Identification Of Duck Down And Goose Down Products By Fluorescent PCR

< p > < strong > 1 Introduction < /strong > < /p >

< p > < a > href= > http://www.91se91.com/news/index_c.asp > > down > /a > refers to the villi growing in the abdomen of the goose and duck, which is a kind of animal protein protein. In feather, cotton, wool and silk, the best thermal insulation property of the four natural thermal insulation materials is [1].

However, at present, the prices of feather and down products in the market are quite different. There are many counterfeit products in the down products. [2-3], at present, there are problems in the down market, such as the filling up of the inferior materials, the inconformities of fluffiness, the unqualified degree of fluffiness and the unqualified degree of cleanliness. Among them, the faking down products made from inferior cotton materials are commonly used: fake feather products made from acrylic cotton, feather products made from raw wool, and [5] down products made from crushed velvet (flying silk).

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< p > in the testing industry, the identification of eiderdown is mainly based on the national standard GB/T 10288 - 2003 "feather and feather inspection method" and the industry standard FZ/T 80001 - 2002, the method of washing feather and down test method. The method is to observe the microscopic structure of the sample with the naked eye and compare the standard description and illustration of the microscopic characteristics of duck feather, goose feather and other birds.

The method is highly demanding, subjective and easy to erroneous, and the schematic diagram and text description are simple. The lack of physical reference is difficult for the actual inspection work [6].

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< p > in view of this, this study developed a fluorescent PCR identification method for eiderdown and < a href= "http://www.91se91.com/news/index_c.asp" > goose down products < /a >, in order to combine with traditional identification methods to improve the objectivity and accuracy of feather down identification.

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< p > < strong > 2 materials and methods < /strong > < /p >

< p > 2.1 materials and reagents < /p >

< p > 2.1.1 test material < /p >

< p > the products of eiderdown, goose down and so on are purchased from the market.

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< p > 2.1.2 reagent < /p >

< p > guanidine thiocyanate, polyvinylpyrrolidone K-40 (PVP K-40), sodium twelve alkyl sulfate (SDS), and beta mercapto ethanol are Amersco products. Salmon essence DNA is Sigma product, Taq enzyme and 2 x premix ExTaq enzyme are purchased from Bao Biological Engineering (Dalian) Co., Ltd., and the leads and probes (table 1) are synthesized by Shanghai bioengineering biotechnology Service Co., Ltd.

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< p > 2.2 method < /p >

< p > 2.2.1 DNA extract < /p >

＜p＞　　挑取帶毛囊的羽絨，剪取毛囊部位約0.03g樣品，加入3mL 2% SDS溶液[2% SDS，50mmol/L Tris·Cl(pH值8.0)，20mmol/L EDTA(pH值8.0)]，65℃水浴1.5h，不時振蕩;12000r/min離心5min，取上清;加入3mL羊毛提取液[4mol/L異硫氰酸胍，0.3mol/L氯化鈉，4% PVPK-40，50mmol/L Tris·Cl(pH值8.0)，20mmol/L EDTA(pH值8.0)]及60μLβ-巰基乙醇，65℃水浴4.5h，不時振蕩;12000r/min離心5min，取上清，加入等體積酚：氯仿：異戊醇(25：24：1)，顛倒混勻，12000r/min離心10min;取上清，加入1μL 10mg/mL鮭精DNA，1/10體積的3mol/L乙酸鈉(pH值5.2)及等體積異丙醇，-20℃沉淀過夜，12000r/min離心15min收集沉淀;小心倒去上清，用70%乙醇洗滌二次，風干;取50μL去離子水溶解沉淀。

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< p > 2.2.2 fluorescence PCR detection system was established < /p >

< p > 2.2.2.1 specific fluorescent primer design for duck down and goose down > /p >

< p > primers and probes were designed according to the complete mitochondrial 12S rDNA sequences of domestic ducks, Muscovy ducks, spotted duck, goose, swan goose, chicken, pigeon, quail and Turkey. Based on the primers and probe specificity, the sequence of the duck was consistent with that of its ancestral species of green duck and spotted duck.

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< p > 2.2.2.2 < a href= "http://www.91se91.com/news/index_c.asp" > fluorescence PCR < /a > amplification and result judgment < /p >

< p > reaction system (20 L):2 x premix Ex Taq 10 L, upstream and downstream primers of 0.2 mol/L, probes 0.1 and mol/L 4 DNA.

Reaction procedure: 95 C 30s, 95 C 5S, 60 C 34S, 40 cycles.

