A kind of lysate and method for extracting meat nucleic acid

Abstract

The invention discloses a lysate and a method for extracting meat nucleic acid. The lysate is obtained by dissolving sodium chloride, sodium dodecylsulfonate, ethylphenyl polyethylene glycol and polyvinylpyrrolidone in water; the concentration of each component in the mixture is: sodium chloride: 0.2~ 1.5M, sodium dodecylsulfonate: 0.01~1g / ml, ethylphenyl polyethylene glycol: 0.01~0.5g / ml, polyvinylpyrrolidone: 0.01~0.1g / ml. The method is as follows: mix the minced meat sample with the lysate at normal temperature, and let it stand for a period of time; centrifuge again, and take the supernatant to obtain the nucleic acid extract. The lysate of the invention is low in cost and non-toxic, and the extraction method can realize rapid extraction of meat nucleic acid, the operation process is simple and easy, and can be applied to PCR or LAMP amplification system for detection.

Description

technical field

[0001] The invention belongs to the technical field of food safety monitoring, and in particular relates to a lysate and a method for extracting meat nucleic acid. Background technique

[0002] At present, in order to obtain economic benefits, many unscrupulous merchants in the market often replace high-priced meat with cheap meat, which seriously damages the rights and interests of consumers. Since the appearance of some similar types of meat (such as pork and beef, mutton and beef) is not very distinguishable, it is difficult for ordinary consumers to distinguish with the naked eye. In order to realize the identification of the type of meat, a common method is to use PCR or LAMP reaction to amplify and detect the unique nucleic acid sequence in the meat. However, before amplification, the nucleic acid of the meat needs to be extracted.

[0003] There are many traditional meat nucleic acid extraction methods, such as phenol-chloroform method, CTAB method, ...

Examples

Embodiment 1

[0044] In order to determine the optimal concentration of the four components (sodium chloride, sodium dodecylsulfonate, ethylphenyl polyethylene glycol, polyvinylpyrrolidone) in the lysate, it was decided to use L 9 (3 4 ) to formulate the experimental scheme, and use the range analysis method to obtain the optimal concentration of the four components in the lysate. See Table 1 for their factor levels.

[0045] Table 1

[0046]

[0047] In the experiment, pork was used as a sample to conduct an orthogonal experiment. The mass volume ratio of pork and lysate was fixed at 2:3g / ml. The sample and lysate were mixed and allowed to stand for 1 min, then fixed at 8000 rpm and centrifuged for 1 min. Finally, 50-fold dilutions were uniformly performed with TE buffer to obtain nucleic acid extracts. C according to the real-time fluorescent PCR reaction t Value (the initial number of cycles that can detect the fluorescent signal in the PCR reaction) to determine the nucleic acid ...

Embodiment 2

[0060] After determining the optimal concentration of the four components in the lysate, it is also necessary to determine the mass-to-volume ratio of the sample to the lysate (unit: g / ml), the resting time after mixing the sample and the lysate, and the ability to separate nucleic acids from impurities Three factors such as the optimal rotational speed of the tester (the dilution factor of the nucleic acid extract is finally determined). In order to reduce the number of trials as much as possible, it is still decided to use L 9 (3 4 ) to formulate the experimental program, and use the range analysis method to obtain the best conditions of these three factors. See Table 3 for their factor levels. The letters A, B, and C in the table represent the mass-volume ratio of the sample to the lysate, the resting time, and the centrifugation speed, respectively.

[0061] table 3

[0062]

[0063] In the experiment, pork was used as a sample to carry out an orthogonal experiment,...

Embodiment 3

[0070] First, mix 100 mg of fresh pork with 150 μL of lysate (0.6M sodium chloride, 0.6 g / ml sodium dodecyl sulfate, 0.1 g / ml ethyl phenyl polyethylene glycol, 0.05 g / ml polyvinylpyrrolidone) and mix well, and let stand at room temperature for 2 minutes; then, centrifuge at 10,000 rpm for 1 minute, absorb the supernatant to obtain nucleic acid extract, and use PCR to determine the nucleic acid extraction effect.

