CN113881758B - LAMP constant temperature amplification method for detecting coconut ingredients in vegetable protein beverage - Google Patents

Abstract

The present invention proposes a method for detecting vegetable protein beverage coconut component LAMP constant temperature amplification method, the steps are as follows: 1, prepare vegetable protein beverage DNA sample and LAMP constant temperature amplification specific primer; plant protein beverage DNA sample is measured by ultra-micro spectrophotometer , the A260/A280 is between 1.7-2.0, and the DNA concentration is >10ng/μL; the LAMP constant temperature amplification specific primer is synthesized by selecting the coconut-specific gene sequence, and the coconut gene adopts the coconut 3a (Hd3a) gene; the specific gene sequence The position in the coconut gene is 451-1300bp; the specific primers for LAMP constant temperature amplification are: outer primer F3/B3, inner primer FIB/BIP, and loop primer LB; The sample is subjected to LAMP constant temperature amplification, the fluorescence signal is detected, and the detection result is obtained. The invention has high sensitivity and high specificity for identifying coconut components, and can be used for rapid detection of coconut components in vegetable protein beverages.

Description

A constant temperature amplification method for detecting vegetable protein beverage coconut component LAMP

technical field

The invention belongs to the technical field of molecular biology, and in particular relates to a constant-temperature amplification method for detecting coconut component LAMP in vegetable protein beverages.

Background technique

Coconut is the fruit of the palm plant coconut tree. Coconut juice and coconut meat contain protein, sugar, oil, vitamins, calcium, phosphorus, iron and other trace elements and minerals, which are deeply loved by consumers. In recent years, coconut-based vegetable protein beverages (such as coconut juice) have developed rapidly by virtue of their advantages of "natural, green, nutritious, and healthy" and occupy a relatively large proportion in the current market. With the surge in market demand and the rise in raw material prices, in order to reduce production costs, some manufacturers use low-priced soybeans or other cheap raw materials to counterfeit coconut beverages and seize the market at low prices. At present, there is no relevant inspection standard for the detection method of vegetable protein beverages for coconut components. The relevant literature is only the ordinary PCR detection method. Due to the large circulation of vegetable protein beverages in the market, the ordinary PCR inspection technology process is cumbersome, and it is necessary to judge the negative and positive by electrophoresis results. The entire detection process cannot be monitored in real time. In addition, the requirements for operators are relatively high, and it is difficult to carry out a large number of popularization and application in the grassroots.

LAMP detection technology, full name Loop-mediated isothermal amplification technology (Loop-mediated isothermal amplification, LAMP), was first published by Japanese scholars in "Nucleic Acids Res" as a constant temperature nucleic acid amplification technology suitable for gene diagnosis. The main principle of the technology is It uses Bst DNA polymerase with strand displacement specificity and 4-6 primers that can specifically recognize multiple specific regions on the target sequence to start a cyclic strand displacement reaction, thereby realizing continuous and rapid reactions under isothermal conditions. This technology has the advantages of strong specificity, high sensitivity, low detection cost, low equipment and personnel requirements, simple operation, short reaction time, etc., and is suitable for rapid detection of large quantities of samples on site.

Therefore, it is very necessary to establish a set of LAMP constant temperature amplification method for plant protein beverage coconut component with good specificity, high sensitivity, suitable for grass-roots and security field applications, and provide an efficient detection method for resisting the adulteration of coconut protein beverages. Provide new technical support to ensure food quality and safety in the beverage industry.

Contents of the invention

The purpose of the present invention is to propose a method for detecting the coconut component of vegetable protein beverage LAMP constant temperature amplification method, which has high sensitivity and high specificity for the identification of coconut components, and can be used for the rapid detection of coconut components in vegetable protein beverages.

