CN116083625A - Microdroplet digital PCR detection primer probe composition for mango allergen component, detection method and application thereof - Google Patents
Microdroplet digital PCR detection primer probe composition for mango allergen component, detection method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of detection of mango allergen components, in particular to a microdroplet digital PCR detection primer probe composition of mango allergen components, a detection method and application thereof. The invention provides a primer probe composition, which comprises the following components: a primer set and a probe; the primer group is any one of the 1 st to 4 th primer groups, and the base sequence of the probe is shown as SEQ ID NO. 9. The primer probe composition provided by the invention is used for detecting mango allergen by a microdroplet digital PCR method, and has the advantages of rapidness, strong specificity and high sensitivity.
Description
Technical Field
The invention relates to the technical field of detection of mango allergen components, in particular to a microdroplet digital PCR detection primer probe composition of mango allergen components, a detection method and application thereof.
Background
Mango (Mangifera indica L.) belongs to the family of lacquer tree (Anagardiaceae), and mango (Mangifera) is a famous tropical fruit, has aromatic and sweet pulp, rich nutrition value and rich beta-carotene and vitamin C, can be used as fresh food, can be processed into fruit juice, ice cream, jam, preserved fruit foods and the like, and is favored by consumers. Meanwhile, the sensitization characteristic of mangoes is not ignored, and researches indicate that after a part of people eat mangoes, cough, asthma, dyspnea, body itch and uncomfortable abdomen symptoms can occur, and even shock can occur to threaten life when the mangoes are eaten seriously. Cross section investigation of food allergy crowd in China shows that the incidence rate of fruit allergens such as mangoes in 1226 allergy crowd is 4.4%, and the fruits allergens are arranged at the 2 nd position of 10 food allergens to be investigated; the results of the intradermal test of food allergens in 1901 allergic disease patients in Kunming area show that the positive rate of the mango fruit allergen skin test is 42.3%.
The best way to avoid mango allergy in allergic patients is to avoid eating mango or food containing mango components. Therefore, the method is particularly important for the allergen identification management of food, and the food allergen detection technology is one of the important components of monitoring means. The existing food allergen detection technology mainly comprises two types, namely detection of protein of an allergen, wherein an ELISA method is widely applied to food allergen detection in the food industry, the principle is that after the allergen is combined with a specific enzyme-labeled antibody, colorimetric reaction is generated to quantitatively or qualitatively analyze the allergen by detecting the enzyme activity, but no product for detecting mango allergen protein is available at home and abroad at present. The other is aimed at nucleic acid detection of the sensitized substance, the common method is a real-time fluorescence PCR method, the method has the advantages of higher accuracy, sensitivity and the like, and a detection standard of SN/T1961 series of detection of the allergen component of export food by using the method is published in China. The emerging microdroplet digital PCR method is based on the principle of water-in-oil, which distributes a DNA reaction system containing a certain concentration into tens of thousands of microdroplets, and performs statistical analysis on an amplified signal result through single molecule amplification, so that accurate detection of a low-concentration nucleic acid sample can be realized. The microdroplet digital PCR method has the advantages of independent amplification efficiency, no influence of PCR inhibitors, higher sensitivity and repeatability and the like, and has been applied to the fields of food-borne pathogenic bacteria, transgenic plants, animal-borne component detection and the like in recent years. At present, no research report on detecting mango allergen components in food based on a microdroplet digital PCR method exists, and the establishment of the method provides a new thought and technical support for detecting the mango allergen components in food.
Disclosure of Invention
The invention provides a microdroplet digital PCR detection primer probe composition for mango allergen components, a detection method and application thereof, which are used for solving the defect that products for detecting mango allergen proteins are absent in the prior art and realizing accurate and sensitive detection of products for mango allergen components.
