CN110317896B - LAMP primer group for detecting corn source component and application thereof - Google Patents
LAMP primer group for detecting corn source component and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biomolecule detection, and particularly relates to an LAMP primer group for detecting corn source components and application thereof. The LAMP primer group for detecting the corn source component comprises the FIP primer, the F3 primer, the BIP primer, the B3 primer and the LB primer, and the LAMP detection system has good specificity and sensitivity, can rapidly, conveniently and efficiently detect whether the corn component is contained in a sample, can be used for rapidly and accurately detecting the quality of sweet potato vermicelli, and can meet the urgent national demand for detecting the authenticity of the sweet potato vermicelli raw material.
Description
Technical Field
The invention belongs to the technical field of biomolecule detection, and particularly relates to an LAMP primer group for detecting corn source components and application thereof.
Background
Corn is a common food crop, and because of its low cost, it can happen that corn is blended into other products to be sub-full in the market.
Taking the vermicelli as an example, the vermicelli is one of traditional foods which are favored by people in China, has the characteristics of convenient and quick eating, reasonable nutrition, rich taste and the like, is favored and popular by consumers, wherein the sweet potato vermicelli always accompanies the daily diet of people, and has the nutrition value and the proper taste. Sweet potatoes (also called sweet potatoes, sweet potatoes and the like) are old tuber crops, have no pesticide pollution in the planting process, are low in chemical fertilizer use, and are the most safe green food raw materials for consumers. Sweet potato is rich in protein, starch, vitamins and various minerals, has the effects of preventing cancer, relaxing the bowels, beautifying and prolonging life, and is praised as a natural green food and a longevity food, and is a first green vegetable. In recent years, sweet potatoes are increasingly regarded as health-care foods, the market consumption demand is continuously increased, along with the continuous improvement of the living standard of people and the natural rising of food and leisure culture, the sweet potatoes are already raised to be main high-quality raw materials of the food industry and internationally enjoy reputation, and a huge market opportunity is brought to the sweet potato vermicelli industry in China. However, the current vermicelli market has a number of problems. Because the prices of the corn starch and the tapioca starch are much lower than those of the sweet potato starch, a plurality of illegal operators add the corn starch and the tapioca starch into the vermicelli in a violent way brought by obtaining the fake goods, and the corn starch is used for manufacturing the so-called pure sweet potato vermicelli, so that the color and luster are similar, the taste is chewy, and even illegal additives such as ink, industrial material paraffin and the like are added. At present, various methods for detecting harmful substances contained in sweet potato vermicelli and judging whether the sweet potato vermicelli is true or false are established, but a method for detecting whether a sweet potato specific gene exists in the sweet potato vermicelli from the gene angle is not established, and the gene detection method has the characteristics of short time, strong specificity, high sensitivity and the like for identifying whether other starch is used for preparing the false sweet potato vermicelli, and has 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, et al, P404-405), primers for detecting corn components using the PCR method are disclosed; CN 101701256a also discloses a PCR identification primer and an identification method for corn, which can specifically amplify the trnL sequence of corn, but it also discloses only an example of amplification using DNA extracted from corn plant tissue as a template; CN 106399540a discloses a corn detection method based on loop-mediated isothermal amplification extinction technology, which can specifically detect intrinsic IVR gene of corn itself, and the transgenic corn does not contain the gene; CN 108277294a discloses specific primers and probes for detecting corn DNA, specifically detecting corn Zein rDNA, zein being the main storage protein of corn endosperm, accounting for 70% of the total protein of endosperm, and being mainly present in corn oil. However, because the corn starch has relatively less DNA, the extraction difficulty is high, and the corn starch needs to be subjected to heat treatment in the processing process of processed foods such as vermicelli, the damage of the DNA is easy to cause, and the difficulty of detecting whether the corn starch processed products contain corn source components is higher.
The edible starch plant source component identification method-real-time fluorescence PCR method-seventh part corn starch (see http:// www.doc88.com/p-9819120496207. Html) discloses a real-time fluorescence amplification PCR primer capable of specifically detecting corn, the specificity is good, DNA extracted from corn is used as a raw material to carry out absolute sensitivity test (a reaction system of 25 mu L and template DNA of 5 mu L), the absolute sensitivity can reach 0.01 ng/mu L, and the corn DNA extracted from a sample can be detected when the corn DNA extracted from the sample is more than 50 pg; corn starch and tapioca starch are mixed, and relative sensitivity is tested, wherein the relative sensitivity can reach 0.1wt% (corn starch content is 0.1g/100 g). However, the standard does not apply the fluorescence amplification PCR primer to detection of sweet potato starch processed foods (such as vermicelli and the like), and the corn starch often needs heat treatment in the processing process, so that DNA in the starch is damaged, and the difficulty of detecting whether the processed products such as vermicelli and the like contain corn source components is greater.
