LMTIA primer group, detection method and kit for potato-derived component detection and application of LMTIA primer group, detection method and kit

Abstract

The invention discloses an LMTIA primer group for potato-derived component detection, a detection method, a kit and application of the LMTIA primer group, and belongs to the technical field of molecular biology nucleic acid detection. The invention designs an LMTIA primer group for detecting the potato-derived components, the LMTIA primer group comprises primers MaLS-24-F12, MaLS-24-B2, MaLS-24-LF and MaLS-24-LB and a probe MaLS-LBPr, a detection method for detecting the potato-derived components is constructed based on an LMTIA ladder melting temperature nucleic acid isothermal amplification technology, and the method has the advantages of short detection time, strong specificity, high sensitivity and the like. The detection method can be used for rapidly, accurately and efficiently identifying the potato-derived components of foods such as starch, vermicelli, vermicelli and potato chips, and a technical means is provided for authenticity identification of the foods.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology nucleic acid detection, and specifically relates to a LMTIA primer set, a detection method, a kit and applications thereof for detecting potato-derived components. Background Art

[0002] Potato (Solanum tuberosum L.), an annual herbaceous plant of the Solanaceae family, is also known as potato, ground egg, yam bean, yam egg, Dutch potato, potato, ground bean, and yam. Potato is the fourth largest food crop in my country. It is rich in nutrition and rich in starch, protein, fat, crude fiber, vitamins, carotenoids, amino acids, and a variety of trace elements. The rich vitamin C content of potatoes far exceeds that of food crops; its high protein and sugar content greatly exceeds that of general vegetables. In the food processing industry, potatoes can be used as raw materials to process various quick-frozen convenience foods and snacks, such as dehydrated products, fried potato chips, quick-frozen French fries, puffed foods, vermicelli and noodles, etc. At the same time, it can also be further processed into fructose syrup and citric acid.

[0003] As an important raw material for the food processing industry, the price of potato starch occupies a unique position in the starch market. At present, the price of potato starch is lower than that of lotus root starch and sweet potato starch, but higher than some other starches such as corn starch and cassava starch. Some starch manufacturers mix low-priced starches such as potato starch, cassava starch and corn starch in the production of sweet potato vermicelli or lotus root starch, or use cassava starch or corn starch to impersonate potato starch for production and sales. Although this type of adulteration of potato starch will not affect food safety, it will affect its performance in application, greatly reduce the taste and appearance of food, and affect the normal order of the market. Therefore, it is particularly urgent to establish a detection method for rapid identification of potato-derived ingredients.

[0004] At present, potato starch food adulteration detection technologies include scanning electron microscopy, laser particle size analyzer, chemical instrument analysis and physical and chemical analysis. Scanning electron microscopy and laser particle size analyzer analyze the ultra-microscopic morphology of potato starch particles for qualitative analysis; rapid viscosity analyzer measures the peak viscosity of different starches for quantitative identification; chemical instrument analysis, such as near-infrared spectroscopy, extracts spectral data and chemometric analysis of potato starch; in physical and chemical analysis, starch shows different color reactions when encountering iodine preparations; rapid viscosity analyzer measures the peak viscosity of different starches for quantitative identification. However, the above detection methods have the disadvantages of cumbersome operation process, expensive instruments, long detection time, high technical requirements for operators, and strong subjectivity.

[0005] Modern molecular biotechnology, represented by PCR, is convenient, fast, and accurate. It does not require large instruments and complex operating systems. It analyzes the sources and characteristics of raw materials and products from nucleic acid molecules and has been widely used in food authenticity identification research such as food type identification and origin tracing. In this regard, a method for detecting potato-derived ingredients in food from the perspective of nucleic acid molecules has been established, which has the characteristics of short time, strong specificity, and high sensitivity, and is of great research value and significance.

