Method for qualitatively detecting bitter gourd component in processed food by using real-time fluorescent PCR (Polymerase Chain Reaction)

Abstract

The invention provides a method for qualitatively detecting a bitter gourd component in processed food by utilizing real-time fluorescent PCR (Polymerase Chain Reaction). The method comprises the following steps: extracting DNA (Deoxyribose Nucleic Acid) in a processed food sample; designing and synthesizing primer and probe sequences of specific genes of the bitter gourds; and carrying out real-time fluorescent PCR reaction, and reading and analyzing a fluorescent signal so as to judge whether the processed food sample contains the balsam pear component or not. The real-time fluorescent PCR method can be used for effectively detecting the specific gene segment of the balsam pear in the processed food, the specificity is good, the detection lower limit is 0.05%, and the method is suitable for qualitative detection of the balsam pear component in the processed food.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology detection, and in particular relates to a method for qualitatively detecting bitter melon components in processed food by using real-time fluorescence PCR. Background Art

[0002] Bitter melon (Momordica charantia L) is a herbaceous plant of the genus Momordica in the Cucurbitaceae family. It is a plant with both medicinal and edible properties and has multiple health functions. The earliest record of bitter melon being used as medicine can be found in the chapter "Bitter Melon" in Volume 2 of "Southern Yunnan Materia Medica": "Bitter melon tastes bitter and is cold in nature. It enters the heart, spleen, and lung meridians. It removes pathogenic heat, relieves fatigue, and clears the heart and improves eyesight." There are records of bitter melon being used for medical treatment and food therapy in traditional Chinese medicine. It is bitter and cold in nature, and has medicinal and health functions such as clearing away heat and detoxifying, nourishing the liver and improving eyesight, nourishing the kidney and moistening the spleen, lowering blood sugar, regulating blood lipids, slimming, anti-oxidation, antibacterial, and improving human immunity.

[0003] In view of the value of bitter melon as both a medicine and food, bitter melon powder as a dietary supplement or health food with bitter melon powder has rich economic benefits and broad market prospects. Bitter melon powder is made by low-temperature drying and ultrafine grinding of bitter melon. It can be taken directly with water, or it can be further made into bitter melon beverages, bitter melon health food, or as one of the ingredients of pasta food. Because the above-mentioned foods need to go through various processing processes such as crushing, high pressure, and stirring during the processing, it is difficult for people to distinguish whether bitter melon is added as a raw material through sensory perception, which invisibly provides a great deal of room for food adulteration. Various economically motivated adulteration behaviors (Economically Motivated Adulteration, EMA) such as passing inferior products as good ones and fake products as genuine ones not only seriously hinder fair trade and harm the interests of consumers, but also pose severe challenges to food safety supervision. Whether it is the adulteration of plant raw materials driven by economic interests or the misuse and mixing of plant raw materials, it is more common in processed foods in powder or juice form that cannot be recognized in their original form. The existing food testing standards do not include any identification and testing methods for bitter melon processed foods. There is an obvious technical gap in identifying the above-mentioned confusion or adulteration behaviors, and it cannot meet the technical needs of relevant food authenticity inspection and supervision.

[0004] A real-time fluorescence PCR qualitative detection method for bitter melon ingredients in processed foods was established, which can be used for label compliance inspection and supervision of bitter melon ingredients in processed foods using bitter melon as raw material, and provide an accurate and reliable technical basis for authenticity identification of related foods. Summary of the invention

[0005] The purpose of the present invention is to provide a method for qualitatively detecting bitter melon components in processed foods with high specificity and effective use of real-time fluorescent PCR in order to solve the above shortcomings and deficiencies.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A method for qualitatively detecting bitter melon components in processed foods using real-time fluorescent PCR, comprising the following steps:

[0008] (1) Extract DNA from processed food samples;

[0009] (2) Design and synthesize primers and probe sequences for bitter melon-specific genes;

[0010] (3) performing a real-time fluorescence PCR reaction, reading and analyzing the fluorescence signal, and thereby determining whether the processed food sample contains bitter melon ingredients.

[0011] Preferably, the bitter melon-specific gene is the ChiA gene.

[0012] Preferably, the upstream primer of the bitter melon-specific gene has a nucleotide sequence as shown in SEQ ID NO.1, the downstream primer has a nucleotide sequence as shown in SEQ ID NO.2, and the probe sequence has a nucleotide sequence as shown in SEQ ID NO.3.

