Human body intestinal canal flora detection parting and quantitative reagent kit

A technology of intestinal flora and kits, which is applied in the fields of material stimulation analysis, microbial measurement/inspection, fluorescence/phosphorescence, etc. It can solve the problems of difficult cultivation, no one is completely ideal, and inaccurate counting

Inactive Publication Date: 2009-08-19
DONGHUA UNIV
View PDF0 Cites 12 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in most cases, a variety of bacteria are often mixed in the sample to be tested. At this time, selective identification medium must be used for differential counting. Various bacteria have different oxygen tolerance, nutritional requirements, antibiotic sensitivity and colony morphology. And the color characteristics constitute the basis of identification. At the same time, the antibiotics added in the culture medium will also have different degrees of inhibition on the bacteria themselves, and the specific culture me

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
  • Human body intestinal canal flora detection parting and quantitative reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0070] Example 1

[0071] Sample pretreatment and DNA extraction

[0072] experiment procedure:

[0073] (1) Pretreatment

[0074] Collect fresh stool samples of healthy adults, each sample weighs about 4g, collected in a sterile plastic bag, and stored in a refrigerator at 4°C for no more than 12h. Each stool specimen was added to a test tube containing 6ml of sterile PBS buffer (0.05mol / L, pH7.4) and shaken well for 5-10min, then centrifuged at 500rpm for 5min, and the supernatant was collected. Repeat this step 3 times. Then the supernatant was centrifuged at 5,000 rpm for 10 min, and the precipitate was collected and placed in an Eppendorf tube. The precipitate was suspended in 1 mL of double distilled water (ddH2O), and the precipitate was collected by centrifugation at 14,000 rpm for 5 min after mixing. Dissolve the precipitate in 200 μL of absolute ethanol (pre-cooled at -20°C), shake and mix well, centrifuge at 14,000 rpm for 2 minutes, discard the supernatant, and repeat ...

Example Embodiment

[0077] Example 2

[0078] 16s rDNA universal primer, LDR and LCR probe design

[0079] experiment procedure:

[0080] Search the 16S rDNA sequence information of each genus in Genebank, and find more than 50 pieces and then compare them together to find the conserved sections at both ends and the middle containing variant regions for design. In order to ensure the consistency of the PCR amplification efficiency and the specificity of the probes, the regions with completely identical sequences at both ends and the intermediate variant regions with specific sequences for each genus were screened out. General primers are designed in the conserved regions at both ends to ensure that the primers are completely matched with the primer binding sequences of each genus. In the middle variant region, the specific sequences of each genus are compared to design specific probes. In order to further ensure the consistency and effectiveness of the amplification efficiency, it was finally confirm...

Example Embodiment

[0081] Example 3

[0082] Universal primer amplification for standard DNA and sample DNA

[0083] experiment procedure:

[0084] (1) The standard DNA is the same as the amplification system used for sample detection.

[0085] (2) Universal primer: upstream, 5'-CAGGATTAGATACCCTGGTAGT-3'

[0086] Downstream, 5′-TTGCGCTCGTTGCGGGACTT-3′

[0087] (3) Amplification system (10μL):

[0088] Upstream primer 0.5μmol / L

[0089] Downstream primer 0.5μmol / L

[0090] 10×Buffer 1×

[0091] dNTP 0.2mmol / L

[0092] Mg 2+ 2.0mmol / L

[0093] Taq polymerase 1U

[0094] Add water to 10μL

[0095] (4) Amplification program:

[0096] 95℃15min

[0097] 94℃30s→55℃30s→72℃60s(40cycles)

[0098] 72℃7min

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 relates to a method for detecting, genotyping and quantifying human intestinal flora, consisting of universal primer PCR amplification and specific probe LDR. In the PCR amplification, the 16sr DNA fragment of selected bacteria genus is amplified by the designed universal primer synchronously; the specific LDR probe of each bacteria genus is utilized to achieve the goal of genotyping through ligation reaction; and LDR signal is detected by ABI Prism 377 type sequencer, and the detected fluorescence value is taken as the quantifying basis. Aiming at the universal primer of intestinal microflora, the detection method of the invention effectively amplifies all target intestinal microflora 16sr DNA sequence, thereby ensuring that each target sequence and the template of low copy therein can be detected, further ensuring the specificity of the specific probe of each bacteria genus, completely identifying each target sequence and realizing multiplex genotyping quantification.

Description

technical field [0001] The invention belongs to the field of microbial nucleic acid diagnosis, and in particular relates to a detection, typing and quantification kit of human intestinal flora. Background technique [0002] Bacteria in the intestinal tract play an important role in human growth and development, immune function, nutrition, drug metabolism, pathogenesis and health. The isolation, identification and quantification of intestinal bacteria are very important for studying the function of intestinal flora and the relationship with the body. At present, several methods for typing rRNA genes of intestinal bacteria have been established at home and abroad, but these methods generally have weaknesses such as unstable results, difficult operation, small throughput, and inability to quantify, which hinder research institutions and clinical units from identifying intestinal bacteria. Carry out effective scientific research and detection of the intestinal flora. [0003] ...

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
IPC IPC(8): C12Q1/68C12Q1/10C12Q1/06G01N21/64
CPCY02A50/30
Inventor 王勤熙李凯周宇荀汤周睿肖君华
Owner DONGHUA UNIV
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