[0006] Since the ladder-shape melting temperature isothermal amplification (LMTIA) was reported in 2021, due to its non-thermal and non-enzymatic single-stranded template production advantages, only one pair of primers is needed to complete amplification within 20 minutes, thus becoming another fast and convenient new nucleic acid amplification technology after PCR and loop-mediated isothermal amplification (LAMP) technology. LMTIA technology solves the problems of long PCR amplification time and expensive instruments, as well as the false positive problems caused by nonspecific amplification, aerosol contamination, and primer dimers in LAMP technology. LMTIA has the advantages of short detection time, low cost, strong specificity and high sensitivity. In recent years, it has been successfully applied to the authenticity identification of starch, meat, edible oil, dairy products, etc., as well as the screening of transgenics, allergens, animal viruses, pathogenic microorganisms, and human genetic disease mutant genes, and has broad application prospects.

[0007] At present, there is no method that uses the Proofman fluorescent probe combined with the LMTIA system to complete the rapid detection of potato-derived components within 20 minutes. There is no report of a method and kit that can quickly, simply, specifically and sensitively detect potato-derived components in starchy food samples at home and abroad.

[0008] Therefore, there is a need in the art for a rapid, highly specific and sensitive method for detecting potato-derived ingredients in food. Summary of the invention

[0009] The present invention intends to adopt the latest LMTIA technology, design LMTIA primers and probes, establish a rapid detection method for potato-derived ingredients, and provide technical support for the identification of potato-derived ingredients in food. One object of the present invention is to provide an LMTIA primer set for rapid detection of potato-derived ingredients, which is used to detect potato-derived ingredients in foods such as starch, vermicelli, vermicelli, and potato chips, and improve food safety supervision and detection technology.

[0010] The present invention is achieved through the following technical solutions:

[0011] The sequences of the LMTIA primer set used for potato-derived component detection are as follows:

[0012] MaLS-24-F12:5`-GGTAGCCGGATTTTGCAAGTGGTGGTTGAAGCTC-3`

[0013] MaLS-24-B2:5`-TCGCGGCTACCTTTTATGGCGCAATCAGGGTCTG-3`

[0014] MaLS-24-LF:5`-CAACGAGAGAGAGTT-3`

[0015] MaLS-24-LB:5`-CGCGCGTCCCGGACTCC-3`

[0016] MaLS-LBPr:5`-BHQ2-CGCGCGTCCCGGACTCT-6-FAM-3`

[0017] The second object of the present invention is to provide a reagent for detecting potato-derived components, and the specific technical scheme is: comprising a universal LMTIA premix, Bst polymerase, LMTIA primers and MaLS-LBPr.

[0018] The third object of the present invention is to provide a kit for detecting potato-derived components. The specific technical solution is: comprising the LMTIA primer set for detecting potato-derived components as described above or the reagent for detecting potato-derived components as described above.

[0019] The fourth object of the present invention is to provide a method for detecting potato-derived components, the specific technical scheme is: comprising the following steps: (1) extracting sample DNA; (2) preparing an LMTIA reaction system using the LMTIA primer set as claimed in claim 1 or the reagent as claimed in claim 3 or the kit as claimed in claim 4; (3) placing the prepared LMTIA reaction system in a real-time fluorescence PCR instrument for constant temperature amplification; (4) observing the amplification curve after 20 minutes of amplification. (5) If an exponential curve appears in the amplification result graph, the sample contains potato-derived components; if no amplification curve appears in the amplification result, the sample does not contain potato-derived components.

[0020] Preferably, the temperature of the isothermal amplification is 62° C., and the time of the isothermal amplification is 20 minutes.

[0021] Preferably, the molar ratio of MaLS-24-F12:MaLS-24-B2:MaLS-24-LF:MaLS-24-LB:MaLS-LBPr in the LMTIA reaction system is 16:16:4:4:1.

[0022] The present invention provides uses of the LMTIA primer set, reagent and kit for detecting potato-derived components in food, which can be used for detecting potato-derived components in food.

[0023] The beneficial effects of the present invention are as follows: the LMTIA primer set for detecting potato-derived components of the present invention has good specificity and sensitivity, a fast detection speed, and can detect potato-derived components in a sample under a constant temperature condition.

[0024] The LMTIA primer set of the present invention has better specificity at 62°C.

[0025] The absolute sensitivity of the LMTIA primer set of the present invention can reach 10 pg / μL, and the relative sensitivity can reach 0.1%.

[0026] The potato-derived component detection method of the present invention is simple and rapid, and the detection result can be obtained by constant temperature reaction for 20 minutes.

