CN110317896B - LAMP primer group for detecting corn source component and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biomolecular detection, and in particular relates to a LAMP primer set for detecting corn source components and its application. The LAMP primer set for detecting corn-derived components of the present invention includes FIP primers, F3 primers, BIP primers, B3 primers and LB primers. The LAMP detection system of the present invention has good specificity and sensitivity, and can be quickly, conveniently and efficiently Detecting whether the sample contains corn ingredients can be used for rapid and accurate detection of the quality of sweet potato vermicelli, so as to meet the urgent needs of the country for authenticity testing of sweet potato vermicelli raw materials.

Description

LAMP primer set for detection of corn-derived components and use thereof

technical field

The invention belongs to the technical field of biomolecular detection, and in particular relates to a LAMP primer set for detecting corn source components and its application.

Background technique

Corn is a commonly used food crop. Due to its low price, there will be situations where corn is mixed with other products in the market to make a shoddy product.

Take vermicelli as an example. Vermicelli is one of the traditional foods that Chinese people like to eat. It has the characteristics of convenient, fast, reasonable nutrition and rich taste. It is loved and welcomed by consumers. Among them, sweet potato vermicelli always accompanies people's daily diet. It is loved for its nutritional value and pleasant taste. Sweet potato (also known as sweet potato, sweet potato, sweet potato, sweet potato, etc.) is an ancient potato crop. There is no pesticide pollution in the planting process, and the use of chemical fertilizers is low. It is the most assured green food raw material for consumers. Sweet potatoes are rich in protein, starch, vitamins and a variety of minerals, and have the effects of anti-cancer, laxative, beauty, and longevity. It is known as "natural green food" and "longevity food", ranking first among green vegetables. In recent years, sweet potatoes have been increasingly recognized as health food, and the market consumption demand has been increasing. With the continuous improvement of people's living standards and the rise of food and leisure culture, sweet potatoes have become the main high-quality raw materials of the food industry. It enjoys a high reputation in the world, which also brings a huge market opportunity to China's sweet potato vermicelli industry. But there are many problems in the current vermicelli market. Since the prices of cornstarch and tapioca starch are much lower than those of sweet potato starch, many unscrupulous businessmen add cornstarch and tapioca starch to vermicelli to make so-called "pure sweet potato vermicelli" in order to obtain huge profits from fake products. Make the color similar, taste chewy, and even add illegal additives such as ink and industrial paraffin. At present, many methods have been established to detect harmful substances contained in sweet potato vermicelli and to judge its authenticity, but no method has been established to detect whether there are sweet potato specific genes in sweet potato vermicelli from the perspective of genes. For fake sweet potato vermicelli, the genetic detection method has the characteristics of short time, strong specificity, and high sensitivity, which is of great research value and significance.

"PCR detection of several plant-derived components in food" ("Food Science", 2006, Vol.27, No.11, Chen Wenbing, Shao Biying, etc., P404-405), discloses primers for detecting corn components by PCR method; CN 101701256A It also discloses PCR identification primers and identification methods for corn. The PCR identification primers can specifically amplify the trnL sequence of corn, but it only discloses the example of using DNA extracted from corn plant tissue as a template for amplification; CN 106399540A A corn detection method based on ring-mediated isothermal amplification extinction technology is disclosed, which can specifically detect the inherent IVR gene of corn itself, which is not contained in transgenic corn; CN 108277294A discloses specific primers for detecting corn DNA And the probe can specifically detect Zein rDNA in maize, Zein is the main storage protein of maize endosperm, accounts for 70% of the total protein of endosperm, and mainly exists in corn oil. However, because the DNA contained in corn starch is relatively small, it is difficult to extract, and corn starch needs to be heat-treated during the processing of vermicelli and other processed foods, which is likely to cause DNA damage. To detect whether corn starch processing products contain corn-derived components It is more difficult.

"Identification Method for Plant Source Components of Edible Starch - Real-time Fluorescent PCR Method - Part Seven Corn Starch" (Industry Standards for Entry-Exit Inspection and Quarantine of the People's Republic of China, see http://www.doc88.com/p-9819120496207.html) published The real-time fluorescence amplification PCR primers that can specifically detect corn have good specificity, and the absolute sensitivity test was carried out by extracting DNA from corn (reaction system 25 μL, template DNA 5 μL), and the absolute sensitivity can reach 0.01ng/μL. The corn DNA extracted from the sample can be detected when it is more than 50pg; and corn starch is mixed with tapioca starch, and the relative sensitivity test is carried out, and the relative sensitivity can reach 0.1wt% (corn starch content 0.1g/100g). However, this standard does not apply fluorescent amplification PCR primers to the detection of processed foods such as sweet potato starch (for example, vermicelli, etc.). Corn starch often needs heat treatment during processing, which will cause damage to the DNA in the starch. The question of whether a product contains corn-derived ingredients is even more difficult.