[0013] Preferably, the PCR reaction system is 25 μL, including: 12.5 μL of 2× real-time fluorescence PCR premix, 1 μL of upstream and downstream primers and probes at a concentration of 12.5 pmol / μL, 5 μL of DNA template, and ddH2O to make up the volume.

[0014] Preferably, the real-time fluorescence PCR reaction conditions are: 50°C for 2 min; pre-denaturation at 95°C for 10 min; 95°C for 15 s, 60°C for 1 min, and 45 cycles.

[0015] Preferably, the detection limit of the method for bitter melon components in processed foods is 0.05%.

[0016] Preferably, in the method, the judgment rule is: when the Ct value is less than 38.0, it is judged that the bitter melon component is detected; when the Ct value is greater than or equal to 38.0, it is judged that the bitter melon component is not detected.

[0017] The present invention can effectively detect bitter melon specific gene fragments in processed foods by using real-time fluorescence PCR method, with good specificity and no cross reaction with other plant components; the detection limit of bitter melon components is 0.05%, which meets the technical requirements and is suitable for the qualitative detection of bitter melon components in processed foods (including bitter melon powder, bitter melon beverages, bitter melon health foods and flour-made foods with added bitter melon powder, etc.), providing a technical basis for the authenticity identification of bitter melon components in processed foods. The method established in this paper can be used for label compliance inspection of bitter melon components in pre-packaged foods and health products, providing a technical basis for the collection and management of customs import and export tariffs, and filling the technical gap in the authenticity identification detection of bitter melon-derived components in my country's existing standard system. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The results of the specificity verification experiment are shown in Figure 1. (a) ChiA gene primer probe amplification curve; (b) GAS1 gene primer probe amplification curve; (c) RIP gene primer probe amplification curve.

[0019] Figure 2 This is a graph showing the optimization experiment results for primer and probe concentrations.

[0020] Figure 3 This is the result of the detection limit verification experiment.

[0021] Figure 4 The stability verification test results are shown in Figure 1. (a) 0.5% bitter melon DNA; (b) 0.1% bitter melon DNA; (c) 0.05% bitter melon DNA. DETAILED DESCRIPTION

[0022] The present invention is further described in detail below in conjunction with specific embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0023] 1. Materials and Methods

[0024] 1.1 Materials and reagents

[0025] There were 17 kinds of vegetables, fruits and grains, including bitter melon, water melon, loofah, winter melon, pumpkin, cucumber, watermelon, cantaloupe, sponge gourd, oatmeal, rice, wheat, monk fruit, peanuts, corn, soybean and kidney bean, 18 kinds of bitter melon powder, 9 kinds of bitter melon processed foods, including bitter melon juice, bitter melon slices, bitter melon tea, bitter melon seed powder, bitter melon buckwheat noodles, bitter melon red bean and barley cakes, bitter melon kudzu root oatmeal, etc., purchased from local supermarkets or e-commerce platforms. There was 1 kind of homemade bitter melon juice (fresh bitter melon juice squeezed with water).

[0026] QuickGene DNA tissue kit S (KURABO, Japan), TaqMan TMFast Advanced Master Mix (Thermo Fisher Scientific, USA), primers and probes (Shanghai Shanjing Molecular Biotechnology Co., Ltd.).

[0027] 1.2 Instruments and Equipment

[0028] ABI 7900HT real-time fluorescence PCR instrument (Thermo Fisher Scientific, USA), Nanospec micro-nucleic acid protein analyzer (Shimadzu, Japan), Centrifuge 5424 small high-speed centrifuge (Eppendorf, Germany), Tube-Mill Control test tube mill (IKA, Germany), MS2 vortex mixer (IKA, Germany), AB204-SMETLER TOLEDO electronic balance (TOLEDO, Switzerland).

[0029] 1.3 Experimental methods

[0030] 1.3.1 Genomic DNA extraction and concentration determination

[0031] First, the experimental samples were homogenized using a test tube grinder, and then the sample DNA was extracted using the genomic DNA extraction kit QuickGeneDNA tissue kit S according to the operating steps in the instruction manual. Finally, the concentration of the sample genomic DNA was determined using a Nanospec micro-nucleic acid protein analyzer, and diluted to 20 ng / μL and placed at 4°C for use.

[0032] 1.3.2 Real-time fluorescence PCR

[0033] Primer Express 5.0 software was used to select three genomic DNA sequences of bitter melon from NCBI GeneBank, and bitter melon-specific primer probes were designed (see Table 1).

[0034] Using 100% Momordica charantia DNA as a positive control, two PCRs were set up in parallel to screen the primers and probes.