[0071] Pork PCR amplification system, amplification program and amplification sequence are the same as in Example 1.

[0072] Dilute the nucleic acid extract 20 times with TE buffer solution as a PCR reaction template for amplification to obtain a fluorescence amplification curve figure 1 .

[0073] The results show: figure 1 The positive samples produced amplification curves, and the negative samples did not produce amplification, indicating that the nucleic acid extraction method can effectively extract the nucleic acid in pork, and can be detected by PCR reactio...

technical field

[0001] The invention belongs to the technical field of food safety monitoring, and in particular relates to a lysate and a method for extracting meat nucleic acid. Background technique

[0002] At present, in order to obtain economic benefits, many unscrupulous merchants in the market often replace high-priced meat with cheap meat, which seriously damages the rights and interests of consumers. Since the appearance of some similar types of meat (such as pork and beef, mutton and beef) is not very distinguishable, it is difficult for ordinary consumers to distinguish with the naked eye. In order to realize the identification of the type of meat, a common method is to use PCR or LAMP reaction to amplify and detect the unique nucleic acid sequence in the meat. However, before amplification, the nucleic acid of the meat needs to be extracted.

[0003] There are many traditional meat nucleic acid extraction methods, such as phenol-chloroform method, CTAB method, ...

Examples

Embodiment 1

[0044] In order to determine the optimal concentration of the four components (sodium chloride, sodium dodecylsulfonate, ethylphenyl polyethylene glycol, polyvinylpyrrolidone) in the lysate, it was decided to use L 9 (3 4 ) to formulate the experimental scheme, and use the range analysis method to obtain the optimal concentration of the four components in the lysate. See Table 1 for their factor levels.

[0045] Table 1

[0046]

[0047] In the experiment, pork was used as a sample to conduct an orthogonal experiment. The mass volume ratio of pork and lysate was fixed at 2:3g / ml. The sample and lysate were mixed and allowed to stand for 1 min, then fixed at 8000 rpm and centrifuged for 1 min. Finally, 50-fold dilutions were uniformly performed with TE buffer to obtain nucleic acid extracts. C according to the real-time fluorescent PCR reaction t Value (the initial number of cycles that can detect the fluorescent signal in the PCR reaction) to determine the nucleic acid ...

Embodiment 2

[0060] After determining the optimal concentration of the four components in the lysate, it is also necessary to determine the mass-to-volume ratio of the sample to the lysate (unit: g / ml), the resting time after mixing the sample and the lysate, and the ability to separate nucleic acids from impurities Three factors such as the optimal rotational speed of the tester (the dilution factor of the nucleic acid extract is finally determined). In order to reduce the number of trials as much as possible, it is still decided to use L 9 (3 4 ) to formulate the experimental program, and use the range analysis method to obtain the best conditions of these three factors. See Table 3 for their factor levels. The letters A, B, and C in the table represent the mass-volume ratio of the sample to the lysate, the resting time, and the centrifugation speed, respectively.

[0061] table 3

[0062]

[0063] In the experiment, pork was used as a sample to carry out an orthogonal experiment,...

Embodiment 3

[0070] First, mix 100 mg of fresh pork with 150 μL of lysate (0.6M sodium chloride, 0.6 g / ml sodium dodecyl sulfate, 0.1 g / ml ethyl phenyl polyethylene glycol, 0.05 g / ml polyvinylpyrrolidone) and mix well, and let stand at room temperature for 2 minutes; then, centrifuge at 10,000 rpm for 1 minute, absorb the supernatant to obtain nucleic acid extract, and use PCR to determine the nucleic acid extraction effect.

[0071] Pork PCR amplification system, amplification program and amplification sequence are the same as in Example 1.

[0072] Dilute the nucleic acid extract 20 times with TE buffer solution as a PCR reaction template for amplification to obtain a fluorescence amplification curve figure 1 .

[0073] The results show: figure 1 The positive samples produced amplification curves, and the negative samples did not produce amplification, indicating that the nucleic acid extraction method can effectively extract the nucleic acid in pork, and can be detected by PCR reactio...

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