Contents of the invention

An object of the present invention is to provide a LAMP primer set for detecting corn-derived components, so as to solve the problem that the genetic detection method in the prior art cannot be used for detecting corn starch processed foods, and the specific technical scheme is as follows:

The sequence of the primer set is:

Yu-F3: 5`-GCGAAAAAGAACCCACGGC-3`

Yu-B3: 5`-GCTTCGGGCGCAACTTG-3`

Yu-FIP: 5`-TAACCGCTGCCCTGGGAGCTTTTCACCAGTACTACCTCCTGCC-3`

Yu-BIP:5`-GCAACGGATATCTCGGCTCTCGTTTTTTTCGCGGGATTCTGCAATT-3`

Yu-LB: 5`-CATCGATGAAGAACGTAGCAAAATG-3`.

The second object of the present invention is to provide reagents for detecting corn-derived components, and the specific technical scheme is: including dNTPs, Mg ²⁺ , Bst enzyme, Buffer, fluorescent dyes and the above-mentioned LAMP primer set.

The third object of the present invention is to provide a kit for detecting corn-derived components, and the specific technical scheme is: including the above-mentioned LAMP primer set for detecting corn-derived components or the above-mentioned LAMP primer set for detecting corn-derived components Components of reagents.

The 4th object of the present invention is to provide the method for detecting corn source component, and concrete technical scheme is: comprise the following steps: (1) extract sample DNA; (2) adopt LAMP primer set as claimed in claim 1 or The reagent as claimed in claim 2 or the kit as claimed in claim 3 prepare the LAMP reaction system; (3) the prepared LAMP reaction system is placed in a real-time fluorescent PCR instrument or a water bath for constant temperature amplification; (4 ) After 1 h of amplification, observe the amplification curve or the color of the reaction solution.

Preferably, the method for extracting sample DNA is CTAB method.

Preferably, the temperature of the isothermal amplification is 60°C.

Preferably, the molar ratio of Yu-FIP:Yu-BIP:Yu-LB:Yu-F3:Yu-B3 in the LAMP reaction system is 8:8:4:1:1.

The purpose of the present invention is also to provide the application of the above-mentioned LAMP primer set for detecting corn-derived components, and the specific technical solution is: application in detecting corn-derived components.

Preferably, the above-mentioned LAMP primer set/reagent/kit for detecting corn-derived components is used in the detection of corn-derived components in starch or starch-processed foods.

Preferably, the above-mentioned LAMP primer set/reagent/kit for detecting corn-derived components is used in the detection of corn-derived components in vermicelli.

The beneficial effect of the present invention is that: the LAMP primer set for detecting corn-derived components of the present invention has good specificity and sensitivity, and can realize the detection of corn-derived components in a sample under constant temperature conditions.

The LAMP primer set of the present invention has better specificity at 60°C.

The absolute sensitivity of the LAMP primer set of the invention can reach 10pg/μL, and the relative sensitivity can reach 5%.

The LAMP primer set of the present invention can be used to detect corn-derived components in vermicelli.

When the LAMP primer set of the present invention is used to detect the corn source components in vermicelli, the CTAB method for extracting sample DNA has better sensitivity.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only These are some embodiments of the present invention. For those skilled in the art, other drawings can also be obtained according to these drawings without any creative effort.

Fig. 1 is the amplification curve of adding sweet potato DNA, cassava DNA, potato DNA, HO and corn DNA to the LAMP reaction system at 59°C for isothermal amplification;

Figure 2 is the amplification curve of isothermal amplification of sweet potato DNA, cassava DNA, potato DNA, H2O and corn DNA respectively added to the LAMP reaction system at 60°C;

Figure 3 is the amplification curve of isothermal amplification of sweet potato DNA, cassava DNA, potato DNA, H2O and corn DNA respectively added to the LAMP reaction system at 61°C;

Figure 4 is the amplification curve of isothermal amplification of sweet potato DNA, cassava DNA, potato DNA, H2O and corn DNA respectively added to the LAMP reaction system at 62°C; wherein, GAN DNA in Figures 1-4 represents sweet potato DNA, MA DNA means potato DNA, MU DNA means cassava DNA, YU DNA means maize DNA;

