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A common screening kit and detection method based on the animal -derived component of the melting curve technology

A technology of animal-derived components and kits, which is applied in biochemical equipment and methods, recombinant DNA technology, and microbial determination/inspection, etc., can solve the problems of complex system composition, high application cost, limited applicability, etc. Good performance, cost-effective, and the effect of avoiding pollution

Active Publication Date: 2022-08-09
宁波市产品食品质量检验研究院(宁波市纤维检验所)
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to achieve the purpose of multiple identification, it is often necessary to conduct multiple experiments or use multiple primer and probe composition detection methods. The workload is heavy, the system composition is complex, the competition is large, and the cost of identification is high.
Although gene chip technology has fast and efficient multiple detection capabilities, it has not been widely used in the field of food inspection and testing due to the high application cost due to the need for professional equipment and special commercial reagents
There are also published detection technologies that use universal primers combined with high-resolution melting curve methods to achieve multiple identification targets, but this type of technology has special requirements for detection equipment, and its applicability is relatively limited

Method used

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  • A common screening kit and detection method based on the animal -derived component of the melting curve technology
  • A common screening kit and detection method based on the animal -derived component of the melting curve technology
  • A common screening kit and detection method based on the animal -derived component of the melting curve technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Design and optimization of universal primers and molecular beacon compositions

[0049] 1. Download 9 species of livestock, poultry and animal mitochondrial genome sequences that can be identified by the kit of the present invention from the National Center for Biotechnology Information (NCBI) genome database, as a reference for the design of universal primers, molecular beacon probes and sample species identification According to, including: pig (Sus scrofa) NC012095, cattle (Bos taurus) NC006853, goat (Caprahircus) NC005044, donkey (Equus asinus) NC001788, horse (Equus caballus) NC001640, camel (Camelus bactrianus) NC009628, dog (Canis lupus familiaris) ) NC002008, Chicken (Gallusgallus) NC001323, Duck (Anas platyrhynchos) EU755252.

[0050] 2. Design and optimize a combination of universal primers and molecular beacon probes with multiple identification capabilities. The principles and methods of target gene sequence screening are: (1) The sequence size is...

Embodiment 2

[0074] Example 2: Detection of Livestock and Poultry Meat Samples

[0075] Using the detection kit comprising the universal primer and probe composition provided by the present invention, real-time fluorescent PCR and melting curve analysis were used to detect 9 kinds of fresh meat samples of livestock and poultry.

[0076]1. Experimental materials: fresh pork, yellow beef, goat meat, donkey meat, horse meat, camel meat, dog meat, chicken, and duck meat. Methods Real-time fluorescent PCR method (GB / T38164-2019) and other standard methods were used for identification and sequence comparison analysis, and the species source was clear.

[0077] 2. Reagents and equipment: Food genome extraction kits and probe-based qPCR detection reagents are commercial general-purpose kits; fluorescence quantitative PCR instrument is AB ViiA of Thermo Fisher Company TM Type 7. The combination of universal primers and molecular beacon probes provided by the kit of the present invention is entrus...

Embodiment 3

[0091] Example 3: Sensitivity and specificity experiments

[0092] 1. Test sample and method: take pig-derived standard as the sample to be tested, and dilute 10-fold gradient to the final template concentration of 10 1 -10 8 copies / μL, using the 20 μL reaction system and detection conditions established by the present invention, real-time fluorescent PCR detection and melting curve analysis are performed to test sensitivity and specificity.

[0093] 2. Analysis of results: According to image 3 The results of the displayed serial dilution of the porcine standard DNA template can be seen at 10 1 -10 8 At the template concentration of copies / μL, the real-time fluorescence PCR reaction amplification curve was good, and the Ct value was in the range of 15 to 35 ( image 3 a), showing a good linear relationship with the initial template concentration (R 2 =0.99), indicating that the universal primer-molecular beacon probe combination provided by the present invention has good...

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Abstract

The invention discloses a general screening kit and detection method for animal-derived components based on melting curve technology, and relates to the technical field of molecular biology detection. The invention designs universal primers and molecular beacon fluorescent probe compositions for the gene fragments with specific structural characteristics in the genomes of 9 kinds of livestock and poultry animals. Taking the genomic DNA of the sample to be tested as a template, applying the kit and method disclosed in the present invention to carry out real-time fluorescent PCR detection and melting curve analysis, by comparing the shape of the melting peak of the sample to be tested and the corresponding characteristic melting temperature (Tm value), Realize the rapid identification of 9 kinds of animal-derived components of livestock and poultry under the condition of a single tube. The system of the invention is simple in structure, low in application cost, high in sensitivity, strong in specificity and good in applicability, and can be used for rapid screening of animal-derived components in food, feed and other samples and identification of adulteration and counterfeiting.

Description

technical field [0001] The invention relates to a general screening kit and detection method for animal-derived components based on melting curve technology, belonging to the technical field of molecular biology detection. Background technique [0002] Livestock and poultry meat and its products have always been one of the important categories in the daily dietary composition of residents because of their delicious flavor and rich nutrition, and their food safety is of great significance to safeguarding the rights and interests of consumers. Due to the high production cost of livestock and poultry meat and its products and the large market demand, adulteration and counterfeiting has become one of the main food safety problems in current meat products. In recent years, major food safety incidents related to it have occurred frequently at home and abroad. report. This kind of illegal and fraudulent manufacturing and marketing behaviors cause economic losses to consumers, and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/6888C12Q1/686C12Q2527/107C12Q2563/107C12Q2561/113
Inventor 邢家溧承海郑睿行沈坚毛玲燕徐晓蓉姚振民
Owner 宁波市产品食品质量检验研究院(宁波市纤维检验所)
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