Disclosure of Invention
The invention aims to provide an LAMP primer group for detecting corn source components, which solves the problem that a gene detection method in the prior art cannot be used for detecting corn starch processed foods, and specifically adopts the following technical scheme:
the primer group has the sequence as follows:
Yu-F3：5`-GCGAAAAAGAACCCACGGC-3`
Yu-B3：5`-GCTTCGGGCGCAACTTG-3`
Yu-FIP：5`-TAACCGCTGCCCTGGGAGCTTTTCACCAGTACTACCTCCTGCC-3`
Yu-BIP：5`-GCAACGGATATCTCGGCTCTCGTTTTTTCGCGGGATTCTGCAATT-3`
Yu-LB：5`-CATCGATGAAGAACGTAGCAAAATG-3`。
the invention provides a reagent for detecting corn source components, which comprises the following specific technical scheme: comprises dNTPs, mg 2+ Bst enzyme, buffer and fluorescent dye and LAMP primer set as described above.
The invention provides a kit for detecting corn source components, which comprises the following specific technical scheme: comprising the LAMP primer group for detecting a corn-derived component as described above or the reagent for detecting a corn-derived component as described above.
The 4 th object of the invention is to provide a method for detecting corn source components, which comprises the following specific technical scheme: the method comprises the following steps: (1) extracting sample DNA; (2) Preparing a LAMP reaction system by using the LAMP primer set as claimed in claim 1 or the reagent as claimed in claim 2 or the kit as claimed in claim 3; (3) Placing the prepared LAMP reaction system in a real-time fluorescence PCR instrument or a water bath kettle for isothermal amplification; (4) after 1h of amplification, observing the amplification curve or the color of the reaction solution.
Preferably, the method for extracting the sample DNA is a CTAB method.
Preferably, the isothermal amplification temperature is 60 ℃.
Preferably, the molar ratio of Yu-FIP to Yu-BIP to Yu-LB to Yu-F3 to Yu-B3 in the LAMP reaction system is 8:8:4:1:1.
The invention also aims to provide the application of the LAMP primer group for detecting corn source components, and the specific technical scheme is as follows: the application in detecting corn source components.
Preferably, the LAMP primer group/reagent/kit for detecting corn-derived components as described above is used for detecting corn-derived components in starch or starch processed foods.
Preferably, the LAMP primer group/reagent/kit for detecting corn-derived components as described above is used for detecting corn-derived components in vermicelli.
The beneficial effects of the invention are as follows: the LAMP primer group for detecting the corn source component has good specificity and sensitivity, and can detect the corn source component in a sample under the constant temperature condition.
The LAMP primer group of the invention has better specificity at 60 ℃.
The LAMP primer group of the invention has an absolute sensitivity of 10 pg/mu L and a relative sensitivity of 5%.
The LAMP primer group provided by the invention can be used for detecting corn source components in vermicelli.
When the LAMP primer group is used for detecting corn source components in vermicelli, the CTAB method is selected to extract sample DNA, so that the sensitivity is better.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.
FIG. 1 is an amplification curve of isothermal amplification by adding sweet potato DNA, cassava DNA, potato DNA, H2O and corn DNA, respectively, to a LAMP reaction system at 59 ℃;
FIG. 2 is an amplification curve of isothermal amplification by adding sweet potato DNA, cassava DNA, potato DNA, H2O and corn DNA, respectively, to a LAMP reaction system at 60 ℃;
FIG. 3 is an amplification curve of isothermal amplification by adding sweet potato DNA, cassava DNA, potato DNA, H2O and corn DNA, respectively, to a LAMP reaction system at 61 ℃;
FIG. 4 is an amplification curve of isothermal amplification by adding sweet potato DNA, cassava DNA, potato DNA, H2O and corn DNA, respectively, to a LAMP reaction system at 62 ℃; in FIGS. 1 to 4, GAN DNA represents sweet potato DNA, MA DNA represents potato DNA, MU DNA represents cassava DNA, and YU DNA represents corn DNA;
FIG. 5 is an amplification curve of an absolute sensitivity test of the LAMP primer group of the present invention, wherein 1pg indicates a concentration of a DNA template of 1 pg/. Mu.L, 10pg indicates a concentration of a DNA template of 10 pg/. Mu.L, and 100pg indicates a concentration of a DNA template of 100 pg/. Mu.L;
FIG. 6 is an amplification curve of a relative sensitivity test of the LAMP primer group of the present invention;
FIG. 7 is a melting curve of the relative sensitivity test of the LAMP primer group of the present invention, wherein 20, 1, 10, 5 in FIGS. 6 and 7 represent the mass fraction of corn starch in a mixed sample of corn starch and tapioca starch, respectively, as 20%, 1%, 10%, 5%;
FIG. 8 is an amplification curve of detection of No. 18-29 vermicelli samples (DNA in vermicelli samples extracted by a kit method) using the LAMP primer group of the present invention;
FIG. 9 is an amplification curve of detection of sample No. 25 vermicelli (DNA in sample vermicelli extracted by CTAB method) using the LAMP primer group of the present invention;
FIG. 10 is a melting curve of detection of sample No. 25 vermicelli (DNA in sample vermicelli extracted by CTAB method) using the LAMP primer group of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The sources of reagents used in the present invention are detailed in Table 1 below
TABLE 1 assay reagent sources
Example 1
(1) Primer design: primers for LAMP were designed using the maize rRNA gene spacer sequence (Internal Transcribed Spacer, ITS) as a target sequence and PrimerExplorer V5, and a large number of primer sets (synthesized by general biosystems (Anhui) Inc.) were obtained by screening, and the specific sequences are shown in Table 2 below.