[0027] The LMTIA primer set of the present invention can be used for detecting potato-derived components in foods such as starch, vermicelli, rice noodles, potato chips, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor.

[0029] Figure 1 The results of temperature optimization of the potato LMTIA primer set of the present invention; the amplification curve of potato sample DNA is above the baseline, and the blank control (ddH2O) is below the baseline; 1 is potato DNA, and 2 is a blank control (ddH2O).

[0030] Figure 2 The specific result of potato LMTIA primer set of the present invention for detecting potato at 62°C; the amplification curve of potato sample DNA is above the baseline, and the DNA of 9 samples including cassava, sweet potato, corn, yam, rice, soybean, mung bean, wheat and taro and a blank control (ddH2O) are below the baseline; 1 represents potato DNA, and 2 represents 9 sample DNAs including cassava and a blank control (ddH2O).

[0031] Figure 3 The figure is an amplification curve of the potato LMTIA primer set of the present invention at 62°C for detecting potato absolute sensitivity test; the line above the amplification curve of potato sample DNA, 1 indicates that the concentration of the DNA template is 1 ng / μL; 2 indicates that the concentration of the DNA template is 100 pg / μL, 3 indicates that the concentration of the DNA template is 10 pg / μL, and 4 indicates a blank control (ddH2O).

[0032] Figure 4 The present invention is a potato LMTIA primer set at 62°C, detecting an amplification curve of a potato relative sensitivity test; the area above the baseline is an amplification curve of a potato sample DNA, and 1, 2, 3, 4, 5, 6, and 7 represent a mixed sample of potato flour and corn flour, wherein the mass fraction of potato flour is 100%, 20%, 10%, 5%, 1%, 0.1%, and a blank control (ddH2O), respectively. DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] The sources of the reagents used in the present invention are shown in Table 1 below.

[0035] Table 1 Sources of reagents

[0036]

[0037] Example 1 LMTIA primer set design and temperature optimization

[0038] (1) Design of potato LMTIA primer set:

[0039] Table 2 Potato LMTIA primer set design

[0040]

[0041] (2) DNA extraction:

[0042] The potato DNA was extracted using a plant genomic DNA extraction kit purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. The specific operation steps are shown in the instruction manual of the kit.

[0043] (3) Temperature Optimization Test of Potato LMTIA Primer Set of the Present Invention

[0044] According to the LMTIA reaction system in Table 3, take 8-tube PCR tubes, add the reaction system prepared as above to the first two and last two wells of each 8-tube, respectively (the system does not include template DNA, 8 μL in each tube), add 2 μL potato DNA (template concentration is 1 ng / μL) to the 8-tube, add 2 μL ddH2O, do two replicates for each sample, add paraffin oil to each well, cover the lid and centrifuge, and place in Gentier 96E fully automatic medical PCR instrument for amplification reaction. The insulation temperatures of each group were 61°C, 62°C, 63°C, and 64°C, respectively, and 40 cycles were set, each cycle was 30s.

[0045] Table 3 Potato LMTIA reaction system (10 μL)

[0046]

[0047] The test results were analyzed using the Gentier 96E fully automated medical PCR analysis system (V1). Figure 1 As shown, at 62°C, potato DNA ( Figure 1 The MLS-DNA in the potato can be amplified in large quantities under the action of primers, with the least number of cycles, good reproducibility of amplification curve and the highest efficiency. Therefore, 62℃ was selected as the optimal temperature for amplification of potato LMTIA primer set.

[0048] Example 2 Specificity of the LMTIA primer set of the present invention

[0049] (1) Sample DNA extraction: DNA from cassava, sweet potato, corn, yam, rice, soybean, mung bean, wheat and taro was extracted using a plant genomic DNA extraction kit purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. The concentration was adjusted to 5 ng / μL as template DNA.

