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

Specific primers and probe by fluorogenic quantitative PCR (Polymerase Chain Reaction) for detecting sheep-derived ingredients in meat products and kit thereof

A fluorescent probe, sheep-derived technology, applied in the field of food inspection and biological detection, can solve the problems of accurate source identification, inability to destroy protein components or immunogenic components, etc., achieves simple operation, shortened detection period, repeatability Good results

Active Publication Date: 2015-11-18
苗丽
View PDF8 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Currently, immunological methods can be used to identify different fresh meats, but for heat-processed meat products, the source cannot be accurately identified due to the destruction of protein components or immunogenic components.

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
  • Specific primers and probe by fluorogenic quantitative PCR (Polymerase Chain Reaction) for detecting sheep-derived ingredients in meat products and kit thereof
  • Specific primers and probe by fluorogenic quantitative PCR (Polymerase Chain Reaction) for detecting sheep-derived ingredients in meat products and kit thereof
  • Specific primers and probe by fluorogenic quantitative PCR (Polymerase Chain Reaction) for detecting sheep-derived ingredients in meat products and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Design and synthesis of primers and probes

[0041] 10 sheep mitochondrial Cytb genes published in GenBank (the accession numbers in GenBank are DQ459341, JX567831. .1, JX010746.1, AB044308.1) for sequence comparison, screen out the sheep Cytb gene conservative sequence, its nucleotide sequence is as SEQ ID NO.4 (wherein, Y, R and M are degenerate bases, Y=C or T, R=A or G, M=A or C), and use Primer5 software to design fluorescent quantitative PCR specific primers and probes used in conjunction with the primers.

[0042] The nucleotide sequence of the preferred primer is:

[0043] Upstream primer: 5'-GCATAATATTCCGMCCAATCAG-3';

[0044] Downstream primer: 5'-AGTTGTCCAATAATRATGTAGGG-3', wherein M and R are degenerate bases, M=A or C, R=A or G.

[0045] The preferred nucleotide sequence of the probe is: 5'-FAM-TCACATGAATTGGAGGMCAGCCAG-BHQ1-3', wherein M is a degenerate base, and M=A or C.

[0046] The primers and probes were synthesized by Shanghai Huirui Bi...

Embodiment 2

[0047] Embodiment 2: the extraction of sample DNA

[0048] The genomic DNA of the sample was extracted by the phenol / chloroform method, and the specific steps were as follows:

[0049] Take fresh meat, remove fat and other tissues, cut into about 1cm 2 Diced meat of different sizes, washed with distilled water, soaked overnight in water at 10°C to remove excess salt and grease, minced and dried in an oven at 80-85°C for 72 hours, then added the dried meat to the tissue homogenate Grinding and homogenizing in the machine to obtain meat samples. Take a 50 mg meat sample, add 700 μL tissue lysate and 20 μL proteinase K (20 mg / mL) to it, vortex to mix, bathe in 56°C water for 2 hours, add an equal amount of Tris to balance phenol, mix up and down, centrifuge at 10,000 rpm for 10 min; For the supernatant, add 1 / 2 volume of Tris balanced phenol and 1 / 2 volume of chloroform:isoamyl alcohol (24:1), mix well, and centrifuge at 10000rpm for 10min; then take the supernatant and transfe...

Embodiment 3

[0050] Example 3: Establishment of a fluorescent quantitative PCR detection method for detecting sheep-derived components in meat products

[0051] (1) Optimization of fluorescent quantitative PCR reaction system

[0052] Refer to the fluorescent quantitative PCR reaction system suggested by 2×PremixExTaq (ProbeqPCR) (25 μL system: 2×PremixExTaq (ProbeqPCR) 12.5 μL; the final concentration of upstream and downstream primers is 0.2 μM; the final concentration of probe is 0.4 μM; DNA template 2 μL, ddH 2 (28.5 μL) was used as the initial system to optimize the fluorescent quantitative PCR reaction system of the present invention, one condition was optimized at a time, and the remaining conditions were fixed. According to the Ct value and the strength of the fluorescent signal, the optimized reaction conditions were judged, and the best conditions were combined as the final optimized system.

[0053] The annealing temperature ranges from 55°C to 63°C, and a total of 9 gradients ...

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 group of primers and a probe by the fluorogenic quantitative PCR (Polymerase Chain Reaction) for detecting sheep-derived ingredients in meat products; nucleotide sequences of the primers and the probe are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3. The invention also provides a detection kit for detecting the sheep-derived ingredients in the meat products by using the primers and the probe by the fluorogenic quantitative PCR. The detection method and the detection kit provided by the invention have the advantages of being accurate in detection, high in sensitivity and specificity, simple and quick.

Description

technical field [0001] The invention relates to the technical fields of food inspection and biological detection, in particular to a fluorescent quantitative PCR primer, a probe and a kit for detecting sheep-derived components in meat products. Background technique [0002] Mutton has a natural nourishing effect and has been favored by consumers for a long time. With the increasingly developed market circulation, mutton has become the main means of modern marketing after being frozen and processed, but mutton is generally frozen, especially cooked mutton after processing. Driven by high profits, some unscrupulous traders used chicken, duck, pork, etc. in the production and sales of meat products to pass off fakes as real ones, which aroused strong indignation among consumers. This not only involves issues such as economy and nutrition, but also directly affects the health of consumers, especially those who are allergic to certain things. [0003] At present, immunological ...

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): C12Q1/68C12N15/11
CPCC12Q1/6851C12Q1/6888C12Q2531/113C12Q2561/113C12Q2563/107
Inventor 苗丽张秀平李珂杨娜王永杰白杰
Owner 苗丽
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

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com