The invention provides a primer probe composition, which comprises the following components: a primer set and a probe;
the primer group is any one of the 1 st to 4 th primer groups,
the base sequences of the 1 st group of primers are shown as SEQ ID NO.1 and SEQ ID NO. 2;
the base sequences of the primers in the group 2 are shown as SEQ ID NO.3 and SEQ ID NO. 4;
the base sequences of the 3 rd group primers are shown as SEQ ID NO.5 and SEQ ID NO. 6;
the base sequences of the 4 th group of primers are shown as SEQ ID NO.7 and SEQ ID NO. 8.
The base sequence of the probe is shown as SEQ ID NO. 9. The primer probe composition provided by the invention is used for detecting mango allergen by a microdroplet digital PCR method, and has the advantages of rapidness, strong specificity and high sensitivity.
Preferably, group 2 primers are selected. The primer of the group 2 is matched with the probe of the invention, the Ct value is small, and the amplification sensitivity is very good.
According to the primer probe composition provided by the invention, the 5 '-end of the probe is marked with FAM, and the 3' -end of the probe is marked with BHQ.
The invention also provides a kit comprising the primer probe composition.
The invention also provides a method for detecting mango allergen, which utilizes the primer probe composition or the kit.
According to the method for detecting mango allergens, the mango allergens are detected by using a microdroplet digital PCR method.
According to the method for detecting mango sensitizers, the optimal annealing temperature for the reaction is 51.5-52.5 ℃. At this temperature, non-specific amplification is reduced.
According to the method for detecting mango sensitizers, the reaction procedure is as follows: 95 ℃ for 10min; 45 cycles were performed at 94℃for 30s,55℃for 30s, and 72℃for 30 s; and 98 ℃ for 10min.
According to the method for detecting mango allergen, when the 1 st group of primers is used, the primer use concentration of the 1 st group of primers is 200nmol/L, and the probe use concentration is 100nmol/L;
when the 2 nd group of primers are used, the primer use concentration of the 2 nd group of primers is 200nmol/L, and the probe use concentration is 75nmol/L;
when the 3 rd group of primers are used, the primer use concentration of the 3 rd group of primers is 200nmol/L, and the probe use concentration is 50nmol/L;
when the 4 th primer set was used, the primer concentration of the 4 th primer set was 100nmol/L and the probe concentration was 60nmol/L.
The primer probe composition, the kit or the method for detecting the mango allergen are applied to detection of the mango allergen in mango or food.
According to the application, the mango content in the system template is detected to be not lower than 4mg/kg; preferably, the mango content is 10000-5 mg/kg. The invention can detect the mango template with low concentration.
The invention has the beneficial effects that:
(1) The primer probe composition provided by the invention is used for detecting mango allergen by a microdroplet digital PCR method, and has the advantages of rapidness, strong specificity and high sensitivity.
(2) The invention uses the microdroplet digital PCR method to detect the mango specific gene, and can realize the detection of mango allergen components.
(3) The invention can realize the accurate detection of the low-concentration nucleic acid mango sample.
Drawings
In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a primer probe screen provided by the present invention;
FIG. 2 shows the primer probe specificity verification result provided by the invention;
FIG. 3 is an annealing temperature optimization result of the present invention;
FIG. 4 is a primer probe optimization result of the present invention;
FIG. 5 shows ddPCR amplification results of mango samples of different contents according to the invention;
FIG. 6 shows the amplification results of ddPCR of different samples according to the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, 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.
1. Materials and methods
1.1 Materials and reagents
Plant genome DNA extraction kit, deep processing extraction kit: TIANGEN company, beijing; microdroplet digital PCR reaction system related reagents: the United states Burley company; primers and probes were synthesized by the company Shanghai, inc. of Biotechnology.