[0035] Real-time fluorescence PCR reaction system (25 μL): 2× real-time fluorescence PCR premix 12.5 μL, upstream and downstream primers (10 pmol / μL) and probe (10 pmol / μL) 1 μL each, DNA template 5 μL (50 ng-500 ng), ddH2O to make up the volume. PCR reaction conditions are: 50℃2min; pre-denaturation 95℃10min; 95℃15s, 60℃1min, 45 cycles.

[0036] Table 1. Primers and probes

[0037]

[0038] 1.3.3 Primer-probe specificity assessment

[0039] Extract genomic DNA from the 17 common vegetables, fruits and grains in 1.1, set up two parallel PCRs for each sample DNA, and verify the specificity of the three sets of primers and probes in Table 1.

[0040] 1.3.4 Optimization of primer-probe reaction concentration

[0041] In order to optimize the PCR reaction system, the final concentrations of primers and probes in the reaction system were set to F / R / P (upstream primer / downstream primer / probe) (nmol / L): 100 / 100 / 100, 100 / 100 / 50, 200 / 200 / 200, 200 / 200 / 100, 300 / 300 / 300, 300 / 300 / 150, 400 / 400 / 400, 400 / 400 / 200, 500 / 500 / 500, 500 / 500 / 250, 600 / 600 / 600, 600 / 600 / 300, a total of 12 combinations, and real-time fluorescence PCR was used to detect bitter melon genomic DNA, and 2 PCRs were set up in parallel.

[0042] 1.3.5 Evaluation of the lower limit of real-time fluorescence PCR detection

[0043] 100% of bitter melon genomic DNA was diluted with soybean DNA (extracted from dry soybean seeds by conventional methods) in gradients, and the concentrations of bitter melon genomic DNA were 100%, 10%, 5%, 1%, 0.5%, 0.1%, 0.05%, and 0.01%, respectively. Real-time fluorescence PCR was performed using bitter melon specific primer probes with optimized reaction concentrations, and two parallel real-time fluorescence PCR reactions were performed for each concentration of DNA to evaluate the detection limit of the bitter melon real-time fluorescence PCR method.

[0044] 1.3.6 Stability Assessment

[0045] In order to evaluate the stability of this method, 10 parallel real-time fluorescence PCR tests were performed on momordica charantia DNA with DNA concentrations of 0.5%, 0.1%, and 0.05%.

[0046] 1.3.7 Testing of Bitter Melon Processed Foods on the Market

[0047] In this paper, 18 samples of bitter melon powder and 10 bitter melon processed foods were collected, and their origins included Guangdong, Anhui, Hebei, Henan, etc. Genomic DNA was extracted from the bitter melon processed foods on the market, and real-time fluorescence PCR was performed using the optimized reaction concentration of bitter melon-specific primer probe (upstream and downstream primers and probes 500nmol / L each) and higher plant chloroplast tRNALeu gene primer probe (tRNA-F:CGRAATYGGTAGACGCTACG; tRNA-R:TTCCATYGAGTCTCTGCACCT; tRNA-P:FAM-TGGGGCAATCCTGAGCCAAATCC-TAMRA (upstream and downstream primers 200nmol / L each, probe 100nmol / L)) to evaluate the applicability of this method.

[0048] 2. Results and Analysis

[0049] 2.1 Primer-probe specificity

[0050] The specificity validation results are shown in Table 2 and Figure 1 . As can be seen from the chart, the three sets of primer probes ChiA, GAS1 and RIP can all amplify the bitter melon gene, with an average Ct value of 21, 21 and 22. There is a weak amplification of kidney beans, with Ct values ​​of 38, 36 and 37, respectively, and no cross reaction to the other 15 common melon and food plants. The ChiA primer probe has the smallest Ct value and the strongest specificity, so the primer probe of the ChiA gene was selected for subsequent research, and the judgment basis of this method is set as follows: when the Ct value is <38.0, it is judged that the bitter melon component is detected; when the Ct value is ≥38.0, it is judged that the bitter melon component is not detected, so as to exclude the interference of nonspecific amplification of other plants.

