阅读说明：本技术 一种多重pcr检测羊肉掺假的方法 (Method for detecting mutton adulteration by multiple PCR ) 是由 苟惠天 曹启航 韩晓梅 刘圆园 孙亚楠 薛慧文 魏衍全 于 2020-07-27 设计创作，主要内容包括：本发明公开了一种一种多重PCR检测羊肉掺假的方法,生物技术领域。本发明通过标准品采集及DNA提取、设计多重PCR引物、引物筛选扩增、检测引物的特异性和稳定性、构建双重PCR扩增体系、对掺杂羊肉样品进行检测这六个步骤来实现多重PCR检测羊肉掺假。本发明利用多重多聚酶链反应依据片段大小可以在一次PCR中检测羊肉及掺杂其中的其他肉类。本发明建立的复式PCR方法操作简便,特异性强,灵敏度高,可以在肉类及产制品鉴定和溯源中应用,提高了检测效率。本发明中以单一引物、两种引物或者四种引物进行总DNA模板进行PCR反应,其特异性片段均能被扩增出。(The invention discloses a method for detecting mutton adulteration by multiple PCR, belonging to the technical field of biology. The method realizes the multiplex PCR detection of mutton adulteration by six steps of standard product collection and DNA extraction, multiple PCR primer design, primer screening and amplification, primer specificity and stability detection, double PCR amplification system construction and mutton adulteration sample detection. The invention can detect mutton and other meat doped therein in one PCR by utilizing multiple polymerase chain reactions according to the size of fragments. The duplex PCR method established by the invention is simple and convenient to operate, has strong specificity and high sensitivity, can be applied to identification and tracing of meat and products, and improves the detection efficiency. In the invention, a single primer, two primers or four primers are used for carrying out PCR reaction on the total DNA template, and specific fragments can be amplified.)

1. A method for detecting mutton adulteration by multiplex PCR is characterized by comprising the following steps:

1) collecting standard products and extracting DNA: respectively mincing mutton, pork, duck meat and rat meat in a small food processor for later use, weighing the mutton, the pork, the duck meat and the rat meat in the same weight into a centrifugal tube, and respectively extracting DNA of 4 pure meat samples of the mutton, the pork, the duck meat and the rat meat through a gene extraction kit;

2) designing a multiplex PCR primer: designing and determining respective specific primers according to mitochondrial 18SrRNA gene sequences of mutton, pork, duck meat and rat meat, wherein the serial numbers of four species are sheep-Y, pig-Z, duck-YA and rat-S respectively, and the product sizes are 276bp,327bp,570bp and 168bp respectively;

3) and (3) primer screening and amplification: taking 10 0.2ml PCR tubes, numbering 1-10 in sequence, adding samples respectively, and putting the samples into a PCR instrument for amplification;

4) and (3) detecting the specificity and stability of the primer: taking a centrifuge tube, sequentially adding 100uL of Tap enzyme, 88uL of water, forw all4uL and revsus4uL, uniformly mixing, and sequentially adding 49uL into 4 PCR tubes; adding the sample sheep, pig, duck and mouse into the 4 tubes in sequence; performing PCR amplification; after the amplification is finished, taking the PCR product, adding sample, and performing electrophoresis;

5) constructing a double PCR amplification system: in the reaction system: amplifying 25uLTap enzyme 12.5uL, water 11uL, forw all0.5uL, rev sheep and mice 0.25uL respectively, and sheep and mouse templates 0.25uL respectively;

6) and (3) detecting the doped mutton sample: the multiplex PCR reaction adopts a 50uL reaction system, 25uL of Tap enzyme, 22uL of water, 1uL of forw all, 1uL of rev duck, sheep, mouse and pig in a certain proportion, 1uL of sample template is doped, and after amplification is finished, a PCR product is sampled and subjected to electrophoresis.

2. The method for detecting mutton adulteration by multiplex PCR according to claim 1, which is characterized in that the amplification conditions in the step 3) are as follows: pre-denaturation: 95 ℃/2min, denaturation: 95 ℃/30S, annealing: the temperature of the No. 1-10 tubes is 50-59 ℃/30S respectively, and the extension is as follows: 72 ℃/1min, extension: and (5) preserving at 72 ℃/8 min.

3. The method for detecting mutton adulteration by multiplex PCR according to claim 2, wherein after the amplification at 4 ℃, the PCR product is sampled and subjected to electrophoresis.