The purpose of the present invention can be achieved through the following technical solutions:

A method for detecting plant protein beverage coconut component LAMP constant temperature amplification, which includes the following steps:

Step 1, preparing a plant protein beverage DNA sample, and preparing LAMP constant temperature amplification specific primers for performing LAMP constant temperature amplification on the plant protein drink DNA sample;

After the DNA sample of the vegetable protein beverage is measured by an ultra-micro spectrophotometer, its A260/A280 is between 1.7-2.0, and the DNA concentration is >10ng/μL, and it is stored at -20°C for use;

The specific primers for the constant temperature amplification of the LAMP are synthesized by selecting the coconut specific gene sequence, and the coconut gene adopts: the coconut 3a (Hd3a) gene, whose accession number is MH092984.1; the specific gene sequence is listed in the The position in the coconut gene is 451-1300bp;

The specific primers for the LAMP constant temperature amplification are:

Outer primer F3: 5'-CTCCAAGTCCAAGTGACC-3'

Outer primer B3: 5'-GTGGAGCTAGTTTTGTTCC-3'

Internal primer FIB: 5'-CATGGCTTCCCGATGGGTACCTCAGGGAGTATTTGCACTG-3'

Internal primer BIP: 5'-GCTATATATTGGGCCGGACTTCGCAATCTCTTCTCTTCCTTGT-3'

Loop Primer LB: 5'-ACTCTTTCAGCGGGTGCTTAC-3'

Step 2. Using the LAMP constant temperature amplification reaction system, perform LAMP constant temperature amplification on the plant protein beverage DNA sample, detect the fluorescence signal of the amplified product, and obtain the detection result; wherein, the LAMP constant temperature amplification reaction system includes the LAMP Isothermal amplification specific primers.

Optimization scheme, when the vegetable protein beverage in step 1 is solid or liquid containing solids, the solid vegetable protein beverage is brewed with warm water according to the instructions of the solid vegetable protein beverage, stirred evenly, and a liquid vegetable protein beverage to be extracted is obtained; the solid vegetable protein beverage contains Grinding the solids of the liquid vegetable protein beverage with a grinding machine to obtain a liquid vegetable protein beverage to be extracted; performing DNA extraction on the vegetable protein beverage to be extracted to obtain the DNA sample of the vegetable protein beverage in step 1.

Optimizing scheme, described LAMP isothermal amplification reaction system is 25 μ L, includes inner primer FIP (100 μ mol/L) and inner primer BIP (100 μ mol/L) each 0.4 μ L, outer primer F3 (100 μ mol/L) and outer primer B3 ( 100 μmol/L) each 0.05 μL, loop primer LB (100 μmol/L) 0.2 μL; other reagent components are: 10×Thermo pol Buffer 2.5 μL, MgSO ₄ (100 mmol/L) 1 μL, dNTPs (10 mmol/L) 2.5 μL, betaine (5mol/L) 6 μL, Bst enzyme (8U/μL) 1 μL, dye (250×) 0.5 μL, DNA template 2 μL, ddH ₂ O 8.4 μL.

In the optimized scheme, in the LAMP constant temperature amplification reaction system, the LAMP constant temperature amplification condition of the constant temperature amplification instrument used is: the amplification temperature is 63°C, 45 cycles are performed, and the amplification time of each cycle is 60s; The amplification conditions of the real-time fluorescent PCR instrument are as follows: the first step is to amplify at 63°C for 30s, the second step is to amplify at 63°C for 15s and perform 45 cycles, and the third step is to amplify at 63°C for 45s and perform 45 loops.

The present invention has the following outstanding substantive features and remarkable progress:

1. The present invention provides a specific gene sequence for identification of coconut components. The sequence has high specificity, can be used for qualitative detection of coconut components, and has commercial development value. The applicant used the LAMP constant temperature amplification specific primers designed by the specific gene sequence and the LAMP constant temperature amplification reaction system to specifically detect 45 kinds of plant and animal material DNA samples, and the results showed that the present invention was positive for coconut DNA sample amplification, However, DNA samples from other species were amplified negatively, indicating that the specific gene sequence has high specificity and can be used as a specific marker for qualitative detection of coconut components. In addition, the applicant has also conducted a sensitivity test and a stability test on the present invention, and the results show that the method of the present invention has the characteristics of high sensitivity and stability.

2. In the LAMP constant temperature amplification method for detecting vegetable protein beverage coconut components of the present invention, a LAMP constant temperature amplification specific primer that can specifically identify coconut components is provided, which has good specificity and can achieve specificity for coconut-derived components detection, but other components other than coconut cannot be specifically amplified; in addition, the present invention also provides a LAMP constant temperature amplification reaction system, which can realize whether the sample contains coconut components within 1 hour, and has The detection is efficient, fast and easy.