[0050] (2) According to the LMTIA reaction system in Table 3, take the PCR eight-tube strip and add the prepared reaction system to the wells of the eight-tube strip (the system does not include template DNA, 8 μL in each well). Add 2 μL of cassava DNA, 2 μL of sweet potato DNA, 2 μL of corn DNA, 2 μL of yam DNA, 2 μL of rice DNA, 2 μL of soybean DNA, 2 μL of mung bean DNA, 2 μL of wheat DNA, 2 μL of taro DNA, 2 μL of ddH2O and 2 μL of potato DNA in order, and repeat three times for each sample. Finally, add paraffin oil to each well, cover the lid, centrifuge, and place in the Gentier 96E fully automatic medical PCR instrument for amplification reaction. The temperature is set to 62°C, and 40 cycles are set, each cycle is 30 seconds.

[0051] The test results were analyzed using the Gentier 96E fully automated medical PCR analysis system (V1). Figure 2 As shown in Figure 2, at 62°C, only potato DNA was amplified in large quantities under the action of LMTIA primers (see Appendix Figure 2 No amplification curves were found in the 9 samples (marked with number 1) such as cassava and blank control (ddH2O) (see Appendix Figure 2 This fully demonstrates that the potato LMTIA primer set designed in this experiment has strong specificity for amplification of potato DNA only.

[0052] Example 3 Absolute sensitivity of the LMTIA primer set of the present invention

[0053] The extracted potato DNA was gradiently diluted to 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL and 0.1 pg / μL. According to the LMTIA reaction system in Table 3, eight PCR tubes were taken, and the reaction system prepared as above was added to the eight tubes (the system did not include template DNA, 8 μL in each tube), and 2 μL ddH2O, 2 μL 0.1 pg / μL potato DNA, 2 μL 1 pg / μL potato DNA, 2 μL 10 pg / μL potato DNA, 2 μL 100 pg / μL potato DNA, 2 μL 1 ng / μL potato DNA were added in sequence, and three replicates were made for each sample. Finally, paraffin oil was added to each well, the lid was closed and centrifuged, and then the well was placed in a Gentier 96E fully automatic medical PCR instrument for amplification reaction. The temperature was set at 62°C, with 40 cycles of 30 s per cycle.

[0054] The test results were analyzed using the Gentier 96E fully automated medical PCR analysis system (V1). Figure 3 As shown in the figure, the numbers 1, 2 and 3 respectively indicate that when the potato DNA concentration is 1ng / μL, 100pg / μL and 10pg / μL, there is obvious amplification; however, when the potato DNA concentration is 1pg / μL and 0.1pg / μL, there is no amplification trend (see Appendix Figure 4 The number 4 in the figure is because the concentration of potato DNA exceeds the sensitivity of the method, making the amplification unstable. Therefore, the absolute sensitivity of this method for potato DNA detection can reach 10pg / μL. In a 10μL reaction system, 10pg of potato DNA extracted from the sample can be detected.

[0055] Example 4 Relative sensitivity of the LMTIA primer set of the present invention

[0056] (1) DNA extraction: Corn flour and potato flour were premixed, with the mass fractions of potato flour being 100%, 20%, 10%, 5%, 1%, and 0.1%, respectively, using Macherey, Germany - Nagel's Nucleo The kit was used to extract DNA from a mixed sample of potato flour and corn flour, and the concentration was adjusted to 1 ng / μL for use as template DNA.

[0057] (2) According to the LMTIA reaction system in Table 3, take eight PCR tubes, add the reaction system prepared as above (the system does not include template DNA, 8 μL in each tube) and 2 μL of potato template DNA of mixed samples with a proportion of 100%, 20%, 10%, 5%, 1% and 0.1% and blank control (ddH2O) to 8 tubes respectively, and perform three replicates for each sample. Finally, add a drop of paraffin oil to each well, cover the lid, centrifuge and put it into the Gentier 96E fully automatic medical PCR instrument for amplification reaction. The temperature is set to 62°C, 40 cycles, and each cycle is 30s.

[0058] The test results were analyzed using the Gentier 96E fully automated medical PCR analysis system (V1). Figure 4 As shown, in the mixed sample of potato flour and corn flour, the numbers 1, 2, 3, 4, 5 and 6 respectively indicate that there is obvious amplification when the mass fraction of potato flour is 100%, 20%, 10%, 5%, 1% and 0.1%, and the number 7 indicates that there is no amplification in the blank control (ddH2O). Therefore, the detection limit (relative sensitivity) of this method can reach at least 0.1%.

