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

Abstract

The invention belongs to the technical field of molecular biology nucleic acid detection, and particularly relates to an LMTIA primer group for lotus root-derived component detection, a detection method, a kit and application of the LMTIA primer group. The invention designs an LMTIA primer group for lotus root-derived component detection, the LMTIA primer group comprises primers Lian-O-F, Lian-O-B, Lian-O-LF and Lian-O-LB and a probe Lian O-LFPr, a lotus root-derived component detection method is constructed based on an LMTIA ladder melting temperature nucleic acid isothermal amplification technology, and the LMTIA detection method has the advantages of high detection speed, strong specificity, high sensitivity and the like. The detection method can be used for rapidly, accurately and efficiently detecting whether the food contains the lotus root source component or not, can be used for rapidly and accurately detecting starch food such as lotus root starch and the like, and provides a technical means for authenticity identification of the food.

Description

Technical Field

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

[0002] Lotus root (Nelumbo nucifera), the rhizome of the genus Nelumbo in the family Nymphaeaceae, is rich in vitamins, trace elements, dietary fiber, minerals, tannins and carbohydrates, etc. It has high nutritional and medicinal value and is often used to clear away heat, cool blood and stop bleeding. Lotus root powder is a powdered food made by peeling, crushing, homogenizing, precipitating, washing and drying fresh lotus roots. It has a fine texture and is easy to dissolve. It is often used to make lotus root powder, desserts, juices or as a thickener.

[0003] However, the starch content in lotus root is only 9.7% to 12.9%, so the price of pure lotus root powder is significantly higher than other common starches, and adulteration is particularly serious. Some lotus root powder manufacturers reduce the content of pure lotus root powder in lotus root powder by adding low-value starches such as cassava, corn, potato, wheat and sweet potato starch, thereby reducing production costs and seriously affecting the nutritional value and quality of the product. Therefore, in order to safeguard the legitimate rights and interests of consumers and the reasonable order of the market, there is an urgent need for fast, low-cost, easy-to-operate and accurate technologies to authenticate the authenticity of lotus root-derived products such as lotus root powder.

[0004] At present, domestic and foreign methods have established scanning electron microscopy detection technology, infrared detection technology, nucleic acid detection technology, liquid chromatography-mass spectrometry detection technology, etc. for the detection of adulteration of lotus root-derived ingredients, which has achieved accurate identification and quantitative detection of lotus root-derived ingredients. Scanning electron microscopy detection technology can distinguish most starch granules, but cannot distinguish the ultramicroscopic morphology of cassava starch or sweet potato starch in adulterated lotus root powder; the national standard GB / T 25733-2022 "General Rules for Quality of Lotus Root Powder" uses optical microscopes and polarizing microscopes to determine lotus root starch granules, but this method can only identify starch granules similar to lotus roots, and cannot identify potato or other large starch granules. Infrared detection technology, liquid chromatography-mass spectrometry detection technology and conventional starch color development tests have the problems of cumbersome operation, expensive instruments, long detection cycle, high technical requirements for operators, and strong subjectivity.

[0005] The development of nucleic acid detection technology has made up for many of the shortcomings of the above detection technologies. It identifies the authenticity and purity of varieties by identifying the differences in the nucleic acid molecule level of species and varieties. It has the advantages of high accuracy, low cost, good repeatability, etc., and is easy to realize automated detection. Since the report of ladder-shape melting temperature isothermal amplification technology (LMTIA) 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 easy new nucleic acid amplification technology after PCR and loop-mediated isothermal amplification technology (LAMP). LMTIA technology solves the problems of long PCR amplification time and expensive instruments, as well as the false positive problems caused by non-specific 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, screening of transgenics, allergens, animal viruses, pathogenic microorganisms, and mutant genes of human genetic diseases, and has broad application prospects.

[0006] At present, there is no method or kit that can quickly, simply, specifically and sensitively detect lotus root-derived components in starchy foods and other samples, which combines Proofman fluorescent probe with LMTIA system to complete the rapid detection of lotus root-derived components within 20 minutes.