(2) Extraction of template DNA: corn DNA was extracted using a plant genomic DNA extraction kit purchased from the root biochemistry technology (beijing) limited. The specific operation steps are shown in the instruction book of the kit.
(3) Specificity test of LAMP primer of the invention:
the reaction system of LAMP is shown in Table 3 below
TABLE 3 LAMP reaction system (10. Mu.L)
The LAMP primer was a mixture of FIP (final concentration 0.8. Mu.M), BIP (final concentration 0.8. Mu.M), LB (final concentration 0.4. Mu.M), F3 (final concentration 0.1. Mu.M) and B3 (final concentration 0.1. Mu.M) at a ratio of 8:8:4:1:1.
Taking PCR eight-joint tubes, adding the reaction system prepared according to the table 3 above (the system does not comprise template DNA, 9 mu L of each tube) into 8 single tubes, wherein 1 mu L of corn DNA is added into two tubes, and 1 mu L of sweet potato DNA, 1 mu L of cassava DNA, 1 mu L of potato DNA and 1 mu L of potato DNA are respectively added into the other six tubes; placing the mixture into a StepOnEPlus real-time fluorescence PCR instrument, setting the temperature at the heat preservation stage to be 5min, setting the temperature at 59 ℃, 60 ℃, 61 ℃, 62 ℃ in each group, setting the temperature at 59 ℃, 60 ℃, 61 ℃, 62 ℃ in the circulation stage, setting the melting curve ratio to be 1.5%, and continuously circulating for 110 circulations;
as can be seen from the observation of the amplification curves (see FIGS. 1-4 of the drawings of the specification): corn DNA can be greatly amplified under the action of the primer at 59 ℃, but one sweet potato DNA is amplified, and the other sweet potato DNA is not amplified, which indicates that the primer has weak specificity to corn DNA at 59 ℃; the corn DNA is obviously amplified at 60 ℃, and other curves have no amplification trend, so that the specificity of the primer to the corn DNA at 60 ℃ is strong, the two curves are basically coincident, and the reproducibility is good; the corn DNA is obviously amplified at 61 ℃, and other curves have no amplification trend, which indicates that the primer has strong specificity and good reproducibility on the corn DNA at 61 ℃; all DNA was not amplified at 62 ℃, indicating that at 62 ℃ it is not the ideal temperature for efficient amplification of primer-bound DNA. In summary, 60℃was chosen as the optimal temperature for LAMP.
EXAMPLE 2 sensitivity of LAMP primer of the present invention
(1) Absolute sensitivity: the extracted corn DNA was subjected to gradient dilution to 1 ng/. Mu.L, 100 pg/. Mu.L, 10 pg/. Mu.L and 1 pg/. Mu.L, PCR octal tubes were taken, and the reaction system prepared according to Table 4 above (excluding the template DNA in the system, 9. Mu.L in each tube) was added to 8 individual tubes, and 1. Mu.L of corn DNA 1 ng/. Mu.L was added to 2 individual tubes, 1. Mu.L of corn DNA 100 pg/. Mu.L was added to 2 individual tubes, 10 pg/. Mu.L of corn DNA was added to 2 individual tubes, and 1 pg/. Mu.L of corn DNA was added to 2 individual tubes, respectively. Setting negative control, taking 2 single PCR tubes, and adding respectively the reaction system (the system does not include template DNA, 9. Mu.L in each tube) and 1. Mu.L ddH prepared according to the above Table 4 2 O; placing the PCR octant and negative control into a StepOnEPlus real-time fluorescence PCR instrument, setting the temperature at the heat preservation stage to be 5min, setting the temperature at 60 ℃ respectively, setting the temperature at the circulation stage to be 60 ℃ respectively, setting the dissolution curve ratio to be 1.5%, and continuously circulating for 110 cycles; the amplification curve was observed (see FIG. 5 of the drawings of the specification for details).
