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Triple fluorescent PCR primer probe set, kit and method for detecting six avian-derived components

A primer-probe and kit technology, which is used in biochemical equipment and methods, recombinant DNA technology, and microbial assay/inspection to achieve the effect of improving specificity and high efficiency

Active Publication Date: 2020-08-18
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 primer probe set, kit and method for detecting six avian-derived components
  • Triple fluorescent PCR primer probe set, kit and method for detecting six avian-derived components
  • Triple fluorescent PCR primer probe set, kit and method for detecting six avian-derived components

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

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

[0056] Table 2 Preparation of 100μmol / L stock solution into 10μmol / L use solution

[0057]

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

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

[0060] ③ Positive control: Take all the primer stock solution and dilute to 10 μmol / L respectively. Extrac...

Embodiment 2

[0076] Example 2 National food safety supervision spot check risk monitoring samples - pickled pepper chicken feet, sweet and spicy duck tongue, fresh foie gras, quail eggs, ecological pigeon (pigeon meat), frozen partridge (chicken chicken), Dengying beef, Detection of air-dried mutton, dried pork with vegetables

[0077] Pickled pepper chicken feet, sweet and spicy duck tongue, fresh foie gras, quail eggs, ecological squab (meat), frozen rock chicken (meat), Dengying beef, air-dried mutton, vegetable pork jerky, respectively numbered For #1, #2, #3, #4, #5, #6, #7, #8, #9 samples. After the sample was ball milled, the sample DNA was extracted on a small magnetic stand (Qiagen MagAttract Magnetic Rack) using an outsourced DNA extraction kit (Qiagen MagAttract Hmw DNA kit), all using 3d H 2 O was diluted (or concentrated in vacuum) to a concentration of about 50ng / μL. The primer set, positive control, negative control, and blank control were all according to the kit component...

Embodiment 3

[0088] Example 3 Provincial Food Supervision and Sampling Inspection Samples—Detection of Spicy Chicken and Crispy Duck

[0089] The provincial sampling samples of spicy chicken and crispy duck are numbered #10 and #20 respectively. Judging from the production methods of the samples, the chicken in the spicy chicken was cooked at high temperature with a lot of seasonings, and the duck in the crispy duck was fried at high temperature. The loss of DNA in both was very serious. DNA extraction: ① The sample was pulverized with a ball mill, and the mass after treatment was 10.0 g; ② Add 10 mL of n-hexane and 20 mL of CTAB extract, homogenize in an air shaker at 120 rpm for 6 hours, and occasionally mix by inversion; ③ Centrifuge at 10 krpm for 10 min to make the aqueous phase Clearly delaminated with the organic phase, remove the organic phase and pipette the aqueous phase; ④ centrifuge the gained aqueous phase at 10krpm for 10min, and pipette the supernatant aqueous phase; ⑤ carry...

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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 in particular relates to a triple fluorescence PCR primer probe set, a kit and a method for detecting six kinds of poultry-derived components. Background technique [0002] At present, the adulteration of animal products occurs frequently, and there are many such reports at home and abroad. For example, in 2013, there were incidents of beef food adulteration that used horse meat as beef in many EU countries. In recent years, our country has also exposed many adulteration incidents of animal products, including various cases seized by food and drug supervision, quality inspection, industrial and commercial offices in various places and published by the news media: duck meat is used as mutton, pork and chicken are used as beef, Using cat meat as rabbit meat and other counterfeiting methods. Because most of the adulterated animal products have added flavors and fragrances, added flavorin...

Claims

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Application Information

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Patent Type & Authority Patents(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
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