[0059] Example 5 Detection of commercially available samples of the LMTIA primer set of the present invention

[0060] (1) DNA extraction from samples: 26 samples of commercially available samples were directly weighed for powdered samples and weighed for non-powdered samples after crushing. The samples were extracted using the Nucleo PCR kit from Macherey-Nagel, Germany. The kit was used to extract DNA from commercially available samples, and the concentration was adjusted to 1 ng / μL as template DNA. The kit was extracted according to the steps in the instruction manual and numbered 1-26.

[0061] (2) The system was prepared according to Table 3 and used to analyze the potato LMTIA primer set to detect whether commercial samples contain potato-derived components. Take eight PCR tubes and add the reaction system prepared according to Table 3 (the system does not include template DNA, 8 μL in each tube) and 2 μL of DNA extracted from commercial samples No. 1-26, 2 μL ddH2O (blank control), and 2 μL potato DNA (positive control). Finally, add a drop of paraffin oil to each well, cover the lid and centrifuge, and then put it into the Gentier 96E fully automatic medical PCR instrument for amplification reaction. The temperature was set to 62°C, 40 cycles, and each cycle was 30 seconds.

[0062] The test results were analyzed using the Gentier 96E fully automatic medical PCR analysis system. The test results are shown in Table 4 ("-" represents no amplification, and "+" represents amplification). Samples No. 9, 13, 14, and 15 showed potato DNA amplification, indicating that the food contains potato-derived ingredients, among which the potato-derived ingredients of samples No. 9 and 13 were not reflected in the label ingredients.

[0063] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

[0064] Table 4 Amplification of potato LMTIA primer set for commercial samples

[0065]

Claims

1. A LMTIA primer set for detecting potato-derived ingredients, characterized in that: The primer set sequence is: MaLS-24-F12:5`-GGTAGCCGGATTTTGCAAGTGGTGGTTGAAGCTC-3` MaLS-24-B2:5`-TCGCGGCTACCTTTTATGGCGCAATCAGGGTCTG-3` MaLS-24-LF:5`-CAACGAGAGAGAGTT-3` MaLS-24-LB:5`-CGCGCGTCCCGGACTCC-3` MaLS-LBPr:5`-BHQ2-CCGGCGTCCGGACTCT-6-FAM-3`.

2. The primer combination according to claim 1, wherein a fluorescence quenching group BHQ2 is connected to the 5' end of the potato probe MaLS-LBPr, and a fluorescence reporter group 6-FAM is connected to the 3' end.

3. A reagent for detecting potato-derived components, characterized in that: It comprises a universal LMTIA premix, Bst polymerase and the LMTIA primer set and probe as claimed in claim 1.

4. A kit for detecting potato-derived ingredients, characterized in that: The method comprises the LMTIA primer set and probe according to claim 1 or the reagent for detecting potato-derived components according to claim 3.

5. A method for detecting potato-derived components, characterized in that: The method comprises the following steps: (1) extracting sample DNA; (2) preparing an LMTIA reaction system using the LMTIA primer set and probe as claimed in claim 1 or the reagent as claimed in claim 3 or the kit as claimed in claim 4; (3) placing the prepared LMTIA reaction system in a real-time fluorescence PCR instrument for constant temperature amplification; (4) observing the amplification curve after 20 minutes of amplification. (5) If an exponential curve appears in the amplification result graph, the sample contains potato-derived components; if no amplification curve appears in the amplification result, the sample does not contain potato-derived components.

6. The method for detecting potato-derived components according to claim 5, characterized in that: The temperature of the isothermal amplification is 62° C., and the time of the isothermal amplification is 20 minutes.

7. The method for detecting potato-derived components according to claim 5, characterized in that: The molar ratio of MaLS-24-F12:MaLS-24-B2:MaLS-24-LF:MaLS-24-LB:MaLS-LBPr in the LMTIA reaction system is 16:16:4:4:

1.

8. The LMTIA primer set probe according to claims 1-2 and the kit according to claim 4, characterized in that: Application in the detection of potato-derived ingredients in foods such as starch, vermicelli, rice noodles, and potato chips.

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