DNA nucleic acid constant value quality control material and preparation method thereof

A quality control material, HIV-2DNA technology, applied in the field of medical testing, can solve problems such as difficulty in ensuring the validity and consistency of measurement results, differences in the accuracy and traceability of quantitative values, and delays in the development of related quality control materials, and achieve stability Good stability and uniformity, accurate target value determination, and the effect of ensuring accuracy and reliability

Inactive Publication Date: 2018-12-25
GUANGZHOU BDS BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The development of relevant quality control substances is relatively lagging behind, making the testing of many products impossible, and we have to rely on foreign imports to meet laboratory testing requirements
Due to the different management systems implemented by the producers of these imported quality control substances, the accuracy and traceability of the measurement values ​​are also different, so it is difficult to ensure the validity and consistency of the measurement results in my country

Method used

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  • DNA nucleic acid constant value quality control material and preparation method thereof
  • DNA nucleic acid constant value quality control material and preparation method thereof
  • DNA nucleic acid constant value quality control material and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1-HIV-2 DNA droplet digital PCR method establishment

[0037] (1) Design of primers and probes:

[0038] According to the relevant information of HIV-2 gene sequence, two pairs of PCR primer probes were designed with the aid of Primer Premier5.0 biological software.

[0039] Table 1 Primers and probes for PCR amplification of HIV-2 gag-gp105 genome

[0040]

[0041]

[0042] (2) Extraction of viral nucleic acid (Tiangen Magnetic Bead Method Viral DNA / RNA Extraction Kit)

[0043] a. Add 20μl proteinase K to a clean 1.5ml centrifuge tube.

[0044] b. Add 200μl of plasma / serum / lymph to the centrifuge tube (the sample needs to be equilibrated to room temperature).

[0045] c. Add 400μl Carrier working solution to the sample, close the cap, and vortex for 10 seconds to mix. d. Incubate at 56°C or room temperature for 10 minutes. Centrifuge briefly to collect the liquid adhering to the tube wall and tube cap.

[0046] e. Add 400μl of absolute ethanol, flocculent precipitation ma...

Embodiment 2

[0073] Example 2-Preparation of HIV-2 standards and establishment of QPCR method

[0074] Take HIV-2 viral nucleic acid as a template and use full-length primers for amplification. The amplified product was ligated to the pEASY-T3 vector, and then transformed into Trans T1 competent cells. Bacteria containing positive recombinant plasmids are screened out and sent to sequencing for identification. Then, using the purified SPE I digestion linearized product as a template, the NanoDrop2000 ultraviolet spectrometer was used to determine the concentration, and the copy number was calculated, and a 10-fold dilution was performed. Calculate the number of DNA copies per microliter volume according to the following formula. y(copies / μL)=[x (g / μL)DNA / (transcript length in nucleotides×340)]×6.02×10 23

[0075] Using the designed primers and probes to establish the QPCR method, the QPCR reaction system is ProbeqPCR dUTP Master Mix 10μL, 10μM primer 0.8μL, 10μM probe 0.5μL, water 5.9μL, te...

Embodiment 3

[0081] Example 3-Preparation of negative and positive samples of DNA-type nucleic acid quality control materials

[0082] (1) Negative plasma (serum) sample

[0083] The appearance properties are screened out and the appearance package is complete, without damage and leakage. There is a clear label, indicating the blood supply unit, blood donor information, preparation time, expiration date, volume, storage conditions, etc. After thawing, it should be a light-yellow homogeneous liquid, without obvious flocculent sediments or large pieces of sediment, and the result of the contamination test (PCR method) is negative.

[0084] After the qualified negative plasma is inactivated at 56°C for 60 minutes, fibrin is removed by filtration with sterile medical gauze in a Class II biological safety cabinet in a Class 10,000 work area, and stored at -20±5°C for use.

[0085] (2) Positive plasma (serum) sample

[0086] The samples were tested using the nucleic acid extraction method of Example 2 a...

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Abstract

The invention aims to provide a DNA nucleic acid constant value quality control material which is accurate in constant value of a target value, has traceability, stability and good uniformity, and hasa higher using value. The DNA nucleic acid constant value quality control material is formed by a clinic positive sample or a microorganism culture and a freeze-drying protective agent. Detection for11 types of infectious microorganism items is contained, and a microdrop digital PCR with high specificity and high sensitivity is used for detecting without a standard curve. Quantification can be directly performed to determine a standard value of the quality control material, and general scientific research and clinic using can be satisfied. The DNA nucleic acid constant value quality controlmaterial has the advantages of a stable measured value, good uniformity, the accurate constant value, the traceability, good intercommunity with a clinic sample and the like.

Description

[0001] Field [0002] The invention belongs to the technical field of medical testing, and belongs to the category of deoxyribonucleic acid (DNA) biological products in the technical field of polymerase chain reaction (PCR), and specifically relates to a quality control substance for DNA nucleic acids of infectious microorganisms and a preparation method thereof. Background of the invention [0003] Digital PCR (Digital PCR, dPCR) technology is a breakthrough quantitative analysis technology that has attracted attention in recent years and has developed rapidly. In 1992, when Sykes et al. detected low-abundance IgH heavy chain mutant genes in a complex background, they used the limited dilution of the sample to obtain only a single template molecule in each well, and calculated the amplified signal after PCR in order to accurately determine it. The number of starting molecules, although the concept of "digital PCR" has not been clearly put forward, the basic experimental procedure ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12Q1/04
CPCC12Q1/6851C12Q2545/113C12Q2563/159
Inventor 李尔华高旭年王清涛华文浩钟凤然吴淑贤
Owner GUANGZHOU BDS BIOLOGICAL TECH CO LTD
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