[0007] Therefore, there is a need in the art for a simple, specific, and sensitive method for detecting lotus root-derived components for rapid detection of lotus root-derived components in food. Summary of the invention

[0008] The present invention intends to adopt the latest LMTIA technology, design LMTIA primers and probes, establish a rapid detection method for lotus root-derived components, and provide technical support for the identification of lotus root-derived components in food. One purpose of the present invention is to provide an LMTIA primer set for rapid detection of lotus root-derived components, which is used to detect lotus root-derived components in foods such as lotus root powder and improve food safety supervision and detection technology.

[0009] The present invention is achieved through the following technical solutions:

[0010] The sequences of the LMTIA primer set used for the detection of lotus root-derived ingredients are as follows:

[0011] Lian-OF:5`-GGGCGCAACTTGTTTTTTGCAGAATCCCGTGAACCA-3`

[0012] Lian-OB:5`-CACGCCTGCCTGGGTTTTATGGGAA-3`

[0013] Lian-O-LF:5`-CGTTCAAAGACTCGA-3`

[0014] Lian-O-LB:5`-CGTCACGCATCGTTG-3`

[0015] LianO-LFPr:5`-BHQ2-CGTTCAAAGACTCGG-6-FAM-3`

[0016] The second object of the present invention is to provide a reagent for detecting lotus root-derived components. The specific technical scheme is: including a universal LMTIA premix, Bst polymerase, LMTIA primers and a probe LianO-LFPr.

[0017] The third object of the present invention is to provide a kit for detecting lotus root-derived components. The specific technical solution is: comprising the LMTIA primer set and probe for detecting lotus root-derived components as described above or the reagent for detecting lotus root-derived components as described above.

[0018] The fourth object of the present invention is to provide a method for detecting lotus root-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 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 lotus root-derived components; if no amplification curve appears in the amplification result, the sample does not contain lotus root-derived components.

[0019] Preferably, the temperature of the isothermal amplification is 60° C., and the time of the isothermal amplification is 20 minutes.

[0020] Preferably, the molar ratio of Lian-OF:Lian-OB:Lian-O-LF:Lian-O-LB:LianO-LFPr in the LMTIA reaction system is 16:16:4:4:1.

[0021] The present invention provides uses of the LMTIA primer set, reagent and kit for detecting lotus root-derived components in food, which can be used for detecting lotus root-derived components in food.

[0022] The beneficial effects of the present invention are as follows: the LMTIA primer set for detecting lotus root-derived components of the present invention has good accuracy, specificity and sensitivity, fast detection speed, and can detect lotus root-derived components in samples under constant temperature conditions.

[0023] The LMTIA primer set of the present invention has better specificity at 60°C.

[0024] 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%.

[0025] The detection method of the lotus root-derived components of the present invention is simple and rapid, and the detection result can be obtained by constant temperature reaction for 20 minutes.

[0026] The LMTIA primer set of the present invention can be used for detecting lotus root-derived components in foods such as lotus root powder. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] 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 description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0028] Figure 1 The results of temperature optimization of the lotus root LMTIA primer set of the present invention; the amplification curve of lotus root DNA is above the baseline, and the blank control (ddH 2 O); 1 is lotus root DNA, 2 is blank control (ddH 2 O).

[0029] Figure 2 The specific results of the lotus root LMTIA primer set of the present invention for detecting lotus root at 60°C; the amplification curve of the lotus root sample DNA is above the baseline, and the amplification curve of 8 samples of cassava, corn, sweet potato, potato, yam, rice, wheat, taro and the blank control (ddH 2 O); 1 represents lotus root DNA, 2 represents 8 kinds of sample DNA including cassava and blank control (ddH 2 O).

[0030] Figure 3 The amplification curve of the lotus root LMTIA primer set of the present invention at 60°C for detecting the lotus root absolute sensitivity test; the amplification curve of the lotus root sample DNA is above the baseline, 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 (ddH2 O).

[0031] Figure 4 The amplification curve of the lotus root LMTIA primer set of the present invention at 60°C is used to detect the relative sensitivity test of lotus root; wherein the amplification curve of the lotus root sample DNA is above the baseline, and 1, 2, 3, 4, 5, 6, and 7 represent the mass fraction of lotus root starch in the mixed sample of lotus root starch and corn flour, which is 100%, 20%, 10%, 5%, 1%, 0.1%, and the blank control (ddH 2 O).