As shown in FIG. 5, both lines were significantly amplified at a concentration of 1 ng/. Mu.L, 100 pg/. Mu.L, and 10 pg/. Mu.L of corn DNA, but only one line had a tendency to amplify at a concentration of 1 pg/. Mu.L, because the concentration of 1 pg/. Mu.L exceeded the detection limit of the method, making amplification unstable. Therefore, the absolute sensitivity of the method can reach 10 pg/mu L, and the corn DNA extracted from the sample can be detected in 10 mu L of reaction system.
(2) Relative sensitivity: mixing corn starch and tapioca starch, wherein the mass fractions of the corn starch are respectively 20%, 10%, 5% and 1%, extracting DNA (plant genome DNA extraction kit, radix kadsurae, extracted according to the steps in the specification) of the mixed sample as template DNA for standby; taking PCR eight-joint tubes, adding the prepared reaction system (the system does not comprise template DNA, and each tube does not comprise 9 mu L) and 1 mu L of template DNA extracted from 20%, 10%, 5% and 1% mixed samples into 8 single tubes respectively, and repeating each sample; setting negative control, taking 2 single PCR tubes, and adding respectively the reaction system (the system does not include template DNA, 9. Mu.L in each tube) and 1. Mu.L ddH prepared according to the above Table 4 2 O; placing the PCR octant and negative control into a StepOnEPlus real-time fluorescence PCR instrument, setting the temperature at the heat preservation stage to be 5min, setting the temperature at 60 ℃ respectively, setting the temperature at the circulation stage to be 60 ℃ respectively, setting the dissolution curve ratio to be 1.5%, and continuously circulating for 110 cycles; the amplification curve (see FIG. 6 of the drawings for details) and the melting curve (see FIG. 7 of the drawings for details) were observed.
As can be seen from FIG. 6, the mixed samples contained 20%, 10% and 5% corn starch, which had a significant tendency to amplify, but the curve when 1% was spiked did not amplify, indicating that 1% exceeded the limit of detection of this method. The relative sensitivity of the method can thus be at least 5%.
Example 3 detection of vermicelli Using LAMP primer of the present invention
(1) Detection of 30 purchased vermicelli samples
Extraction of vermicelli DNA: pulverizing vermicelli, extracting with plant genome DNA extraction kit and radix et rhizoma Tiandi according to the steps in the kit instruction, and respectively numbering 1-30.
Preparing an LAMP reaction system: adding a reaction system (the system does not comprise template DNA, and 9 mu L of each tube) prepared according to the table 3 and 1 mu L of DNA extracted from No. 1-30 vermicelli samples into PCR tubes respectively, and carrying out a set of repeated experiments on each sample; placing the PCR tube into a StepOnEPlus real-time fluorescence PCR instrument, setting the temperature at the heat preservation stage to be 5min, setting the temperature at 60 ℃ respectively, setting the temperature at the circulation stage to be 60 ℃ respectively, setting the dissolution curve ratio to be 1.5%, and continuously circulating for 110 cycles; the amplification curve was observed.
Detection result: no. 1-24 and No. 26-30 vermicelli samples were not amplified linearly, indicating that the vermicelli was free of corn starch adulteration; the sample No. 25 vermicelli showed amplification (see FIG. 8 of the drawings), but the reproducibility was poor, indicating that sample No. 25 could have a small amount of corn starch adulterated.
