CN102839212A - Kit and detection method for quickly screening mango-derived component in food and beverage - Google Patents

Abstract

The invention belongs to a kit and detection technology for quickly screening a mango-derived component in food and beverage. The invention is particularly characterized by using a real-time fluorescent PCR (polymerase chain reaction) technology to detect gene segments of a mango ITS2 gene. By using the real-time fluorescent PCR technology, the gene contained in a mango-derived component can be quickly, sensitively and specifically detected, so that whether the mango-derived component is contained in food and beverage can be screened. The quick screening kit and detection technology can be used for detecting the allergen mango component possibly contained in a compound sample, and can be also used for detecting whether a sample is free of the mango component. The method is quick, high in specificity, high in sensitivity, convenient to operate and good in reproducibility.

Description

Mango derived component fast screening reagent kit and detection method in food and the beverage

Technical field

The invention belongs to plant-derived composition mango DNA detection technology in food and the beverage; The method that specifically is to use the real-time fluorescence PCR reaction technology that mango in food and the beverage is carried out rapid screening and detection, method comprise that extraction, real-time fluorescence PCR amplification and the result of DNA judge.Its advantage be fast, high specificity, highly sensitive, easy and simple to handle, good reproducibility.

Background technology

Mango mango, formal name used at school Mangifera indica Linn., another name: fruit, face fruit and Buddhist nunnery POLO fruit are hoped, hoped to mango, vast and boundless fruit, vexed fruit, honey, belongs to Anacardiaceae (Anacardiaceae) Mangifera.Mango not only pulp is abundant sweet, and helps beautifying face and moistering lotion and fat-reducing, has the reputation of " torrid zone fruit king " to claim.The whole world has more than 1000 mango kind approximately, and mango often is processed to Sucus Mangiferae indicae, food, ice cream, sour milk, sugar etc.The mango meat fragrant and sweet good to eat be the tropical fruit that is loved by the people.Though mango is delicious nutritious, damp and hot because of its property, many foods are not only unhelpful harmful on the contrary, and especially some people has responsive symptom to mango, so mango also is one of common fruit sensibiligen.More and more because of mango patient hypersensitive in recent years, aphtha appears, the fruit rash, the oral cavity fiber crops itch, and symptoms such as asthma and anaphylactic shock appear in serious meeting.Mango food-processing article after carrying out processing treatment in, often lose the identifiable morphological specificity of mango, differentiating for mango food makes troubles.Along with mango product and beverage constantly enlarge, illegal manufacturers adopts food to add raw material and adulterates for practicing thrift cost, pretends to be pure natural fruit juice to make the processing fruits article, upsets processing market.In order to protect public's rights and interests, the detection method of setting up reliable mango is extremely urgent.

Whether be mixed with the mango derived component in the food, still none decision method fast.Most of consumer demand is judged the method that contains the mango derived component, and few consumers is owing to the food-processing article that the irritated demand of mango do not contained the mango composition.Human consumer's quality of life needs detection method really up to the mark and foundation to guarantee.

Summary of the invention

The objective of the invention is to mango ITS2 gene design one group-specific primers; Set up a kind of in food and beverage rapid screening with detect mango derived component real-time fluorescence PCR detection method; To overcome, detect the method that rapid detection is provided for mango product in food and the beverage based on the limitation of formalness detection method in the food-processing product detects.

Cardinal principle of the present invention is: design the one group of primer that can discern the mango sequence specifically to and probe, improve experimental system and reaction conditions, make target DNA be accumulated to 10 fast through the real-time fluorescence PCR reaction ⁹～10 ¹⁰Copy is differentiated amplification through amplified fluorescence curve, Ct value.

The sophisticated experimental technique of setting up among the present invention can guarantee in fabricated products such as food, beverage, to detect the mango derived component.This method has been omitted processes such as agarose electrophoresis, can directly observe amplification curve with fluorescent PCR and carry out interpretation, simply and fast is specially adapted to the unit that some detect a line.