[0032] Figure 5 The lotus root LMTIA primer set of the present invention is used for the amplification curve of the commercial lotus root powder sample detection at 60°C; wherein the amplification curve of the lotus root DNA is above the baseline, 1 represents the lotus root positive control, 2, 3, 4 represent the lotus root powder samples No. 1, 2, 3 respectively, 5 represents 4-10 (lotus root powder No. 4, 5, 6, 7, 8, 9, 10) samples and blank control (ddH 2 O). 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 lotus root LMTIA primer set:

[0039] Table 2 Design of primer set for lotus root LMTIA

[0040]

[0041] (2) DNA extraction:

[0042] The DNA of lotus root 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 Lotus Root LMTIA Primer Set of the Present Invention

[0044] According to the LMTIA reaction system in Table 3, take eight PCR tubes, add the reaction system prepared as above to the first two and last two wells of each eight-tube, respectively (the system does not include template DNA, 8 μL in each tube), add 2 μL lotus root DNA (template concentration is 1 ng / μL) to the eight-tube, add 2 μL ddHO 2 O, each sample was repeated twice, paraffin oil was added to each well, the lid was closed and centrifuged, and the well was placed in the Gentier96E fully automatic medical PCR instrument for amplification reaction. The insulation temperatures of each group were 59°C, 60°C, 61°C, 62°C, and 40 cycles were set, each cycle was 30s.

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

[0046]

[0047] The test results were analyzed using the Gentier 96E fully automated medical PCR analysis system. Figure 1 As shown in the figure, lotus root DNA can be amplified in large quantities under the action of primers at 60℃, with the least number of cycles, good curve reproducibility and the highest efficiency. Therefore, 60℃ is selected as the optimal temperature for lotus root LMTIA primers.

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

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

[0050] (2) According to the LMTIA reaction system in Table 3, take eight PCR tubes and add the prepared reaction system to the wells of the eight tubes (the system does not include template DNA, 8 μL in each well). Add 2 μL cassava DNA, 2 μL corn DNA, 2 μL sweet potato DNA, 2 μL potato DNA, 2 μL yam DNA, 2 μL rice DNA, 2 μL wheat DNA, 2 μL taro DNA and 2 μL ddH in order. 2 Finally, paraffin oil was added to each well, the lid was covered, and the well was centrifuged and placed in a Gentier 96E fully automatic medical PCR instrument for amplification reaction. The temperature was set to 60°C, 40 cycles were set, and each cycle was 30 seconds.

[0051] The test results were analyzed using the Gentier 96E fully automated medical PCR analysis system (V1). Figure 2As shown in Figure 2, at 60°C, only lotus root DNA was amplified in large quantities under the action of LMTIA primers (see Appendix Figure 2 Figure 1), 8 samples including cassava and blank control (ddH 2 O) did not show any amplification curve (Appendix Figure 2 Figure 2) fully demonstrates that the primer probe designed in this experiment has strong specificity for amplifying lotus root DNA only.

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

[0053] The extracted lotus root DNA was diluted to lng / μL, 100pg / μL, 10pg / μL, 1pg / μL and 0.1pg / 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, 8μL in each tube), and 2μL ddHO was added in sequence. 2 O, 2μL 0.1pg / μL lotus root DNA, 2μL 1pg / μL lotus root DNA, 2μL 10pg / μL lotus root DNA, 2μL 100pg / μL lotus root DNA, 2μL 1ng / μL lotus root DNA, each sample was repeated three times, and finally paraffin oil was added to each well, the lid was closed and centrifuged, and then placed in the Gentier 96E fully automatic medical PCR instrument for amplification reaction. The temperature was set to 60℃, 40 cycles, and each cycle was 30s.

[0054] The test results were analyzed using the Gentier 96E fully automated medical PCR analysis system. Figure 3 As shown in the figure, when the lotus root DNA concentration is 1ng / μL, 100pg / μL, and 10pg / μL, there is obvious amplification, but there is no amplification trend when the DNA concentration is 1pg / μL and 0.1pg / μL. This is because the concentration exceeds the sensitivity of the method, making the amplification unstable. Therefore, the absolute sensitivity of this method can reach 10pg / μL. In a 10μL reaction system, the lotus root DNA extracted from the sample can be detected when it reaches 10pg.