3. The present invention utilizes the specific primers for constant temperature amplification of LAMP to quickly and high-throughput detect whether there is coconut ingredient in the vegetable protein beverage, and the required instruments and equipment are cheap, easy to operate, high in efficiency, and have no requirements for inspectors. High, suitable for rapid detection of grassroots and on-site support.

4. The LAMP constant temperature amplification method for detecting vegetable protein beverage coconut components of the present invention is applicable to the detection of various vegetable protein beverage coconut components, including solid beverages, liquid beverages, semi-solid beverages, etc., and can be popularized and applied in large quantities to ensure the quality of products , to protect the rights and interests of consumers, to provide technical support for maintaining the normal order of the beverage industry, and to provide technical support for food market supervision and management departments.

Description of drawings

Fig. 1 is to verify adopting the method of the present invention to carry out 20 kinds of plant protein drinks coconut component LAMP amplification spectrum.

Figure 2 is a LAMP-specific amplification spectrum for verifying the detection of coconut components by the method of the present invention.

Figure 3 is a LAMP sensitivity amplification spectrum for verifying the detection of coconut components by the method of the present invention.

FIG. 4 is a LAMP stability amplification spectrum for verifying the detection of coconut components by the method of the present invention.

Detailed ways

The present invention will be further described below in conjunction with accompanying drawing.

Example 1

A method for detecting plant protein beverage coconut component LAMP constant temperature amplification, which includes the following steps:

Step 1, preparing a plant protein beverage DNA sample, and preparing LAMP constant temperature amplification specific primers for performing LAMP constant temperature amplification on the plant protein drink DNA sample;

In this step, when the vegetable protein beverage to be detected is a solid or a liquid containing solids, the solid vegetable protein beverage is brewed with warm water according to the instructions of the solid vegetable protein beverage, and stirred evenly to obtain a liquid vegetable protein beverage to be extracted; For liquid vegetable protein drinks with solids, use a grinder to grind the solids sufficiently to obtain liquid vegetable protein drinks to be extracted;

Use "magnetic bead method to extract plant protein beverage DNA" (Liang Meidan, Xiao Jian, Chen Kai, Huang Zhishen, Lao Jiaqian. Journal of Food Safety and Quality Inspection, 2021, 12(13):5363-5368.) on the vegetable protein beverage The sample to be extracted is subjected to DNA extraction, the extraction method is detailed in literature, and the DNA sample of the vegetable protein beverage is obtained; after the DNA sample of the vegetable protein beverage is measured by an ultra-micro spectrophotometer, its A260/A280 is between 1.7-2.0, DNA concentration > 10ng/μL, store at -20°C for later use;

The specific primers for constant temperature amplification of LAMP are obtained by designing and synthesizing coconut specific gene sequences. Specifically: download the coconut (Cocos nucifera) 3a (Hd3a) gene (accession number: MH092984.1) from the NCBI website (https://www.ncbi.nlm.nih.gov/), select the specific gene sequence, and the position is 451-1300bp, use PrimerExplorer Version 4 (http://primerexplorer.jp/e/v4_manual/index.html) to design a set of LAMP constant temperature amplification primers:

Outer primer F3: 5'-CTCCAAGTCCAAGTGACC-3'

Outer primer B3: 5'-GTGGAGCTAGTTTTGTTCC-3'

Internal primer FIB: 5'-CATGGCTTCCCGATGGGTACCTCAGGGAGTATTTGCACTG-3'

Internal primer BIP: 5'-GCTATATATTGGGCCGGACTTCGCAATCTCTTCTCTTCCTTGT-3'

Loop Primer LB: 5'-ACTCTTTCAGCGGGTGCTTAC-3'

Step 2. Using the LAMP constant temperature amplification reaction system, perform LAMP constant temperature amplification on the plant protein beverage DNA sample, detect the fluorescence signal of the amplified product, and obtain the detection result; wherein, the LAMP constant temperature amplification reaction system includes the LAMP Specific primers for constant temperature amplification, see Table 1 for details.