The test kit of rapid detection mango derived component comprises pcr amplification reaction liquid, two kinds of characteristic parts of positive control dna;

The reaction system TV is 25 μ L; Wherein contain: sample DNA (10 μ g/mL-100 μ g/mL) 2 μ L, the mango primer is to (10 μ mol/L) each 0.5 μ L, mango probe (10 μ mol/L) 1 μ L; Taq archaeal dna polymerase (5U/ μ L) 0.5 μ l; DNTP (10 μ mol/L) 2 μ L, 10 * PCR damping fluid, 2.5 μ L, water complement to TV 25 μ L.

Also can adopt commercial real-time fluorescence PCR amplification system, reaction premixed liquid Real time Mix 10 μ L, 10 μ mol/L upstream primers, 0.5 μ L, 10 μ mol/L downstream primers, 0.5 μ L, 10 μ mol/L probes, 1 μ L and template composition 2 μ l add ddH ₂O (sterilization distilled water) complements to 25 μ l.

Primer and probe that the real-time fluorescence PCR augmentation detection that the present invention relates to is used, its sequence is following:

(1) upstream primer: 5 '-TCTGAGTTCTCGGTGACGCTTTC-3 ';

(2) downstream primer: 5 '-CCGGTCTCTAGGGTCGAAGAGC-3 ';

(3) probe sequence: 5 '-FAM-ATCCTGTCGTGCGGTTGCGTTCTCC-TAMRA-3 '.

In practical application, the volume ratio of upstream primer, downstream primer and probe is: 1: 1: 2.

Positive control dna is the DNA extraction liquid that contains the mango derived component.

Real-time fluorescence PCR detects the method for mango composition in the food-processing product, comprises the following steps successively (1)-(3):

(1) extraction of sample DNA to be checked

A. take by weighing the 0.1g sample, go in the 1.5mL centrifuge tube.The lysis buffer that adds 600 μ L preheatings, gently behind the mixing, 65 ℃ of water bath heat preservation 10min;

B. in pipe, add equal-volume phenol/chloroform 600 μ L, the abundant mixing that turns upside down, extracting 2min;

C.12000g centrifugal 5min draws in the new centrifuge tube of supernatant to, adds and the isopyknic precipitated liquid of supernatant, and mixing, after normal temperature was placed 10min, the centrifugal 5min of 12000g removed supernatant, kept deposition;

D. in deposition, add 60 μ L RNA enzymes, 37 ℃ place 2min after, with the rifle head with its abundant mixing, in 37 ℃ of dissolution precipitations, adding 300 μ L damping fluids behind the 5min, mixing 10 times turns upside down;

E. take out centrifugal post, centrifugal post is placed on the sleeve pipe of 1 2mL, solution is joined in the centrifugal post, place 2min;

F. centrifugal post and 2mL sleeve pipe one are reinstated the centrifugal 30sec of 8000g, discard solution in the sleeve pipe, in centrifugal post, add 200 μ L washing lotions, the centrifugal 30sec of 8000g discards solution; Repeat this step once;

G. in centrifugal post, add 200 μ L70% ethanol, the centrifugal 30sec of 8000g discards solution; Repeat this step once;

H.12000g centrifugal 30sec removes trace residue solution in the centrifugal post;

I. centrifugal post is placed in the new 1.5mL centrifuge tube, the central 50 μ L elution buffers that add in centrifugal column bottom, behind 37 ℃ of placement 2min, the centrifugal 30sec of 12000g.Solution in the centrifuge tube promptly can be used as the template of PCR reaction.

(2) pcr amplification

A. in the amplified reaction pipe, add Real time Mix 10 μ L, 10 μ mol/L upstream primers, 0.5 μ L, 10 μ mol/L downstream primers, 0.5 μ L, 10 μ mol/L probes, 1 μ L, ddH ₂O (sterilization distilled water) complements to 25 μ l, mixing;

B. in the amplified reaction pipe, add sample DNA 2 μ L to be checked (about 200ng), mixing;

C. in the fluorescent PCR appearance, carry out 40-45 circulation of PCR reaction, program is:

Preparatory 95 ℃ of 3min of sex change, 40-45 circulation: 95 ℃ of 15s, 60 ℃ of 30s.

(3) result detects

After the real-time fluorescence PCR reaction finished, the automatic analytical data of instrument drew Ct value and amplification curve.See that totally knee point is clear, it is obvious for index, and the whole collimation of amplification curve is good, and baseline does not have the phenomenon of raising up, and the side is by Ct value judged result.

