Example 3. Independent internal standard quality control detection

[0083] When the internal standard in the sample needs to be detected separately, take 1ul sample and perform PCR reaction with a total reaction volume of 10 microliters (1μL 10× PCR buffer ( Qiagen company), 0.2μL 25mmol magnesium chloride, 1μL detection internal standard primer, 0.8μL 2.5mmol dNTP, 0.04 µL HotStarplus Taq enzyme ( Qiagen company), 5.96 μL wxya 2 o ). The PCR reaction conditions were set as follows: 95°C for 10 minutes; 7 cycles of Touchdown program (94°C for 20 seconds, 65°C-1 for 40 seconds and 72°C for 2 min), 28 cycles of amplification (94°C for 20 seconds, 63 ℃ 30 seconds and 72°C 2min), extended at 72°C for 2 minutes; the sequence of the missing internal standard primer is shown in Seq No.16.17:

[0084] IMMLABF: 5′-TTTTATCATGGCGCCCTCACTT-3′,

[0085] IMMLABR5'-CCTTAGCGAAGGGCGTAGGAGT-3'.

[0086] After the PCR reaction is completed, dilute the PCR amplific...

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Abstract

The invention provides a molecular internal standard quality control method and a kit for biological sample nucleic acid detection. The molecular internal standard quality control method and the kit can realize sample quality control such as sample identification and detection analysis. Quality control molecular internal standards are specific exogenous nucleotide sequences different from nucleotide sequences of tested samples and have homology less than or equal to 99% with the tested samples. The specific exogenous nucleotide sequences are mixed with the tested samples uniformly, and corresponding records are made and simultaneously, follow-up treatment is carried out. Through DNA synthesis, site-directed mutagenesis and gene cloning technologies, the quality control molecular internal standards are prepared. The quality control molecular internal standards are used in an identifier quality control system for biological sample nucleic acid detection. Through common technologies of amplification, fragment length analysis, SNP typing, multiple fluorescent polymerase chain reaction (PCR), capillary electrophoresis and sequencing, quality control molecular internal standard information can be obtained and be used for sample verification so that quality control processes of identification and acceptance inspection of a sample are realized simply and easily.

Description

[0001] technical field [0002] The invention relates to the fields of biochemical detection technology and clinical molecular diagnosis, in particular to a molecular internal standard quality control method for nucleic acid detection of biological samples. Background technique [0003] Since the birth of molecular biology, the structure and function of biological macromolecules have been studied at the molecular level to reveal the essence of life. With the continuous upgrading and improvement of exploration technology, the achievements of the Institute of Molecular Biology have made outstanding contributions to human beings' understanding of the origin of life, genetic evolution, and mastering the laws of inheritance. Nucleic acid, as a class of biological macromolecules, plays a pivotal role in biological genetic evolution, and has attracted much attention from scientific researchers. Nowadays, there are many kinds of research on nucleic acid, and the research techniques...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 姜正文李才华张希
Owner GENESKY DIAGNOSTICS SUZHOU
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