Fig. 5 is the amplification curve of the absolute sensitivity test of LAMP primer set of the present invention, and wherein 1pg represents that the concentration of DNA template is 1pg/μL, and 10pg represents that the concentration of DNA template is 10pg/μL, and 100pg represents that the concentration of DNA template is 100pg/μL. µL;

Fig. 6 is the amplification curve of the relative sensitivity test of LAMP primer set of the present invention;

Fig. 7 is the melting curve of the relative sensitivity test of LAMP primer set of the present invention, wherein, 20,1,10,5 in Fig. 6 and Fig. 7 represent respectively in corn starch and tapioca starch mixing sample, the massfraction of corn starch is 20%, 1%, 10%, 5%;

Fig. 8 is the amplification curve that adopts the LAMP primer set of the present invention to detect No. 18-29 vermicelli samples (using the kit method to extract the DNA in the vermicelli samples);

Fig. 9 is the amplification curve that adopts the LAMP primer set of the present invention to detect the No. 25 vermicelli sample (using the CTAB method to extract the DNA in the vermicelli sample);

Fig. 10 is the melting curve of the No. 25 vermicelli sample (DNA in the vermicelli sample was extracted by CTAB method) using the LAMP primer set of the present invention.

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

The source of reagent used in the present invention, see table 1 below for details

Table 1 Sources of test reagents

Example 1

(1) Primer design: Using the maize rRNA intergenic spacer sequence (Internal Transcribed Spacer, ITS) as the target sequence, PrimerExplorerV5 was used to design LAMP primers, and a primer set with the following sequence was obtained after a large number of screenings (synthesized by General Biosystems (Anhui) Co., Ltd. ), the specific sequence is shown in Table 2 below.

(2) Extraction of template DNA: Maize DNA was extracted using a plant genome DNA extraction kit purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. Please refer to the instruction manual of the kit for specific operation steps.

(3) specificity test of the LAMP primer of the present invention:

The reaction system of LAMP is shown in Table 3 below

Table 3 LAMP reaction system (10μL)

Wherein, the LAMP primer is FIP (final concentration 0.8 μ M), BIP (final concentration 0.8 μ M), LB (final concentration 0.4 μ M), F3 (final concentration 0.1 μ M), B3 (final concentration 0.1 μ M) according to 8:8: 4:1:1 ratio mixed liquid mixture.

Take eight PCR tubes, and add the reaction system prepared according to the above table 3 into the eight single tubes (template DNA is not included in the system, 9 μL in each tube), add 1 μL corn DNA to two tubes, and add 1 μL corn DNA to the other six tubes. Add 1 μL of sweet potato DNA, 1 μL of sweet potato DNA, 1 μL of cassava DNA, 1 μL of cassava DNA, 1 μL of potato DNA, and 1 μL of potato DNA; put them into the StepOnePlus real-time fluorescent PCR instrument, set the temperature of the holding stage to 5 min, and set the holding temperature of each group to 59 °C, 60 °C, 61 °C, 62 °C, the temperature of the cycle stage is also set to 59 °C, 60 °C, 61 °C, 62 °C respectively, the melting curve ratio is 1.5%, and the continuous cycle is 110 cycles;

Observing the amplification curve (see Figure 1-4 of the accompanying drawings for details), it can be seen that at 59°C, corn DNA can be amplified in large quantities under the action of primers, but sweet potato DNA has one amplification and the other does not amplify, indicating that at 59°C The specificity of the primers to corn DNA was not strong; at 60°C, the corn DNA was significantly amplified, and there was no amplification trend in other curves, indicating that the primers had strong specificity to corn DNA at 60°C, and the two curves basically overlapped. The reproducibility is also good; at 61°C, corn DNA was significantly amplified, and other curves had no amplification trend, indicating that the primers had strong specificity and good reproducibility for corn DNA at 61°C; There was no amplification, indicating that 62°C is not an ideal temperature for efficient amplification of primer-bound DNA. In summary, 60°C is chosen as the optimum temperature for LAMP.