4. The method for detecting mutton adulteration by multiplex PCR according to claim 1, which is characterized in that the amplification conditions in the step 5) are as follows: pre-denaturation: 95 ℃/2min, denaturation: 95 ℃/30S, annealing: 52 ℃/30S, extension: 72 ℃/1min, extension: 72 ℃/8min, preservation: PCR products were collected at 4 ℃ and subjected to 150V electrophoresis for 30min on 2.0% agarose gel.

5. The method for detecting mutton adulteration by multiplex PCR according to claim 1, wherein in the step 6), the adulteration and meat adulteration are judged according to the bands appearing in the gel imaging system.

6. The method for detecting mutton adulteration by multiplex PCR according to claim 4, wherein the downstream primer needs to be adjusted at least 2 times until 2 clear bands appear.

7. The method for detecting mutton adulteration by multiplex PCR according to claim 1, wherein the primer is specific when only the corresponding mouse band is existed in the step 4).

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for detecting mutton adulteration by multiple PCR.

Background

The food is taken by people as the day since ancient times, and the food is an indispensable part in daily life of people all the time. With the development of socio-economic, meat and its products become important sources and components of people's daily food. Nowadays, the abundant diversification of food and the network sale channel also bring an opportunity for meat adulterants. This not only involves problems of the consumer's economic, nutritional value and food safety, but also disturbs the order of the meat distribution market. In order to seek self benefits, some illegal merchants or individuals impersonate meat with high price and high quality by using low-price and low-quality meat, particularly the counterfeiting behavior of adulterated meat is the most common.

The traditional meat morphology identification means relying on sense and experience is far from meeting the requirements of controlling and monitoring adulteration of meat products. At present, the meat identification methods mainly comprise: the source of the meat is judged by a microscopic observation method through the taste, the color, the texture and the like of the meat product; protein detection methods, such as isoelectric focusing electrophoresis, immunization, enzyme-linked immunosorbent assay, chromatography; DNA detection methods such as nucleic acid probe hybridization, restriction fragment length polymorphism polymerase chain reaction analysis, DNA fingerprinting, and PCR-specific amplification. The identification method based on the microscope and the protein needs abundant experience and has great subjectivity, and is mainly used for identifying fresh meat. In cooked meat products, proteins are susceptible to high temperatures, and their fleshy seed source cannot be sensitively detected by protein detection methods. In addition, these methods have the disadvantages of high cost, large workload, high requirements for detection objects, and the like. The DNA hybridization method in molecular biological identification is time-consuming, expensive and complex, while the PCR method has become the most common method in meat product variety identification due to the short reaction time, high sensitivity, specificity and identifiability. At present, a great number of reports exist on PCR and real-time fluorescence PCR methods established by designing species-specific primers according to mitochondrial genome DNA sequence differences, and the methods enter the detection technical standard of Chinese authority. The PCR method can solve the problem of incapability caused by protein denaturation in the traditional method, so that the identification of the types of meat and products thereof becomes necessary, and more accurate information is provided for clinical or food safety detection.

According to the invention, specific primers are designed according to the difference sites of the 16S rRNA genes of animal mitochondria, a PCR reaction system with common forward primers and specific reverse primers is established by optimizing the PCR reaction system, and specific fragments of four different species can be amplified simultaneously by one-time PCR reaction. The method has high sensitivity and accuracy, is simple and easy to operate, and is expected to become an effective method for conventional detection.

Disclosure of Invention

The invention aims to provide the method for detecting the mutton adulteration by the multiplex PCR, which is simple to operate, improves the sensitivity and the accuracy and can simultaneously amplify specific fragments of four different species by one-time PCR reaction.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for detecting mutton adulteration by multiplex PCR comprises the following steps:

1) collecting standard products and extracting DNA: respectively mincing mutton, pork, duck meat and rat meat in a small food processor for later use, weighing the mutton, the pork, the duck meat and the rat meat in the same weight into a centrifugal tube, and respectively extracting DNA of 4 pure meat samples of the mutton, the pork, the duck meat and the rat meat through a gene extraction kit;

2) designing a multiplex PCR primer: designing and determining respective specific primers according to mitochondrial 18SrRNA gene sequences of mutton, pork, duck meat and rat meat, wherein the serial numbers of four species are sheep-Y, pig-Z, duck-YA and rat-S respectively, and the product sizes are 276bp,327bp,570bp and 168bp respectively;