(2) Repeated verification is carried out on the No. 25 vermicelli sample by DNA extracted by adopting a CTAB method:
the DNA in the No. 25 vermicelli sample is extracted by adopting a CTAB method: crushing a No. 25 vermicelli sample by a universal crusher, weighing 0.1g of powder into a clean 2.0ml centrifuge tube, adding 1.5ml of CTAB lysate (1 g of CTAB, 4.0908g of NaCl, 0.37224g of disodium ethylenediamine tetraacetate, 0.6057g of Tris are dissolved in a beaker, and then fixed in volume by deionized water in a 50ml volumetric flask), and carrying out water bath at 65 ℃ for one hour and reversing the steps upside down; centrifuging at 8000rpm for 15min, collecting 1ml supernatant, adding 700 μl chloroform, mixing, centrifuging at 14500rpm for 10min; taking 650 mu L of supernatant to a clean centrifuge tube, adding 1300 mu LCTAB precipitation liquid (0.25 g CTAB, 0.11688g NaCl are dissolved in a beaker and then fixed in a 50ml volumetric flask by deionized water), uniformly mixing for 30s, and standing at room temperature for one hour; centrifuging at 14500rpm for 10min, removing supernatant, adding 350 μL of 1.2M/L NaCl (3.5064 g NaCl is dissolved in a beaker and fixed in a 50ml volumetric flask with deionized water), shaking vigorously for 30s, adding 350 μL chloroform, mixing well for 30s, centrifuging at 14500rpm for 10min; taking 320 mu L of supernatant, adding 256 mu L of isopropanol, uniformly mixing, standing at-20 ℃ for one hour, centrifuging at 14500rpm for 20min, discarding the supernatant, adding 500 mu L of 70% ethanol, uniformly mixing, centrifuging at 14500rpm for 20min, and discarding the supernatant; air-drying, adding 20 μLddH 2 O is dissolved.
Adding a reaction system (the system does not comprise template DNA, 9 mu L of each tube) prepared according to the table 3 and 1 mu L of DNA extracted from a No. 25 vermicelli sample by a CTAB method into a PCR tube respectively, and performing a set of repeated experiments; placing the PCR tube into a StepOnEPlus real-time fluorescence PCR instrument, setting the temperature at the heat preservation stage to be 5min, setting the temperature at 60 ℃ respectively, setting the temperature at the circulation stage to be 60 ℃ respectively, setting the dissolution curve ratio to be 1.5%, and continuously circulating for 110 cycles; the amplification curve (FIG. 9 of the specification) and the melting curve (FIG. 10 of the specification) were observed.
As can be seen from FIGS. 9 and 10, the DNA extracted from sample No. 25 vermicelli by CTAB method was significantly amplified, indicating that sample No. 25 did have corn starch adulterated.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Sequence listing
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Claims (8)
1. The LAMP primer group for detecting corn source components is characterized in that the sequence of the primer group is as follows:
Yu-F3：5`-GCGAAAAAGAACCCACGGC-3`
Yu-B3：5`-GCTTCGGGCGCAACTTG-3`
Yu-FIP：5`-TAACCGCTGCCCTGGGAGCTTTTCACCAGTACTACCTCCTGCC-3`
Yu-BIP：5`-GCAACGGATATCTCGGCTCTCGTTTTTTCGCGGGATTCTGCAATT-3`
Yu-LB：5`-CATCGATGAAGAACGTAGCAAAATG-3`。
2. a reagent for detecting corn-derived components, which is characterized by comprising dNTPs and Mg 2+ Bst enzyme, buffer and fluorescent dye and LAMP primer set according to claim 1.
3. A kit for detecting a corn-derived component, comprising the LAMP primer set according to claim 1 or the reagent for detecting a corn-derived component according to claim 2.
4. A method for detecting a corn source component comprising the steps of: (1) extracting sample DNA; (2) Preparing a LAMP reaction system by using the LAMP primer set as claimed in claim 1 or the reagent as claimed in claim 2 or the kit as claimed in claim 3; (3) Placing the prepared LAMP reaction system in a real-time fluorescence PCR instrument or a water bath kettle for isothermal amplification; (4) observing the color of an amplification curve or a reaction solution after amplification for 1 h;
the isothermal amplification temperature is 60 ℃;
in the LAMP reaction system, the molar ratio of Yu-FIP to Yu-BIP to Yu-LB to Yu-F3 to Yu-B3 is 8:8:4:1:1.
5. The method for detecting corn source components according to claim 4, wherein the method for extracting sample DNA is a CTAB method.
6. The use of LAMP primer set according to claim 1, characterized by the use in detecting corn-derived components.
7. The use of the LAMP primer set as claimed in claim 6, characterized by the use for detecting corn-derived components in starch or starch processed foods.
8. The use of the LAMP primer group according to claim 6, characterized by the use in detecting corn-derived components in vermicelli.
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| CN112662797B (en) * | 2020-11-09 | 2023-12-15 | 许昌学院 | LMCP primer group and kit for detecting plant source components and application thereof |
| CN112626181B (en) * | 2020-12-07 | 2023-10-24 | 许昌学院 | LMCP primer group for detecting walnut source components, detection method and application thereof |
| CN117363784A (en) * | 2023-11-09 | 2024-01-09 | 许昌学院 | LMTIA detection method for corn component in food using ITS gene as target sequence and application thereof |
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