The sensitivity of embodiment 2 LAMP primers of the present invention

(1) Absolute sensitivity: gradiently dilute the extracted corn DNA to 1ng/μL, 100pg/μL, 10pg/μL and 1pg/μL, take PCR eight tubes, and add them to 8 single tubes according to the above Table 4 The prepared reaction system (template DNA is not included in the system, 9 μL in each tube), and 1 μL of 1ng/μL corn DNA was added to two single tubes, and 1 μL of 100pg corn DNA was added to the other two single tubes. /μL corn DNA, add 10pg/μL corn DNA to the other 2 single tubes, respectively add 1pg/μL corn DNA to the last 2 single tubes; The reaction system prepared in Table 4 above (the system does not include template DNA, 9 μL in each tube) and 1 μL ddH ₂ O; put the PCR eight-tube tube and negative control into the StepOnePlus real-time fluorescent PCR instrument, and set the temperature of the incubation stage to For 5 minutes, the incubation temperature was 60°C, the cycle stage temperature was also 60°C, the melting curve ratio was 1.5%, and the continuous cycle was 110 cycles; the amplification curve was observed (see Figure 5 of the accompanying drawing for details).

As shown in Figure 5, when the DNA concentration of corn is 1ng/μL, 100pg/μL, and 10pg/μL, the two lines have obvious amplification, but when the DNA concentration is 1pg/μL, only one line has a tendency to amplify, which is Amplification was unstable because the concentration of 1 pg/μL exceeded the detection limit of the method. Therefore, the absolute sensitivity of this method can reach 10pg/μL, and in a 10μL reaction system, the maize DNA extracted from the sample can be detected when it reaches 10pg.

(2) Relative sensitivity: cornstarch is mixed with tapioca starch, wherein the mass fraction of cornstarch is 20%, 10%, 5% and 1% respectively, and the DNA of the above-mentioned mixed sample is extracted (Plant Genomic DNA Extraction Kit, Tiangen , extracted according to the steps in the manual), and used as template DNA for later use; take eight PCR tubes, and add the reaction system prepared according to the above table 4 into 8 single tubes (the system does not include template DNA, 9 μL in each tube ) and 1 μL of template DNA extracted from 20%, 10%, 5% and 1% mixed samples, and each sample was repeated; set up a negative control, take 2 PCR single tubes, and add them respectively according to the preparation in Table 4 Reaction system (template DNA is not included in the system, 9 μL in each tube) and 1 μL ddH ₂ O; put the eight PCR tubes and the negative control into the StepOnePlus real-time fluorescent PCR instrument, set the temperature of the incubation stage to 5 min, and the incubation temperature is 60°C, the cycle stage temperature is also 60°C, the melting curve ratio is 1.5%, and the continuous cycle is 110 cycles; observe the amplification curve (see Figure 6 of the accompanying drawing for details) and the melting curve (see Figure 7 of the accompanying drawing for details).

It can be seen from Figure 6 that when the mixed sample contains 20%, 10%, and 5% corn starch, there is an obvious amplification trend, but the curve when 1% is adulterated has no amplification, indicating that 1% exceeds the detection limit of this method . Therefore, the relative sensitivity of the method can reach at least 5%.

Embodiment 3 uses LAMP primer of the present invention for the detection of vermicelli

(1) Detect 30 vermicelli samples purchased

Extraction of Vermicelli DNA: after the vermicelli is crushed, it is extracted with a plant genome DNA extraction kit, Tiangen, according to the steps in the kit instruction manual, and numbered 1-30 respectively.

Preparation of the LAMP reaction system: Add the reaction system prepared according to the above Table 3 into the PCR tube (the system does not include template DNA, 9 μL in each tube) and 1 μL of the DNA extracted from vermicelli samples No. 1-30, Do a set of repeated experiments for each sample; put the PCR tube into the StepOnePlus real-time fluorescent PCR instrument, set the temperature of the holding stage to 5min, the holding temperature is 60°C, the cycle stage temperature is also 60°C, and the melting curve ratio is 1.5%. Cycle continuously for 110 cycles; observe the amplification curve.

Test results: No. 1-24 and No. 26-30 vermicelli samples had no amplification linearity, indicating that there was no corn starch adulteration in the above-mentioned vermicelli; No. 25 vermicelli sample had amplification phenomenon (see Figure 8 of the accompanying drawing for details), However, the reproducibility is not good, indicating that sample No. 25 may have a small amount of cornstarch adulteration.