3) and (3) primer screening and amplification: taking 10 0.2ml PCR tubes, numbering 1-10 in sequence, adding samples respectively, and putting the samples into a PCR instrument for amplification;

4) and (3) detecting the specificity and stability of the primer: taking a centrifuge tube, sequentially adding 100uL of Tapase, 88uL of water, 4uL of forwall and 4uL of revsus4uL, uniformly mixing, and sequentially adding 49uL into 4 PCR tubes; adding the sample sheep, pig, duck and mouse into the 4 tubes in sequence; performing PCR amplification; after the amplification is finished, taking the PCR product, adding sample, and performing electrophoresis;

5) constructing a double PCR amplification system: in the reaction system: amplifying 25uLTap enzyme 12.5uL, water 11uL, forw all0.5uL, rev sheep and mice each 0.25uL, and sheep and mouse templates each 0.25 uL;

6) and (3) detecting the doped mutton sample: the multiplex PCR reaction adopts a 50uL reaction system, 25uL of Tap enzyme, 22uL of water, 1uL of forw all, 1uL of rev duck, sheep, mouse and pig in a certain proportion, 1uL of sample template is doped, and after amplification is finished, a PCR product is sampled and subjected to electrophoresis.

Further, the amplification conditions in the step 3) are as follows: pre-denaturation: 95 ℃/2min, denaturation: 95 ℃/30S, annealing: the temperature of the No. 1-10 tubes is 50-59 ℃/30S respectively, and the extension is as follows: 72 ℃/1min, extension: and (5) preserving at 72 ℃/8 min.

Further, after the amplification at 4 ℃ is completed, the PCR product is sampled and subjected to electrophoresis.

Further, the amplification conditions in step 5): pre-denaturation: 95 ℃/2min, denaturation: 95 ℃/30S, annealing: 52 ℃/30S, extension: 72 ℃/1min, extension: 72 ℃/8min, preservation: PCR products were collected at 4 ℃ and subjected to 150V electrophoresis for 30min on 2.0% agarose gel.

Further, in the step 6), whether the meat is doped or not and the doped meat are judged according to the strip appeared in the gel imaging system.

Further, the downstream primer requires at least 2 adjustments until 2 distinct bands appear.

Further, the primer of step 4) is specific when only the corresponding mouse band is present.

The invention has the beneficial effects that:

1) the invention can detect mutton and other meat doped therein in one PCR by utilizing multiple polymerase chain reactions according to the size of fragments.

2) The duplex PCR method established by the invention is simple and convenient to operate, has strong specificity and high sensitivity, can be applied to identification and tracing of meat and products, and improves the detection efficiency.

3) In the invention, a single primer, two primers or four primers are used for carrying out PCR reaction on the total DNA template, and specific fragments can be amplified.

Drawings

FIG. 1 shows the genome amplification bands of four meat products according to the present invention; wherein, M is DNA Marker 2000; 1, sheep; 2, a mouse; 3, duck; 4, the pig is bred.

FIG. 2 is a temperature gradient of a duck genome according to the present invention; wherein the temperatures of 1-8 are 50-57 ℃.

FIG. 3 shows the temperature gradient of the genome of sheep of the present invention.

FIG. 4 shows the specificity of the pig primers of the present invention, wherein 2 is a pork PCR product band; 1. 3 and 4 are mutton, duck meat and rat meat respectively.

FIG. 5 shows the specificity of the primers for ducks of the present invention; wherein, M is DNA Marker 2000; 3 is a duck PCR product strip; 1. 2 and 4 are mutton, pork and rat respectively.

FIG. 6 shows the mutton of the present invention doped with rat meat; wherein, M is DNA Marker 2000; 1 is a positive control of pure mutton; 2 is positive control of pure rat meat; 3. 4 is the amplification product of mutton mixed with rat meat.

FIG. 7 shows the mutton of the present invention mixed with duck meat; wherein, M is DNA Marker 2000; 1 is a positive control of pure mutton; 2 is positive control of pure duck meat; 3. 4 and 5 are amplification products of mutton mixed with duck meat.

FIG. 8 shows the mutton of the present invention mixed with rat meat and pork; m is DNA Marker 2000; 1 is a positive control of pure pork; 2. 3 is the amplification product of mutton and pork.

FIG. 9 shows the mutton of the present invention doped with pork, duck and mouse meat; wherein, M is DNA Marker 2000; 1 is positive control of pure duck meat; 2. 3 is the product of mutton mixed with the amplification products of pork, duck and rat.

Detailed Description

The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.