1.2 instruments and apparatus
Q×200 droplet digital PCR instrument: the United states Burley company; PCR instrument, fluorescent PCR instrument: siemens Fei Co., USA; high-speed centrifuge, heating mixing appearance: eppendorf, germany; an electronic balance:
1.3 method
1.3.1 sample preparation
Breaking fresh mango into small pieces, placing the small pieces in an oven for drying at 70 ℃ for 4 hours, weighing 100mg of sample, adding 1ml of deionized water into a 2ml centrifuge tube, adding about 10 steel balls, and placing the small pieces on a tissue breaker for breaking. Instrument parameters: frenqueue 30/s; time 2min, total break for 10min, break mango samples into homogeneous liquid. The sample was made into mango samples of different dilutions for mango allergen sensitivity testing.
1.3.1 nucleic acid extraction methods
1.3.1.1 plant sample nucleic acid extraction
The nucleic acid extraction was performed on the plant samples according to the instructions of the extraction kit for plant genomic DNA from TIANGEN company, beijing.
1.3.1.2 nucleic acid extraction methods for food samples
100mg of food sample was weighed, and nucleic acid was extracted according to the instructions using a deep-processed food DNA extraction kit from TIANGEN, beijing.
1.3.2 fluorescent PCR detection method
Preparing a fluorescent PCR reaction system, wherein the system is as follows: 2 Xprobe method fluorescence PCR buffer solution 10 u L, will be the primer and probe diluted to 10 u M, each take 0.5 u L upstream, downstream primer and 0.3 u L probe added system, add 1 u L template, finally with sterilization double distilled water to make up 20 u L. The reaction condition is 95 ℃ for 10min; 45 cycles were performed at 94℃for 30s and 55℃for 1 min. The Ct value of the detection result is automatically determined by software of a fluorescence PCR instrument.
1.3.3ddPCR reaction system and detection method
(1) Preparing a ddPCR system, wherein the system is as follows: 2 Xprobe method ddPCR premix 10. Mu.L, the primers and probes were diluted to 10. Mu.M, 0.5. Mu.L each of the upstream and downstream primers and 0.3. Mu.L probe were added to the system, 1. Mu.L of template was added, and finally 20. Mu.L was made up with sterilized double distilled water.
(2) The well-mixed ddPCR system was transferred to a droplet-generating card, and 70. Mu.L of a droplet-generating dedicated oil was added to the droplet-generating card, which was placed in a droplet generator for reaction. Subsequently, 40. Mu.L of the resulting microdroplet was transferred to a 96-well plate for ddPCR, and the 96-well plate was subjected to membrane sealing by a membrane sealer to prepare for PCR.
(3) Reaction procedure for ddPCR: 95 ℃ for 10min; 45 cycles were performed at 94℃for 30s,55℃for 30s, and 72℃for 30 s; and 98 ℃ for 10min.
(4) After ddPCR, the 96-well plate was placed in a QX200 Droplet Reader instrument, sample information was sequentially entered, and the final result (copy/20. Mu.L) was calculated according to Quantasoft software.
1.3.4 mango allergen specific primer probe design and screening
And selecting chloroplast gene trnH-psbA to design primer probe sequences, and designing 4 pairs of specific primers and 1 probe, wherein the 5 '-end of the probe is marked with FAM, and the 3' -end of the probe is marked with BHQ as shown in table 1. And (3) taking mango DNA as a template, respectively carrying out fluorescent PCR detection with a designed specific probe PM1 (FAM-5'-TGGTACCTTACTTATCAATCTCGAAATG-3' -BHQ1, SEQ ID NO. 9), and screening a mango allergen specific primer probe.
TABLE 1 primer sequences
1.3.5 mango allergen primer probe specificity verification
And selecting mango DNA as a target species, selecting 10 non-target species such as banana, grape, orange, hawthorn, strawberry, peach, apple, apricot, papaya, pear and the like, and performing primer probe specificity verification by using a microdroplet digital PCR method.