[0051] Table 2. Specificity verification experiment results

[0052] Sample No. Sample name ChiA Results GAS1 Results RIP Results 1 Water Melon Not detected Not detected Not detected 2 Loofah Not detected Not detected Not detected 3 Winter melon Not detected Not detected Not detected 4 pumpkin Not detected Not detected Not detected 5 Cucumber Not detected Not detected Not detected 6 watermelon Not detected Not detected Not detected 7 Cantaloupe Not detected Not detected Not detected 8 Pear Not detected Not detected Not detected 9 oat Not detected Not detected Not detected 10 Rice Not detected Not detected Not detected 11 wheat Not detected Not detected Not detected 12 Monk Fruit Not detected Not detected Not detected 13 peanut Not detected Not detected Not detected 14 corn Not detected Not detected Not detected 15 Soybean Not detected Not detected Not detected 16 kidney bean Detection, Ct38 Detection, Ct36 Detection, Ct37 17 Momordica charantia Detection, Ct21 Detection, Ct21 Detection, Ct22

[0053] 2.2 Optimization of primer-probe reaction concentration

[0054] The optimization results of primer-probe reaction concentrations are shown in Figure 2 When the final concentrations of primers and probes in the PCR reaction system were both 500 nmol / L, the average Ct value was 21, the Ct value was the smallest and the fluorescence signal value was the highest.

[0055] Therefore, the reaction concentration of the primers and probes in this method was determined to be 500 nmol / L.

[0056] 2.3 Real-time fluorescence PCR detection limit

[0057] The results of the detection limit verification are shown in Figure 3In the figure, curves 1 to 8 are amplification curves of bitter melon genomic DNA concentration percentages of 100%, 10%, 5%, 1%, 0.5%, 0.1%, 0.05% and 0.01%, respectively.

[0058] With Ct value <38.0 as the judgment condition of "detection", this method can detect bitter melon DNA with DNA concentration percentages of 100%, 10%, 5%, 1%, 0.5%, 0.1%, and 0.05%, but cannot detect bitter melon DNA with a DNA concentration percentage of 0.01%. Therefore, the lower limit of real-time fluorescence PCR detection of bitter melon genes by this method is 0.05% (DNA concentration percentage).

[0059] 2.4 Stability Verification Experiment Results

[0060] The experimental results are shown in Figure 4 As can be seen from the figure, 10 parallel real-time fluorescence PCRs with DNA concentration percentages of 0.5%, 0.1%, and 0.05% can all detect bitter melon DNA, so this method can stably detect bitter melon DNA with a DNA concentration percentage as low as 0.05%.

[0061] 2.5 Testing of commercially available processed bitter melon foods

[0062] The actual sample test results are shown in Tables 3 and 4. The experimental results show that tRNALeu can be detected in 18 commercially available bitter melon powder samples and 10 bitter melon processed foods, indicating that this method can effectively extract plant genomic DNA in bitter melon processed foods. Bitter melon components can be detected in 18 bitter melon powder samples and 10 bitter melon processed foods. The above test results show that this method can effectively identify bitter melon components in processed foods, providing a technical basis for the authenticity identification of bitter melon processed foods.

[0063] Table 3. Bitter melon powder actual sample verification test results

[0064]

[0065]

[0066] Table 4. Bitter melon processed food validation test results

[0067]

Claims

1. A method for qualitatively detecting bitter melon components in processed foods using real-time fluorescence PCR, characterized in that The following steps are involved: (1) Extract DNA from processed food samples; (2) Design and synthesize primers and probe sequences for bitter melon-specific genes; (3) performing a real-time fluorescence PCR reaction, reading and analyzing the fluorescence signal, and thereby determining whether the processed food sample contains bitter melon ingredients.

2. The method according to claim 1, characterized in that The bitter melon-specific gene is the ChiA gene.

3. The method according to claim 1, characterized in that The upstream primer of the bitter melon-specific gene has a nucleotide sequence as shown in SEQ ID NO.1, the downstream primer has a nucleotide sequence as shown in SEQ ID NO.2, and the probe sequence has a nucleotide sequence as shown in SEQ ID NO.

3.

4. The method according to claim 1, characterized in that: The PCR reaction system was 25 μL, including: 12.5 μL of 2× real-time fluorescence PCR premix, 1 μL of upstream and downstream primers and probes at a concentration of 12.5 pmol / μL, 5 μL of DNA template, and ddH2O to make up the volume.

5. The method according to claim 1, characterized in that The real-time fluorescence PCR reaction conditions are: 50°C for 2 min; pre-denaturation at 95°C for 10 min; 95°C for 15 s, 60°C for 1 min, and 45 cycles.

6. The method according to claim 1, characterized in that The detection limit of the method for bitter melon components in processed foods is 0.05%.

7. The method according to claim 1, characterized in that In the method, the judgment rule is: when the Ct value is less than 38.0, it is judged that the bitter melon component is detected; when the Ct value is greater than or equal to 38.0, it is judged that the bitter melon component is not detected.

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