The reaction procedure of the LAMP constant temperature amplification reaction system is:

The LAMP constant temperature amplification condition of the constant temperature amplification instrument used is: the amplification temperature is 63°C, 45 cycles are performed, the amplification time of each cycle is 60s, and the total amplification time is 45min;

The amplification conditions of the real-time fluorescent PCR instrument used are: the first step is to amplify at 63°C for 30s, the second step is to amplify at 63°C for 15s, and perform 45 cycles, and the third step is to amplify at 63°C for 45s, 45 cycles were performed, and the total amplification time was about 50 min.

In this example, 20 kinds of vegetable protein beverage DNA samples were tested for coconut components. The names of the 20 kinds of vegetable protein beverage DNA samples and the detection results of coconut components are shown in FIG. 1 and Table 2.

It can be seen from Figure 1 and Table 2 that there are 6 vegetable protein beverages containing coconut ingredients, and the test results of the present invention are completely consistent with the ingredients indicated in the product instructions; the remaining 12 samples are identified as containing walnuts, sesame seeds, hazelnuts, and almonds. , peanuts, soybean ingredients, the detection results of the present invention are all negative results.

Conclusion: 6 samples of vegetable protein beverages claiming to contain coconut ingredients, the present invention is positive for coconut ingredients, 12 samples of plant protein beverages claiming to contain no coconut ingredients, the present invention detects coconut ingredients as negative, and the detection results are 100% consistent. Simultaneously, it also shows that the detection method of the present invention can be used to detect whether there is a coconut component in vegetable protein beverages in large quantities.

In summary, the LAMP constant temperature amplification specific primers and LAMP constant temperature amplification reaction system for coconut components designed by the present invention have good specificity and high sensitivity, and can quickly realize the qualitative detection of coconut components in vegetable protein beverages. Relying on high-end and expensive instruments and equipment, low detection cost, and low requirements for professional and technical personnel, it is suitable for rapid detection at the grassroots level and on-site support, and has a good application prospect. In addition, the specific primers for constant temperature amplification of LAMP can be further made into a coconut component LAMP detection kit, which is applied to the detection kit of coconut components in food and other samples, and has a wide range of applications and strong practicability.

Example 2

This embodiment is to detect the specificity of the LAMP constant temperature amplification reaction system and the LAMP constant temperature amplification specific primer of the present invention, specifically: use the LAMP constant temperature amplification reaction system of Example 1 to test the 45 kinds of The DNA samples of plant and animal materials are detected, and the fluorescent signals of the amplified products are detected. The test results are shown in Figure 2 and Table 3.

As can be seen from Fig. 2 and Table 3, the present invention is positive to the coconut DNA sample amplification, and obvious S-type amplification curve is arranged, and all amplifies negatively to other multiple species DNA samples, without obvious S-type amplification curve.

Conclusion: The detection method of the present invention has good species specificity.

Example 3

This embodiment is to detect the sensitivity of the LAMP constant temperature amplification reaction system of the present invention and the LAMP constant temperature amplification specific primers, specifically: using mung beans as the substrate, prepare coconut mixed samples with different mass fractions in proportion, and the coconut mixed samples The mass fraction of coconut DNA includes: 50%, 10%, 5%, 1%, 0.1%, 0.01%, wherein 0.01% of the coconut mixed sample is obtained by diluting 10 times the coconut mixed sample DNA with a mass fraction of 0.1%, The LAMP constant temperature amplification was carried out according to the LAMP constant temperature amplification reaction system and conditions in Example 1, and the experiment was repeated 6 times.

Results: Coconut mixed samples with mass fractions of 50%, 10%, 5%, 1%, and 0.1% of coconut LAMP fluorescence amplification samples had obvious S-type amplification curves, and the mass fraction of coconut mixed samples was 0.01%. See the S-type amplification curve, indicating that the sensitivity of this method is 0.1%, and the amplification map is shown in Figure 3.