1.3.6ddPCR reaction condition optimization
1.3.6.1 primer probe concentration optimization
Setting 4 groups of upstream and downstream primer and probe concentrations to be optimized, as shown in table 2, using mango DNA as a template, performing ddPCR detection on the 4 groups of concentration by using the pair of specific primer probes obtained by screening according to the description of 1.3.2 part, and screening out the optimal primer probe ddPCR reaction concentration according to the result generated by the droplet detector.
TABLE 2 primer sequences
Note that: annealing temperature optimization in nmol/L1.3.6.2
In designing the reaction procedure for ddPCR amplification, the annealing temperature of ddPCR was optimized based on the ddPCR reaction system described in section 1.3.2 using the pair of primer probes and the corresponding concentrations obtained by the above screening with mango DNA as a template. The annealing temperatures were set to be gradient ddPCR at 51, 52, 53, 54, 55, 56℃and the optimum annealing reaction temperature was selected based on the results generated by the droplet detector.
1.3.7 sensitivity and stability verification of ddPCR method
Mango solution is obtained according to the 1.3.1 part, diluted with deionized water in proportion to obtain diluted solution with mango content of 10000, 5000, 1000, 500, 100, 50, 10, 5 and 1mg/kg, DNA is extracted by using the 1.3.1.1 part, detection is carried out by using an established ddPCR method, and each gradient is subjected to 3 times of parallel detection.
1.3.8 actual sample ddPCR detection
10 batches of food samples were purchased from the market, DNA was extracted according to section 1.3.1.2, and the actual samples were tested using the established ddPCR mango allergen test method, and the applicability of the method was verified.
2 results and analysis
2.1 mango allergen specific primer probe design and screening
Screening experiments were performed on 4 pairs of primer probes in table 2 using a fluorescent PCR method, and as can be seen from fig. 1, ct values of the 4 primer probes are respectively: 37.1 The Ct value of the primer probe group 2 is the smallest, 31.0, 36.2 and 35.3, and the amplification sensitivity of the primer probe group is the best, so that the primer probe group is selected to carry out subsequent experiments.
2.2 mango allergen primer probe specificity verification
The method uses mango and other 10 fruit DNAs as templates, uses the bifidobacterium primer probes in the table 1 to carry out ddPCR amplification, and the result is shown in the figure 2, wherein only mango DNA has blue positive oil drop signals, and other 10 fruits are not amplified and negative and are gray oil drop signals, so that the selected mango specific primer probes have good specificity and can meet the detection of mango sensitizers.
2.3ddPCR reaction condition optimization
2.3.1 annealing temperature optimization
As shown in figure 3, the annealing temperatures 51, 52, 53, 54, 55 and 56 ℃ of the ddPCR amplification system are selected for optimization experiments, fluorescent signals are detected in the range of the annealing temperatures, yin-yang droplets at 51-53 ℃ have obvious boundaries, yin-yang droplets at 54-56 ℃ have no obvious boundaries, the amplitude of the yin-yang droplets at 52 ℃ is larger than that of the direct amplitudes of the yin-yang droplets at other amplification temperatures, and the quantity of dispersed droplets among the yin-yang droplets is moderate under the temperature condition, so that ddPCR nonspecific amplification is less, and 52 ℃ is selected as the optimal annealing temperature for reaction.
2.3.2 primer probe concentration optimization
The ddPCR amplification is carried out on the concentration of 4 groups of primer probes by taking mango DNA as a template, and the result is shown in fig. 4, the ddPCR of 4 groups of concentrations has amplified signals, the fluorescence amplitude difference between the yin and yang microdroplets is obvious, and the fluorescence signal amplitude of the 1 st group of concentration is the largest, so that the primer probe concentration which is most suitable for practical application is selected as the 1 st group: 200. 200, 100nmol/L.
