Unlock instant, AI-driven research and patent intelligence for your innovation.

High-throughput detection method for strains and risk genes in feed microbial product

A detection method and microbial technology, which is applied in the agricultural field, can solve the problems of heavy workload, discrepancy between the strains used in the product and the logo, low content of target bacteria, etc., and achieve the effect of low cost and more targeted risks

Pending Publication Date: 2022-01-28
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
View PDF9 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the microbial products for feeding have the following problems: (1) Some products contain pathogenic bacteria, which cause risks in use; (2) The purity of some products is not enough, and the content of target bacteria is low; (3) The strains used in some products do not match the labels, There are potential safety hazards; (4) The production strains and products contain risk genes such as virulence genes, drug resistance genes, and migratable drug resistance genes
[0005] Existing detection methods and corresponding standards cannot effectively analyze unknown pathogenic bacteria, and do not have the function of non-target detection
The traditional method has great limitations: the purity is detected by plate counting, the workload is large, and the accuracy is poor
[0006] In summary, in the prior art, there is no non-targeted detection method for miscellaneous bacteria or pathogenic bacteria in feed microbial products and a quantitative method for the 36 species of bacteria in the "Catalogue of Feed Additives (2013)", and there is no specific method for feed A method for analyzing drug resistance genes, transferable drug resistance genes and virulence genes in microbial products, explaining the risk of target genes in Chinese and tracing the source of low-cost strains

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
  • High-throughput detection method for strains and risk genes in feed microbial product
  • High-throughput detection method for strains and risk genes in feed microbial product
  • High-throughput detection method for strains and risk genes in feed microbial product

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] Example 1 extracts the microbial nucleic acid in the feed additive

[0105] 1. Take 200 μL of the sample from the PCR tube and add it to a 2 mL tube;

[0106] 2. Add 1mL 10xTE to the sample obtained in step (1), shake the grinder for 1min, and wash the bacteria twice; add physiological saline to the bacteria, shake to suspend the precipitate, then add 50mg / mL lysozyme mother solution and mix well , after reacting at 37°C for 1 hour, centrifuge, discard the supernatant, and collect the bacteria;

[0107] 3. Take 200 μL of zirconium beads from the PCR tube, add them to the sample, then add 1 mL of lysate, and shake the grinder for 3 minutes;

[0108] 4. Crack at 70°C for 15 minutes, and invert 6 times every 5 minutes during this period;

[0109] 5. Centrifuge at 13000g for 15min at 4°C;

[0110] 6. Take the supernatant to a new 1.5mL tube, add 40μL RNase A and incubate at 37°C for 15min;

[0111] 7. Add 40 μL proteinase K, incubate at 70°C for 10 minutes, and incubate...

Embodiment 2

[0128] Example 2 High-throughput detection

[0129] 2.1 Construction of metagenomic library

[0130] (1) Fragmentation of DNA

[0131] Ultrasonic method: Add pure water to the genomic DNA extracted in Example 1, the total volume is about 400 μL, and the nucleic acid concentration is greater than 10 ng / μL. Place the 1.5mL centrifuge tube on an ice box, and use an ultrasonic breaker to fragment the DNA. The ultrasonic parameters are set as follows: ultrasonic 4s, interval 6s, power 25%, time 5min. Take 10 μL of the sonicated genomic DNA for 1.5% TAE agarose gel electrophoresis detection, and the fragment position should be 100-1000 bp. Take 1 μL of sonicated genomic DNA for Qubit quantification.

[0132] Genomic DNA enzymatic method after ultrasound, DNA end repair and A reaction, adapter ligation of DNA fragments, adapter ligation of DNA fragments, purification of DNA fragment ligation products, library amplification, purification of library amplification products and fragme...

Embodiment 3

[0188] Embodiment 3 determines the detection limit of the inventive method

[0189] In order to determine the noise of the risk microorganism detection method, the present invention selects the standard strains of the most common microbial feed additive products in the market for pure culture, so as to determine the misjudgment rate of species annotation in the method of the present invention.

[0190] Test strains: ATCC 23857 (Bacillus subtilis), ATCC 14580 (Bacillus licheniformis), ATCC 31284 (Bacillus coagulans), ATCC 53671 (Lactobacillus acidophilus), ATCC 19398 (Clostridium butyricum), ATCC 19434 (Intestinal bacteria cocci), ATCC 10241 (Lactobacillus plantarum) and ATCC 39392 (Lactobacillus casei) standard strains.

[0191] Test medium: ATCC Medium 415 medium for the cultivation of Bacillus subtilis; ATCC Medium 3 medium for the cultivation of Bacillus licheniformis; ATCC Medium 18 medium for the cultivation of Bacillus coagulans; ATCC Medium 416 medium for the cultivatio...

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 belongs to the technical field of agriculture, and particularly relates to a high-throughput detection method for strains and risk genes in a feed microbial product. The high-throughput detection method for the strains in the feed microbial product comprises the steps of extracting genome DNA of microorganisms in a sample to be detected; performing high-throughput sequencing; performing data analysis on a high-throughput sequencing result to obtain the abundance of each microorganism; and performing result display on the abundance of the microorganisms, wherein the display result is displayed in Chinese. The method provided by the invention can effectively extract the total DNA in the feed microbial product, accurately screen the non-targeted pathogenic bacteria, and quantitatively analyze the content of strains allowed to be used in the Catalogue of feed additives; and the drug-resistant gene, the transferable drug-resistant gene and the virulence gene in the feed microbial product can be analyzed, the risk and strain traceability of the target gene are explained in Chinese, and the problems of the feed microbial product are effectively solved.

Description

technical field [0001] The invention belongs to the field of agricultural technology, and in particular relates to a high-throughput detection method for strain and risk gene analysis and traceability of strains in microbial products for feeding. Background technique [0002] China is the largest breeding country in the world, and the scale is on the rise. With the stable development of animal husbandry and the acceleration of antibiotic-free breeding, feed microbial products, as one of the important alternatives to antibiotics, have a good market prospect. However, the rapid development of the market has also brought new problems. The production level of some enterprises is low, which causes their products to contain pathogenic microorganisms. The large-scale production of miscellaneous bacteria other than the used strains as fermentation strains may not only cause huge losses in the breeding industry due to animal diseases, but also cause serious threats to people's lives...

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): C12Q1/6806C12Q1/6869
CPCC12Q1/6806C12Q1/6869C12Q2527/125C12Q2521/327C12Q2521/537C12Q2523/308C12Q2535/122
Inventor 李明饶正华孟庆石冯潇慧刘娜焦京琳谢秀兰
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
Features
  • R&D
  • Intellectual Property
  • Life Sciences
  • Materials
  • Tech Scout
Why Patsnap Eureka
  • Unparalleled Data Quality
  • Higher Quality Content
  • 60% Fewer Hallucinations
Social media
Patsnap Eureka Blog
Learn More

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

© 2025 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com