Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Mycobacterium tuberculosis spacer oligonucleotide typing method based on melting point coding

A Mycobacterium tuberculosis and oligonucleotide technology, applied in the field of Mycobacterium tuberculosis spacer oligonucleotide typing, can solve the problems of long detection time, multiple manual operations, PCR product contamination, etc., to meet clinical needs, Improve the detection flux, the effect of high detection flux

Active Publication Date: 2015-08-26
XIAMEN ZEESAN BIOTECH
View PDF3 Cites 6 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the traditional solid-phase membrane hybridization technology has the advantages of mature detection process and low reagent cost, it requires more manual operations, long detection time, and the sample detection throughput cannot meet clinical needs.
These two technical bottlenecks have seriously hindered the clinical promotion of Spoligotyping. For this, scientists have proposed the use of gene chips [6,7] , encoded microspheres [8,9] and mass detection [10,11] and other technologies to replace the solid-phase membrane hybridization technology. Although the reported improved Spoligotyping technology has increased the throughput and greatly shortened the detection time, these technologies have not got rid of the detection mode of post-processing of PCR tubes, which may easily cause contamination of PCR products. , and due to the need for some large instruments, it is difficult to be widely used clinically

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Mycobacterium tuberculosis spacer oligonucleotide typing method based on melting point coding
  • Mycobacterium tuberculosis spacer oligonucleotide typing method based on melting point coding
  • Mycobacterium tuberculosis spacer oligonucleotide typing method based on melting point coding

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: System Design of a Mycobacterium tuberculosis spacer oligonucleotide typing method coded by melting point

[0048] The technical principle of a melting point coded Mycobacterium tuberculosis spacer oligonucleotide typing method is as follows figure 1 Shown: There is a 36bp DNA fragment in the genome of Mycobacterium tuberculosis, which repeats in the genome, called direct repeat sequence, denoted as DR, these fragments are separated by 35-41bp spacers, these The sequence of the spacer is almost the same in all strains, but the insertion or deletion of a certain spacer may occur in different strains. Therefore, Mycobacterium tuberculosis can be typed and identified by detecting the presence or absence of the spacer. First, design a pair of universal primers on the direct repeat sequence in the genome of Mycobacterium tuberculosis, denoted as DR-F and DR-R, use this pair of universal primers to amplify the PCR product of the spacer contained; A pair of amplifi...

Embodiment 2

[0051] Example 2: A method for genotyping H37Rv and BCG standard strains by a melting point coded Mycobacterium tuberculosis spacer oligonucleotide typing method

[0052] The present invention is used to carry out 6 repeated experiments on the DNA samples of the Mycobacterium tuberculosis H37Rv standard strain and the BCG standard strain, so as to investigate the repeatability of the Tm value detected by the system. Among them, the DNA samples of Mycobacterium tuberculosis H37Rv standard strain and BCG standard strain were provided by China National Institute for the Control of Pharmaceutical and Biological Products.

[0053] In this example, SLAN96 real-time fluorescent PCR instrument was used to complete the amplification curve and melting curve analysis.

[0054] reaction system:

[0055] Reaction A: The total reaction volume is 25 μL, including 1×PCR buffer (750mM Tris-HCl, 200mM (NH 4 ) 2 SO 4 , 0.1% (v / v) Tween 20), 4mmol / L MgCl 2 , each dNTP 0.5mmol / L, 0.5mmol / L dUTP...

Embodiment 3

[0066] Example 3: Sensitivity analysis of a melting point-coded Mycobacterium tuberculosis spacer oligonucleotide typing method

[0067] The invention is used to carry out experiments on the DNA sample of the tuberculosis mycobacterium H37Rv standard strain to investigate the effective template concentration range of the system for genotyping tuberculosis specimens. Among them, the DNA sample of the standard strain of Mycobacterium tuberculosis H37Rv was provided by the National Institute for the Control of Pharmaceutical and Biological Products.

[0068] In this example, SLAN96 real-time fluorescent PCR instrument was used to complete the amplification curve and melting curve analysis.

[0069] The experimental procedure is as follows:

[0070] (1) Gradient dilution of the H37Rv standard strain: the genomic DNA template of the H37Rv standard strain was diluted 10 times, so that the concentration of genomic DNA added to each gradient was 1×10 6 copies / μl, 1×10 5 copies / μl, ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a mycobacterium tuberculosis spacer oligonucleotide typing method based on melting point coding and relates to mycobacterium tuberculosis. The method comprises the following steps: (1) designing a pair of universal primers on a direct repeat sequence to amplify all spacers, and designing a pair of primers in a GyrA conserved region; (2) designing four groups of double-tagging fluorescent probes specifically hybridized therewith according to 43 spacer sequences and GyrA sequences, and manually controlling the welding point value of each probe by adjusting the probe length and / or a base composition, wherein each probe is provided with the same fluorescent group; (3) distributing the fluorescent probes in three reaction tubes, wherein by the combination of the reaction tubes, the fluorescent groups and the welding point values, each spacer forms a three-dimensional code, and software automatically reads a result. The method has the advantages of simple and convenient one-step operation, high specificity, high repeatability, low time consumption and relatively low price, and a powerful tool is provided for exploration and research of the prevalence and propagation rule of tuberculosis in China.

Description

technical field [0001] The invention relates to mycobacterium tuberculosis, in particular to a typing method for mycobacterium tuberculosis spacer oligonucleotides coded by melting point. Background technique [0002] Mycobacterium tuberculosis is the pathogenic bacterium that causes tuberculosis, which can invade the whole body organs, among which tuberculosis is the most common, and has become a major global public health problem. The M. tuberculosis complex includes M. tuberculosis hominis, M. bovis, M. africanum, and M. microti. Traditionally, the classification of Mycobacterium tuberculosis complex is based on the differences in growth, morphology, and biochemical characteristics of different species of mycobacteria, but this classification method is far from meeting the needs of modern tuberculosis prevention and control work. With the advancement of science and technology and the completion of the genome sequencing of Mycobacterium tuberculosis, people have a deep un...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/32
CPCC12Q1/6813C12Q2563/107C12Q2527/107
Inventor 李庆阁曾小红许晔
Owner XIAMEN ZEESAN BIOTECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products