2.4 sensitivity and stability verification of ddPCR method
The DNA is extracted by 1.3.1.1 parts of the dilutions of 10000, 5000, 1000, 500, 100, 50, 10, 5 and 1mg/kg mango content, and the established ddPCR method is used for detection, as shown in figure 5, positive and negative oil drops can be detected by the dilutions of 10000-5 mg/kg mango content, the number of positive oil drops generated by ddPCR is reduced along with the reduction of the concentration of the DNA content, and the concentration of the template cannot be estimated by an instrument because the concentration of the template is too low and reaches the lower limit of detection, and no positive oil drops exist in the dilution of 1 mg/kg. As shown in Table 3, the established ddPCR detection method has better stability on the detection result of the diluent with the mango content of 10000-5 mg/kg, CV values of 2.10-12.15 and less than 25 percent, and better repeatability.
TABLE 3 mango ddPCR detection with different concentration gradients
2.5 actual sample ddPCR detection
The established ddPCR method is used for detecting 10 batches of food samples, and the positive oil drop amplification of samples 1-7 is shown by fig. 6, which shows that the samples 8-10 have no positive oil drop amplification, which shows that the samples have no mango allergen components, and the information of the samples in table 4 is consistent, which shows that the method is stable and reliable, and has good application value in the mango allergen of the food samples.
Table 4 food sample information
3. Results and discussion
According to the method, a specific primer probe is designed by taking mango chloroplast trnH-psbA as a target gene, ddPCR reaction conditions are optimized, the specificity, sensitivity and repeatability of the method are examined, the stability of an established microdroplet digital PCR method result is good, the CV value is less than 25%, the detection sensitivity can reach 5ppm, and the method has no cross reaction with 10 other common fruits. The method has the advantages of high speed, strong specificity and high sensitivity, and has certain application value in the detection of mango sensitizers in foods.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A primer probe composition comprising: a primer set and a probe;
the primer group is any one of the 1 st to 4 th primer groups,
the base sequences of the 1 st group of primers are shown as SEQ ID NO.1 and SEQ ID NO. 2;
the base sequences of the primers in the group 2 are shown as SEQ ID NO.3 and SEQ ID NO. 4;
the base sequences of the 3 rd group primers are shown as SEQ ID NO.5 and SEQ ID NO. 6;
the base sequences of the 4 th group of primers are shown as SEQ ID NO.7 and SEQ ID NO. 8;
the base sequence of the probe is shown as SEQ ID NO. 9.
2. The primer probe composition of claim 1, wherein the probe is FAM-labeled at the 5 'end and BHQ-labeled at the 3' end.
3. A kit comprising the primer probe composition of any one of claims 1-2.
4. A method for detecting mango allergens, characterized in that a primer probe composition according to any of the claims 1-2 or a kit according to claim 3 is used.
5. The method for detecting mango allergens according to claim 4, wherein the mango allergens are detected by a digital micro-droplet PCR method.
6. The method for detecting mango allergens according to claim 5, wherein the optimal annealing temperature for the reaction is 51.5-52.5 ℃.
7. The method for detecting mango sensitizers according to claim 5 or 6, wherein the reaction procedure is: 95 ℃ for 10min; 45 cycles were performed at 94℃for 30s,55℃for 30s, and 72℃for 30 s; and 98 ℃ for 10min.
8. The method for detecting mango sensitizers according to any of the claims 4-7, wherein when the 1 st set of primers is used, the 1 st set of primers are used at a concentration of 200nmol/L and the probe is used at a concentration of 100nmol/L;
when the 2 nd group of primers are used, the primer use concentration of the 2 nd group of primers is 200nmol/L, and the probe use concentration is 75nmol/L;
when the 3 rd group of primers are used, the primer use concentration of the 3 rd group of primers is 200nmol/L, and the probe use concentration is 50nmol/L;
when the 4 th primer set was used, the primer concentration of the 4 th primer set was 100nmol/L and the probe concentration was 60nmol/L.
9. Use of the primer probe composition of any one of claims 1-2, the kit of claim 3 or the method of detecting mango allergens of any one of claims 4-8 for detecting mango allergens in mango or food.