(2) The DNA extracted by the CTAB method was repeatedly verified on the No. 25 vermicelli sample:

The DNA in the No. 25 vermicelli sample was extracted by the CTAB method: the No. 25 vermicelli sample was crushed with a universal grinder, and 0.1g of the powder was weighed in a clean 2.0ml centrifuge tube, and 1.5ml of CTAB lysate (1gCTAB, 4.0908gNaCl , 0.37224g disodium edetate, 0.6057g Tris were dissolved in a beaker and then fixed to volume with deionized water in a 50ml volumetric flask), in a water bath at 65°C for one hour and turned upside down; after centrifugation at 8000rpm for 15min, 1ml of the supernatant was taken in another Add 700 μL of chloroform to a clean centrifuge tube, mix for 30 seconds, and centrifuge at 14500 rpm for 10 minutes; take 650 μL of the supernatant to a clean centrifuge tube, add 1300 μL of LCTAB precipitation solution (0.25 g CTAB, 0.11688 g NaCl in the beaker after dissolving in deionized water) 50ml volumetric flask to volume), mix for 30s, and stand at room temperature for one hour; centrifuge at 14500rpm for 10min, discard the supernatant, add 350μL 1.2M/L NaCl (3.5064gNaCl is dissolved in a beaker, and use deionized water to volume up in a 50ml volumetric flask ), shake vigorously for 30 s, add 350 μL chloroform, mix for 30 s, and centrifuge at 14500 rpm for 10 min; take 320 μL of supernatant, add 256 μL of isopropanol, mix well and then place it at -20°C for one hour, centrifuge at 14500 rpm for 20 min, discard the supernatant , add 500 μL of 70% ethanol, mix well, centrifuge at 14500 rpm for 20 min, discard the supernatant; air dry, add 20 μL ddH ₂ O to dissolve.

Add the reaction system prepared according to the above Table 3 into the PCR tube (the system does not include template DNA, 9 μL in each tube) and 1 μL of DNA extracted from vermicelli sample No. 25 by CTAB method, and do a set of repeated experiments ;Put the PCR tube into the StepOnePlus real-time fluorescent PCR instrument, set the temperature of the holding stage to 5min, the holding temperature is 60°C, the temperature of the cycle stage is also 60°C, the melting curve ratio is 1.5%, and the continuous cycle is 110 cycles; observe the expansion Increase curve (accompanying drawing 9 of description) and melting curve (accompanying drawing 10 of description).

It can be seen from Figures 9 and 10 that the DNA extracted from the vermicelli sample No. 25 by the CTAB method was significantly amplified, indicating that there was indeed corn starch adulteration in the No. 25 sample.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of the present invention. within the scope of protection.

sequence listing

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Claims (8)

1. be used to detect the LAMP primer set of corn source composition, it is characterized in that, the sequence of described primer set is: Yu-F3: 5`-GCGAAAAAGAACCCACGGC-3` Yu-B3: 5`-GCTTCGGGCGCAACTTG-3` Yu-FIP: 5`-TAACCGCTGCCCTGGGAGCTTTTCACCAGTACTACCTCCTGCC-3` Yu-BIP:5`-GCAACGGATATCTCGGCTCTCGTTTTTTTCGCGGGATTCTGCAATT-3` Yu-LB: 5`-CATCGATGAAGAACGTAGCAAAATG-3`. 2. A reagent for detecting corn-derived components, characterized in that it comprises dNTPs, Mg ²⁺ , Bst enzyme, Buffer, fluorescent dye and the LAMP primer set as claimed in claim 1. 3. A kit for detecting corn-derived components, characterized in that it comprises the LAMP primer set as claimed in claim 1 or the reagent for detecting corn-derived components as claimed in claim 2. 4. detect the method for corn source composition, it is characterized in that, comprise the steps: (1) extract sample DNA; (2) adopt LAMP primer set as claimed in claim 1 or reagent as claimed in claim 2 or as claimed in claim Prepare the LAMP reaction system with the kit described in Requirement 3; (3) Place the prepared LAMP reaction system in a real-time fluorescent PCR instrument or a water bath for constant temperature amplification; (4) Observe the amplification curve or reaction after amplification for 1 hour the color of the liquid; The temperature of the isothermal amplification is 60°C; The molar ratio of Yu-FIP:Yu-BIP:Yu-LB:Yu-F3:Yu-B3 in the LAMP reaction system is 8:8:4:1:1. 5. The method for detecting corn source components according to claim 4, characterized in that, the method for extracting sample DNA is the CTAB method. 6. The use of the LAMP primer set according to claim 1, characterized in that it is used in the detection of corn source components. 7. The use of the LAMP primer set according to claim 6, characterized in that it is used in the detection of corn source components in starch or starch processed foods. 8. The use of the LAMP primer set according to claim 6, characterized in that it is used in the detection of corn source components in vermicelli.

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