Results: when the Ct value is less than 35, the result can be positive. When Ct value is >35, the result can be judged to be negative.

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< p > 2.2.2.3 method specificity and detection limit < /p >

< p > the extracted duck, Muscovy duck, duck feather, goose, goose feather, chicken, quail, cow, buffalo, pig, sheep, goat, horse, rabbit, white mouse and salmon sperm DNA were used as amplification templates. The fluorescence PCR reaction was conducted using duck down and goose specific primers and probes to verify the specificity of the method. The fluorescence PCR reaction was performed on the template of 100ng, 10NG, 1ng, 100PG, 10pg and 1Pg to detect the detection limit of the method, and each gradient was made in three parallel.

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< p > < --EndFragment-- > 2.2.3 sample detection < /p >

< p > 18 eiderdown products were selected, DNA was extracted according to 1.2.1 steps, and the fluorescence PCR reaction was performed according to 2.2.2, and the samples used were shown in Table 2.

As goose down products are scarce, no goose products can be bought on the market, and goose down products are not included in the sample test.

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< p > < strong > 3 Results < /strong > < /p >

< p > 3.1 the DNA extraction method for duck down and goose down products was established by visible visible hair follicle in feather and goose down products. In the experiment, the area of duck's hair and goose hair follicle was cut out. Guanidinium SDS- thiocyanate - [7] was used to extract eiderdown and goose down DNA.

The DNA concentration was 20ng to 200ng, and the Ct values of DNA and goose down DNA were 25 and 26 respectively (PCR 1), indicating that the DNA quality extracted by this method could meet the fluorescence PCR requirement.

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< p > 3.2 method specificity test results < /p >

< p > results, duck feather specific fluorescent primers can only amplify duck, Muscovy duck and duck down DNA, and have no specific amplification with other species DNA. Goose specific fluorescent primers can only amplify goose and goose down DNA, and have no specific amplification with other species DNA, indicating that the designed primers have good specificity.

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< p > 3.3 the detection limit of the method is < /p >.

< p > results, using this method to extract 100% duck feather and 100% goose material DNA, the detection limit of DNA was 10NG (containing salmon sperm DNA) when PCR was reacted.

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< p > 3.4, the test result of samples is < /p >.

< p > detection results showed that 18 duck samples could be detected duck DNA ingredients, and no goose DNA components were detected. The test results were consistent with the product's express ingredients (Table 1).

The duck's specific primers and Muscovy Duck primers were used for fluorescent PCR reaction alone. The DNA components of domestic ducks were detected in 18 samples. The DNA component of Muscovy duck was not detected, indicating that all of the 18 duck products were from eiderdown.

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< p > < strong > 4 discusses < /strong > < /p >.

< p > DNA in animal fiber is mainly located in the hair follicle part, and there are also a small amount of mitochondrial DNA[8] in the hair shaft. Because feather products contain the hair follicles visible to the naked eye, the hair follicle part is selected in the test process. When SDS- is extracted by DNA isothiocyanate guanidine beta mercapto ethanol DNA, the hair follicle area is basically dissolved, and the hair stem is broken in a wide range. Through the sample test, it is shown that this method can successfully extract the eiderdown and goose down DNA.

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< p > the mitochondrial 16S rDNA gene sequence was selected as the primer to design the target site. Primer design must consider that the designed primers can cover all the different duck and goose strains in the world.

Theoretically, because the differentiation time of ducks and geese and their ancestral species is longer than the differentiation time of different strains of ducks and geese, as long as a sequence of DNA sequences is conserved in duck geese and their ancestral species, it can be conserved in all domesticated strains, thus theoretically ensuring that the primer can detect all feather and down products of duck goose strains.

According to Wikipedia and related literature, [9-10], except for Cairina moschata, commonly known as muscovy duck, duck originates from Anas Platyrhynchos and A. poecilorhyncha, and Muscovy ducks are introduced from central and South America.

The geese are formed by the long-term domestication of wild geese in China, < /p >

< p > goose is domesticated from Anser cygnoides, and European goose is domesticated by Hui Yan (A.anser).

Therefore, we downloaded the domestic duck, Muscovy duck, goose and its ancestral species, and downloaded the similar varieties to carry out primer design. The primers designed by NCBI database BLAST comparison and specificity test showed that the specificity was good.

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< p > the identification of down fiber is mainly dependent on microscope. This method is easy to operate, but it relies too much on the quality and experience of the experimenters and is too subjective. The method developed in this study can serve as a powerful supplement to the traditional microscopic identification method and further improve the objectivity and accuracy of the identification of feather and down.

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