10. The use according to claim 9, wherein the mango content in the template of the detection system is not less than 4mg/kg; preferably, the mango content is 10000-5 mg/kg.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102839212A (en) * | 2012-07-18 | 2012-12-26 | 山东出入境检验检疫局检验检疫技术中心 | Kit and detection method for quickly screening mango-derived component in food and beverage |
| CN106596929A (en) * | 2016-12-27 | 2017-04-26 | 斯德润(北京)医疗诊断用品有限公司 | Kit for detecting IgG antibodies of allergens of 30 types of foods and preparation method |
| CN106676189A (en) * | 2017-02-23 | 2017-05-17 | 珠海出入境检验检疫局检验检疫技术中心 | Quantitative detection method of bovine-derived and porcine-derived components based on droplet digital PCR (polymerase chain reaction) as well as primer, probe and kit |
| CN109182471A (en) * | 2018-10-12 | 2019-01-11 | 上海市农业科学院 | A kind of the ddPCR detection method and primer of duck derived component |
| CN111041121A (en) * | 2020-01-08 | 2020-04-21 | 重庆海关技术中心 | Real-time fluorescent PCR (polymerase chain reaction) detection kit for silver fir and products thereof and application |
| JP2020150900A (en) * | 2019-03-22 | 2020-09-24 | 日清食品ホールディングス株式会社 | Primer, and detection method of mango |
| AU2020103596A4 (en) * | 2020-11-22 | 2021-02-04 | Jiangxi Agricultural University | standard gene database and molecular identification method for molecular identification of Coptis chinensis |
| TW202221139A (en) * | 2020-11-27 | 2022-06-01 | 國立中興大學 | Method of detecting mango allergen |
-
2022
- 2022-11-30 CN CN202211525218.XA patent/CN116083625A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102839212A (en) * | 2012-07-18 | 2012-12-26 | 山东出入境检验检疫局检验检疫技术中心 | Kit and detection method for quickly screening mango-derived component in food and beverage |
| CN106596929A (en) * | 2016-12-27 | 2017-04-26 | 斯德润(北京)医疗诊断用品有限公司 | Kit for detecting IgG antibodies of allergens of 30 types of foods and preparation method |
| CN106676189A (en) * | 2017-02-23 | 2017-05-17 | 珠海出入境检验检疫局检验检疫技术中心 | Quantitative detection method of bovine-derived and porcine-derived components based on droplet digital PCR (polymerase chain reaction) as well as primer, probe and kit |
| CN109182471A (en) * | 2018-10-12 | 2019-01-11 | 上海市农业科学院 | A kind of the ddPCR detection method and primer of duck derived component |
| JP2020150900A (en) * | 2019-03-22 | 2020-09-24 | 日清食品ホールディングス株式会社 | Primer, and detection method of mango |
| CN111041121A (en) * | 2020-01-08 | 2020-04-21 | 重庆海关技术中心 | Real-time fluorescent PCR (polymerase chain reaction) detection kit for silver fir and products thereof and application |
| AU2020103596A4 (en) * | 2020-11-22 | 2021-02-04 | Jiangxi Agricultural University | standard gene database and molecular identification method for molecular identification of Coptis chinensis |
| TW202221139A (en) * | 2020-11-27 | 2022-06-01 | 國立中興大學 | Method of detecting mango allergen |
Non-Patent Citations (2)
| Title |
|---|
| M. SANKARAN等: "Morphological, cytological, palynological and molecular characterization of certain Mangifera species，M. Sankaran", 《CURRENT SCIENCE》, vol. 115, no. 7, 10 October 2018 (2018-10-10), pages 1379 - 1386 * |
| 曹会;蔡德丰;许晓璇;杨平常;刘志刚;夏立新;: "儿童芒果果实过敏原研究", 南昌大学学报(医学版), no. 04, 28 August 2017 (2017-08-28), pages 22 - 24 * |
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