Example 4

This embodiment is to detect the stability of the LAMP constant temperature amplification reaction system of the present invention and the LAMP constant temperature amplification specific primer, specifically: the coconut mixed sample DNA whose mass fraction is 0.1% in embodiment 3, according to embodiment 1 The LAMP constant temperature amplification reaction system and conditions were carried out for LAMP constant temperature amplification, and repeated 10 times. As a result, the mixed coconut samples with a coconut mass fraction of 0.1% could amplify an obvious S-shaped curve, proving that the method has good stability. The amplification map is shown in Figure 4.

sequence listing

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Claims (4)

1. An LAMP isothermal amplification method for detecting coconut ingredients in vegetable protein beverage is characterized by comprising the following steps:

step 1, preparing a plant protein beverage DNA sample and preparing LAMP constant temperature amplification specific primers for performing LAMP constant temperature amplification on the plant protein beverage DNA sample;

after the DNA sample of the vegetable protein beverage is measured by an ultramicro spectrophotometer, the A260/A280 of the DNA sample is between 1.7 and 2.0, the DNA concentration is more than 10 ng/mu L, and the DNA sample is stored at the temperature of minus 20 ℃ for later use;

the LAMP constant-temperature amplification specific primer is obtained by selecting a coconut specific gene sequence for design and synthesis, wherein the position of the specific gene sequence in a coconut Hd3a gene is 451-1300bp, and the accession number of the coconut Hd3a gene is MH092984.1;

the LAMP constant temperature amplification specific primer is as follows:

an outer primer F3:5'-CTCCAAGTCCAAGTGACC-3'

An outer primer B3:5'-GTGGAGCTAGTTTTGTTCC-3'

Inner primer FIB:

5’-CATGGCTTCCCGATGGGTACCTCAGGGAGTATTTGCACTG-3’

the inner primer BIP:

5’-GCTATATATTGGGCCGGACTTCGCAATCTCTTCTCTTCCTTGT-3’

the loop primer LB:5'-ACTCTTCAGCGGGTGCTTAC-3'

Step 2, performing LAMP isothermal amplification on the plant protein beverage DNA sample by adopting an LAMP isothermal amplification reaction system, and detecting a fluorescent signal of an amplification product to obtain a detection result; the LAMP isothermal amplification reaction system comprises the LAMP isothermal amplification specific primer.

2. The LAMP isothermal amplification method for detecting the vegetable protein beverage coconut ingredients according to claim 1, characterized in that: when the vegetable protein beverage is solid or liquid containing solid in the step 1, the solid vegetable protein beverage is mixed with warm water and stirred uniformly to obtain a liquid vegetable protein beverage sample to be extracted; fully grinding the solid matter in the liquid vegetable protein beverage containing the solid matter by using a grinder to obtain a liquid vegetable protein beverage sample to be extracted; and (3) carrying out DNA extraction on the plant protein beverage sample to be extracted to obtain the plant protein beverage DNA sample in the step (1).

3. The LAMP isothermal amplification method for detecting the vegetable protein beverage coconut ingredients according to claim 1 or 2, characterized in that: the LAMP constant-temperature amplification reaction system is 25 mu L, and comprises 0.4 mu L of each of 100 mu mol/L inner primer FIP and 100 mu mol/L inner primer BIP, 0.05 mu L of each of 100 mu mol/L outer primer F3 and 100 mu mol/L outer primer B3, and 0.2 mu L loop primer LB of 100 mu mol/L; the other reagent components are respectively as follows: 10 × Thermo pol Buffer2.5 μ L, mgSO ₄ 100 mmol/L1. Mu.L, dNTPs 10 mmol/L2.5. Mu.L, betaine 5 mol/L6. Mu.L, bstase 8U/. Mu.L 1. Mu.L, 250 Xdye 0.5. Mu.L, DNA template 2. Mu.L, ddH ₂ O 8.4μL。

4. The LAMP isothermal amplification method for detecting vegetable protein beverage coconut ingredients according to claim 3, characterized in that: in the LAMP isothermal amplification reaction system, the LAMP isothermal amplification conditions of the isothermal amplification instrument are as follows: the amplification temperature was 63 ℃ and 45 cycles were performed, with an amplification time of 60s per cycle; the fluorescent signal amplification conditions of the real-time fluorescent PCR instrument are as follows: the first step is amplification for 30s at 63 ℃, the second step is amplification for 15s at 63 ℃ and 45 cycles, and the third step is amplification for 45s at 63 ℃ and 45 cycles.

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