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

[0056] (1) DNA extraction: Lotus root starch and corn flour were premixed, with the mass fraction of lotus root starch being 100%, 20%, 10%, 5%, 1% and 0.1%, and the DNA was extracted using the Nucleo DNA Extractor from Macherey-Nagel, Germany. The kit was used to extract DNA from a mixed sample of corn flour and lotus root starch, and the concentration was adjusted to 1 ng / μL as template DNA.

[0057] (2) According to the LMTIA reaction system in Table 3, eight PCR tubes were taken and the reaction system prepared as above (the system does not include template DNA, 8 μL in each tube) and 2 μL of template DNA of mixed samples with lotus root proportions of 100%, 20%, 10%, 5%, 1% and 0.1% and blank control (ddH 2 O), and each sample was repeated three times; finally, a drop of paraffin oil was added to each well, the lid was closed, centrifuged, and then placed in a Gentier 96E fully automatic medical PCR instrument for amplification reaction. The temperature was set to 60°C, 40 cycles, and each cycle was 30 seconds.

[0058] The test results were analyzed using the Gentier 96E fully automated medical PCR analysis system (V1). Figure 4 As shown in the figure, in the mixed sample of lotus root starch and corn flour, the numbers 1, 2, 3, 4, 5 and 6 respectively indicate that there is a significant amplification when the mass fraction of lotus root starch is 100%, 20%, 10%, 5%, 1% and 0.1%, and the number 7 indicates the blank control (ddH 2 O) No amplification, so 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: Ten samples of commercially available lotus root starch were extracted using Nucleo DNA 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 1-10.

[0061] (2) The system was prepared according to Table 3 and used for analyzing the primer set to detect whether the commercially available samples contained lotus root-derived ingredients. The reaction system prepared as above (the system does not include template DNA, 8 μL in each tube) and 2 μL of DNA extracted from samples 1-10, 2 μL of ddHO were added to eight PCR tubes respectively. 2 O (blank control), 2 μL lotus root DNA (positive control), and finally a drop of paraffin oil was added to each well. After centrifugation with the lid closed, the well was placed in a Gentier 96E fully automatic medical PCR instrument for amplification reaction. The temperature was set to 60°C, 40 cycles, and each cycle was 30 seconds.

[0062] The test results were analyzed using the Gentier 96E fully automated medical PCR analysis system. Figure 5As shown in Table 4 ("-" indicates no amplification, "+" indicates amplification), only lotus root DNA amplification occurred in lotus root powder samples No. 1, 2, and 3, and no lotus root DNA amplification occurred in lotus root powder samples No. 4 to 10. This indicates that although some lotus root powder products contain lotus root-derived ingredients in the label ingredients, no lotus root-derived ingredients were detected, which is inconsistent with the labeled 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 results of LMTIA lotus root primer set and probe for detecting commercially available samples

[0065]

Claims

1. The LMTIA primer set for detecting lotus root-derived components is characterized in that: The sequence of the primer set is: Lian-OF:5`-GGGCGCAACTTGTTTTTTGCAGAATCCCGTGAACCA-3` Lian-OB:5`-CACGCCTGCCTGGGTTTTATGGGAA-3` Lian-O-LF:5`-CGTTCAAAGACTCGA-3` Lian-O-LB:5`-CGTCACGCATCGTTG-3` LianO-LFPr:5`-BHQ2-CGTTCAAAGACTCGG-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 lotus root probe LianO-LFPr, and a fluorescence reporter group 6-FAM is connected to the 3' end.

3. A reagent for detecting lotus root-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 lotus root-derived ingredients, characterized in that: It comprises the LMTIA primer set and probe as claimed in claim 1 or the reagent for detecting lotus root-derived components as claimed in claim 3.

5. A method for detecting lotus root-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 lotus root-derived components; if no amplification curve appears in the amplification result, the sample does not contain lotus root-derived components.

6. The method for detecting lotus root-derived components according to claim 5, characterized in that: The temperature of the isothermal amplification is 60° C., and the time of the isothermal amplification is 20 minutes.

7. The method for detecting lotus root-derived components according to claim 5, characterized in that: The molar ratio of Lian-OF:Lian-OB:Lian-O-LF:Lian-O-LB:LianO-LFPr 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 lotus root-derived ingredients in starchy foods such as lotus root powder.

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