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TaqMan fluorescent quantitative PCR method for identifying pork components by utilizing CACA gene and application thereof

A technology for pork and purpose, applied in the field of molecular biology, can solve problems such as being unsuitable for quantitative detection, and achieve the effect of improving trust, avoiding pollution and strong specificity

Active Publication Date: 2020-03-24
SOUTH CENTRAL UNIVERSITY FOR NATIONALITIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the different number of mitochondria in different cells, the mitochondrial DNA copy number will also vary in different tissues, so it is not suitable for quantitative detection

Method used

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  • TaqMan fluorescent quantitative PCR method for identifying pork components by utilizing CACA gene and application thereof
  • TaqMan fluorescent quantitative PCR method for identifying pork components by utilizing CACA gene and application thereof
  • TaqMan fluorescent quantitative PCR method for identifying pork components by utilizing CACA gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1D

[0043] Example 1DNA Extraction and Amplification Experiment

[0044] 1. Experimental materials

[0045] 1.1 Animal material

[0046] Eight different breeds of pigs (Sus scrofa) (Enshi black pig (male), Enshi black pig (female), Enshi native pig, Huangpi native pig, Shuanghui pork, COFCO pork, wild boar, hybrid wild boar) were used for the experiment. The other 13 different animal materials are: cattle (Bovine), sheep (Caprinae), donkeys (Equus asinus), camels (Camelus bactrianus), chickens (Gallus domesticus), ducks (Anatinae), dogs (Canis lupus familiaris), rats (Muroidea), Fish (Piscium), Squid (Todarodes pacificus), Shrimp (Fenneropenaeuschinensis), Scallop (Patinopectenyessoensis), Goose (Ansercygnoidesorientalis).

[0047] 1.2 Enzymes and Reagents

[0048] Hieff qPCR SYBR Green Master Mix (real-time fluorescent quantitative PCR amplification premix solution) was purchased from Shanghai Yisheng Biotechnology Co., Ltd., the catalog number is 11201ES03.

[0049] Chlorofo...

Embodiment 2

[0085] Example 2 Primer Screening for Specific Amplification of Pig-derived Components

[0086] 1. Experimental materials

[0087] 1.1 Pig genomic DNA

[0088] 1.2 Enzymes and Reagents

[0089] Molecular biology reagents, Hieff qPCR SYBR Green Master Mix (real-time fluorescent quantitative PCR amplification premix solution) were purchased from Shanghai Yisheng Biotechnology Co., Ltd., the catalog number is 11201ES03.

[0090] PCR primers were synthesized by Wuhan Sangon Bioengineering (Shanghai) Co., Ltd.

[0091] 1.3 Experimental Instruments

[0092] Real-time fluorescence quantitative PCR instrument: CFX Connect (BIO-RAD);

[0093] Ultra-micro-volume ultraviolet spectrophotometer: NanoPhotometer-N80 (Implen);

[0094] Other instruments include: constant temperature water bath, centrifuge, vortex instrument, ultrapure water instrument, etc.

[0095] 2. Experimental method

[0096] 2.1 Design of primers

[0097] Using the pig-specific gene (GenBank accession number XM_...

Embodiment 3

[0107] Example 3 Analysis of Intraspecific and Interspecific Characteristics of Primers CACAF21-CACAR162-CACAP69 for Specific Amplification of Porcine Components

[0108] 1. Experimental materials

[0109] 8 different breeds of pigs (Sus scrofa) (wild boar, hybrid wild boar, Enshi black pig (male), Enshi black pig (female), Enshi native pig, Huangpi native pig, Shuanghui pork, COFCO pork). The other 18 different animal and plant materials are: cattle (Bovine), sheep (Caprinae), donkeys (Equus asinus), camels (Camelus bactrianus), chickens (Gallus domesticus), ducks (Anatinae), dogs (Canis lupus familiaris), Rat (Muroidea), Fish (Piscium), Squid (Todarodes pacificus), Shrimp (Fenneropenaeus chinensis), Scallop (Patinopectenyessoensis), Goose (Anser cygnoides orientalis), Soybean (Glycine max), Sesame (Sesamumindicum), Corn (Zea mays) , Arabidopsis thaliana, Nicotiana tabacum.

[0110] 2. Experimental method

[0111] 2.1. Extraction of plant genomic DNA

[0112] Plant genomi...

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PUM

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Abstract

The invention discloses a TaqMan fluorescent quantitative PCR method for identifying pork components and an application thereof. Through a real-time fluorescent quantitative PCR technology based on aTaqMan probe, a single-copy gene CACA in pig cell nucleuses is used as a marker gene, and a pork component detection method with strong specificity and high sensitivity is established. The method established by the invention can be used for accurately distinguishing pigs from other 13 animals and 5 plants, carrying out actual detection on 6 kinds of meat products on the market and accurately judging whether the meat products contain pig-derived components or not. The pig-specific real-time fluorescent quantitative PCR detection method established by the invention can provide a technical reference for qualitative and quantitative detection of pig-derived components in mixed meat products.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a fluorescent quantitative PCR method for identifying pork components in food and an application thereof. Background technique [0002] Pigs (Sus scrofa) are one of the most widespread mammalian species worldwide. Belonging to Animalia, Chordate, Mammalia, Artiodactyla, Suidae, Porcine, it is closer to primates than Rodentia. According to different geographical regions, the pig species can be classified into more than 16 different subspecies. As a subspecies of pigs, domestic pigs are the main source of pork and an important economic animal. For example, in Jianli, Hubei Province County Jianli pigs, Qingping pigs from Dangyang City, Hubei Province, black pigs from Enshi City, Hubei Province, etc. However, most of the domestic pigs sold in the domestic market now use three imported pigs, namely Duroc, Landrace and Large White, to produce three-way hybrid commercial p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6851C12N15/11
CPCC12Q1/6888C12Q1/6851C12Q2600/156C12Q2531/113C12Q2561/101C12Q2563/107
Inventor 张丽严钰澳刘彦刘婷覃瑞方莹王海英
Owner SOUTH CENTRAL UNIVERSITY FOR NATIONALITIES
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