One group of nucleic acid aptamers capable of specifically identifying Beijing genotypes tuberculosis bacterial strain antigen and application of nucleic acid aptamers

A Beijing genotype and nucleic acid aptamer technology, applied in the biological field, can solve problems such as unusable monitoring, difficult popularization, and high cost, and achieve the effects of overcoming low sensitivity, simple implementation, and low cost

Active Publication Date: 2015-03-25
武汉顺可达生物科技有限公司
View PDF2 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows us to screen and prepare DNA molecules called probablocks from natural sources like mannequinolide deoxyribitaminuxin ribosides. These small pieces are attached to other proteins found on certain organisms such as yeast cell walls by covalently linking them together through their sugar chains. They have been shown to specifically recognize various types of microorganism including gram negative rods caused by Mtb strain BCG-19, which makes up about 70% of all cases of tularemicoids worldwide. By comparing these genetic material against traditional methods based upon immunological techniques, we discovered new ways to make better tools for identifying pathogenicity associated with diseases like pulmonary tubercle disease.

Problems solved by technology

This patent describes different ways to diagnose and treat infectious disease due to Bacillus tulansilagene isolates called pseudomycetospora cassettes. These isolated sequences contain unique repeating segments that allow them to attach themselves to proteins like ribbon structures within host systems such as phylaxis gamma cycloviran monoferrato fevere serograms. However, current approaches require complicated procedures and involve subjective interpretation based upon qualities of individual peptoids.

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
  • One group of nucleic acid aptamers capable of specifically identifying Beijing genotypes tuberculosis bacterial strain antigen and application of nucleic acid aptamers
  • One group of nucleic acid aptamers capable of specifically identifying Beijing genotypes tuberculosis bacterial strain antigen and application of nucleic acid aptamers
  • One group of nucleic acid aptamers capable of specifically identifying Beijing genotypes tuberculosis bacterial strain antigen and application of nucleic acid aptamers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] [Example 1] Screening of aptamers

[0040] 1. Extraction, purification and identification of the surface lipid sugar ManLAM of Mycobacterium tuberculosis Beijing genotype strain

[0041] (1) Culture in 7H9 liquid medium (Weigh 4.79 grams of Mycobacterium tuberculosis medium Middlebrook 7H9 and dissolve in 900ml of deionized water, add 2ml of glycerin and mix well, sterilize at 121°C for 10 minutes, add in a volume ratio of 9:1 Bacteria enrichment solution (Middlebrook OADC, spare) to OD about 1.8, inactivated in a 65°C water bath for 1 hour, then centrifuged at 10,000 rpm for 10 minutes to collect Mycobacterium tuberculosis Beijing genotype bacteria, added a small amount of PBS to wash twice, and freeze-dried;

[0042] (2) Resuspend the dried H37Rv in 10 times the volume of chloroform / methanol (v / v=2:1), and degrease in a water bath at 37°C for 12 hours;

[0043] (3) Collect the precipitate by centrifugation at 10,000 rpm for 10 minutes, resuspend the precipitate in ch...

Embodiment 2

[0126] [Example 2] ELONA (adapter) method detection specificity

[0127] 1. Coated with Beijing genotype Mycobacterium tuberculosis, Mycobacterium smegmatis, BCG, Mycobacterium avium, Escherichia coli DH5α, Staphylococcus aureus, Bacillus denatus, Enterococcus, Klebsiella pneumoniae, Staphylococcus epidermidis, Pseudomonas aeruginosa, Streptococcus, Neisseria, H37Rv Mycobacterium tuberculosis (10 6 cfu), were respectively coated on 96-well plates, placed in a 37°C wet box for coating for 1 hour, and the microwells were washed 3 times with PBST;

[0128] 2. Block the microwells with 1% BSA (200 μl / well) at 37°C for 1 hour, and wash the microwells 3 times with PBST;

[0129] 3. After measuring the concentration of the above five aptamers of biotin-labeled ManLAM obtained by asymmetric PCR, add 30 pmol, incubate at 37°C for 1 hour, and wash the microwells 3 times with PBST;

[0130] 4. Add HRP-labeled streptavidin (diluted 1:2000), 100 μl / well, act at 37°C for 45 minutes, wash ...

Embodiment 3

[0134] [Example 3] Aptamer ELONA (aptamer) method detects the sensitivity of ManLAM lipid sugar

[0135] 1. Coating with different concentrations of ManLAM lipid sugar (100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3 μg / mL, 1.5 μg / mL, 0.75 μg / mL, 0.37 μg / mL mL, 0.18 μg / mL) in a 96-well microplate, coated at 37°C for 1 hour, and washed with PBST for 3 times;

[0136] 2. Block the microwells with 1% BSA (200 μl / well) at 37°C for 1 hour, and wash the microwells 3 times with PBST;

[0137] 3. After measuring the concentration of the five biotin-labeled aptamers obtained by asymmetric PCR, add 30 pmol, incubate at 37°C for 1 hour, and wash the microwells 3 times with PBST;

[0138] 4. Add HRP-labeled streptavidin (diluted 1:2000), 100 μl / well, act at 37°C for 45 minutes, wash the microwells 3 times with PBST;

[0139] 5. Add TMB substrate, react at 37°C for 1min to 3min, and observe the color change. Then stop with 2M concentrated sulfuric acid;

[0140] 6. Use a micro...

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 one group of nucleic acid aptamers capable of specifically bonding with tuberculosis mycobacterium Beijing genotype strain surface lipolysaccharide namely mannose modified mannosylated lipoarabinomannan (ManLAM) and application of the nucleic acid aptamers. The aptamers are specific to small-molecular single-chain DNA (ssDNA) of the ManLAM, and have a nucleotide sequence as shown in the SEQ ID No.1-5. The ssDNA has functions similar to those of a monoclonal antibody, and can be directly and specifically bonded with ManLAM. By adopting an enzyme-linked oligonucleotide assay (ELONA) method, the ManLAM antigen in serum of patients with pulmonary tuberculosis, extrapulmanory tuberculosis and the like can be quickly effectively detected in 2 hours, the ManLAM antigen in sputum of an active pulmonary tuberculosis patient can be directly identified, and the detection sensitivity on the Man LAM antigen can be 0.035mu g/mL. The aptamers are simple to prepare and low in cost, and can be used as an effective novel reagent for diagnosing the mycobacterium tuberculosis antigen of early and latent tuberculosis.

Description

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

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
Owner 武汉顺可达生物科技有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap