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Triple fluorescent PCR (Polymerase Chain Reaction) primer probe groups for detecting six avian-derived ingredients, kit and method

A technology of primer probes and kits, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., to achieve high efficiency and improve specificity

Active Publication Date: 2018-02-06
贵州省产品质量检验检测院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no literature report on the high-efficiency detection of simultaneous multiplex PCR for ingredients derived from chicken, duck, goose, quail, pigeon, and chubby chicken. Therefore, a triple fluorescent PCR primer-probe set for detecting six kinds of avian-derived ingredients is provided. and related methods are important

Method used

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  • Triple fluorescent PCR (Polymerase Chain Reaction) primer probe groups for detecting six avian-derived ingredients, kit and method
  • Triple fluorescent PCR (Polymerase Chain Reaction) primer probe groups for detecting six avian-derived ingredients, kit and method
  • Triple fluorescent PCR (Polymerase Chain Reaction) primer probe groups for detecting six avian-derived ingredients, kit and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Construction and verification of a triple fluorescence kit for detecting six avian-derived components

[0055] ① Primer probe set: as shown in Table 1, it can be synthesized by a company that has the ability to synthesize primers and probes. In this example, Shanghai Shenggong Bioengineering Service Co., Ltd. was selected to synthesize, and the dry powder of primers and probes was diluted to 100μmol / L was used as a stock solution, and 10 μmol / L was prepared as a working solution according to Table 2.

[0056] Table 2 100μmol / L stock solution is prepared as 10μmol / L use solution

[0057]

[0058] Note: * refers to packaging with 1.5mL transparent centrifuge tube; # refers to packaging with 1.5mL black centrifuge tube.

[0059] ② Fluorescent PCR reagents: common commercially available fluorescent PCR reagents, selected in this example Path-ID TM qPCRMaster Mix.

[0060] ③Positive control: Take all the primer stock solutions and dilute them to 10μmol / L. Extract the...

Embodiment 2

[0076] Example 2 National Food Safety Supervision Random Inspection Risk Monitoring Samples-Pickled Chicken Feet, Sweet and Spicy Duck Tongue, Fresh Foie Gras, Quail Eggs, Ecological Pigeon (Pigeon Meat), Frozen Partridge (Stone Chicken), Dengying Beef, Detection of dried lamb, vegetable and pork

[0077] Retrieving the nationally smoked risk samples Pickled chicken feet, sweet and spicy duck tongue, fresh foie gras, quail eggs, ecological pigeon (meat), frozen stone chicken (meat), Dengying beef, dried lamb, vegetable and pork breast, respectively numbered For #1, #2, #3, #4, #5, #6, #7, #8, #9 samples. After pulverizing the sample by ball milling, use the purchased DNA extraction kit (Qiagen MagAttract Hmw DNA kit) to extract the sample DNA on a small magnetic rack (Qiagen MagAttract Magnetic Rack), all using 3d H 2 O diluted (or vacuum concentrated) to a concentration of about 50ng / μL, the primer set, positive control, negative control, blank control, etc. are all according to...

Embodiment 3

[0088] Example Three provincial-level food supervision samples-detection of spicy chicken and crispy duck

[0089] Collect the provincial samples of spicy chicken and crispy duck, which are numbered #10 and #20 samples respectively. From the sample preparation method, the chicken in the spicy chicken is cooked at a high temperature and there are many seasonings, and the duck in the crispy duck is fried at a high temperature. The DNA loss of both is very serious. Choose the reagent method and the large-volume DNA extraction kit method, manual Extract DNA: ①The sample is crushed by a ball mill, and the processed mass is 10.0g; ②Add 10mL n-hexane, 20mL CTAB extract, in an air shaker at 120rpm, homogenize for 6h, and occasionally mix upside down; ③10krpm, 10min centrifugation to make the water phase Separately with the organic phase, remove the organic phase and remove the water phase; ④ Centrifuge the obtained water phase at 10krpm for 10 min, and remove the supernatant water phase;...

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Abstract

The invention discloses triple fluorescent PCR (Polymerase Chain Reaction) primer probe groups for detecting six avian-derived ingredients. The triple fluorescent PCR primer probe groups comprise triple fluorescent PCR primer probe groups for chicken, ducks and gooses; the triple fluorescent PCR primer probe group for quails, doves and chukars are specially shown in Seq. ID No.1 to Seq. ID No.14.The triple fluorescent PCR primer probe groups for the chicken, the ducks and the gooses belong to the technical field of gene engineering detection; all primers and probes cannot cause mutual interference, amplification only on specific target sequences is carried out and fluorescence signals are excited while amplification on non-target signals is avoided and fluorescence signals are not produced, so that accurate detection on the six avian-derived ingredients can be realized; the triple fluorescent PCR primer probe groups have the advantages of good specificity, small standard errors, shortdetection time, and the cost of reagents is reduced; moreover, the triple fluorescent PCR primer probe groups are suitable for daily detection on poultry products.

Description

Technical field [0001] The invention belongs to the technical field of gene detection, and particularly relates to a triple fluorescent PCR primer probe set, kit and method for detecting six avian-derived components. Background technique [0002] At present, adulteration of animal products occurs frequently, and there are frequent reports of this kind at home and abroad. For example, in 2013, many countries in the European Union had adulterated beef products that used horse meat as beef. In recent years, my country has also successively exposed many adulteration incidents of animal products, including various cases found by food and drug supervision, quality inspection, industrial and commercial offices and published by the news media: using duck to pretend to be lamb, and using pork and chicken to pretend to be beef. Counterfeiting methods such as passing cat meat as rabbit meat. Since most adulterated animal products have some confusing measures such as adding flavors and fragr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2600/16C12Q2537/143C12Q2563/107
Inventor 孙端方孙棣肖洋董睿李春宇田志强张谦黄家瑞
Owner 贵州省产品质量检验检测院