Ct value according to fluorescent signal judges, when the gene test Ct of testing sample value more than or equal to 45, positive control and blank result are normal, then can be judged as and not detect the mango gene in the sample, do not contain the mango derived component in the sample.

Be less than or equal to 36 when the structure specific gene of testing sample detects the Ct value, positive control and blank result are normal, then can be judged as and detect the mango gene in the sample, contain the mango derived component in the sample.

When detected sample structure specific gene detects Ct value greater than 36 less than 45, the real-time fluorescence PCR of should reforming detects, and afterwards the Ct value is still less than 45 if reform, and positive control and blank result are normal, then can be judged as and detect the mango gene in the sample; Back Ct value is greater than 45 if reform, and positive control and blank result are normal, then can be judged as and not detect the mango gene in the sample.

Description of drawings

The ITS2 gene fragment real-time fluorescence PCR collection of illustrative plates of a plurality of kind mango of Fig. 1.

The real-time fluorescence PCR amplification figure of the different extent of dilution platform of Fig. 2 awns dna solution and 2 kinds of unknown concentration mango nectar amplification figure.

Embodiment

Below in conjunction with embodiment the present invention is further specified.

Embodiment 1

Detect according to following program:

(1) extraction that possibly contain mango composition sample DNA to be measured

A. take by weighing the 0.1g sample, go in the 1.5mL centrifuge tube.The lysis buffer that adds 600 μ L preheatings, gently behind the mixing, 65 ℃ of water bath heat preservation 10min;

B. in pipe, add equal-volume phenol/chloroform 600 μ L, the abundant mixing that turns upside down, extracting 2min;

C.12000g centrifugal 5min draws in the new centrifuge tube of supernatant to, adds and the isopyknic precipitated liquid of supernatant, and mixing, after normal temperature was placed 10min, the centrifugal 5min of 12000g removed supernatant, kept deposition;

D. in deposition, add 60 μ L RNA enzymes, 37 ℃ place 2min after, with the rifle head with its abundant mixing, in 37 ℃ of dissolution precipitations, adding 300 μ L damping fluids behind the 5min, mixing 10 times turns upside down;

E. take out centrifugal post, centrifugal post is placed on the sleeve pipe of 1 2mL, solution is joined in the centrifugal post, place 2min;

F. centrifugal post and 2mL sleeve pipe one are reinstated the centrifugal 30sec of 8000g, discard solution in the sleeve pipe, in centrifugal post, add 200 μ L washing lotions, the centrifugal 30sec of 8000g discards solution; Repeat this step once;

G. in centrifugal post, add 200 μ L70% ethanol, the centrifugal 30sec of 8000g discards solution; Repeat this step once;

H.12000g centrifugal 30sec removes trace residue solution in the centrifugal post;

I. centrifugal post is placed in the new 1.5mL centrifuge tube, the central 50 μ L elution buffers that add in centrifugal column bottom, behind 37 ℃ of placement 2min, the centrifugal 30sec of 12000g.Solution in the centrifuge tube promptly can be used as the template of PCR reaction.

(2) the mango composition beverage pcr amplification that contains to be measured

A. in the amplified reaction pipe, add fluorescent PCR reaction premixed liquid Real time Mix 10 μ L, 10 μ mol/L upstream primers, 0.5 μ L, 10 μ mol/L downstream primers, 0.5 μ L, 10 μ mol/L probes, 1 μ L, ddH ₂O (sterilization distilled water) complements to 25 μ l, mixing;

B. in the amplified reaction pipe, add sample DNA 2 μ L to be checked (about 200ng), mixing;

C. in ABI 7900 fluorescent PCR appearance, carry out the PCR reaction, program is:

95 ℃ of 3min, 45 circulations: 95 ℃ of 15s, 60 ℃ of 30s.

(3) result detects

After the real-time fluorescence PCR reaction finished, the automatic analytical data of instrument drew Ct value and amplification curve.See that totally knee point is clear, it is obvious for index, and the whole collimation of amplification curve is good, and baseline does not have the phenomenon of raising up, by Ct value judged result.

Ct value according to fluorescent signal judges that the gene test Ct value of testing sample is between 20-36, and positive control and blank result are normal, are judged as and detect the mango gene in the sample.

Claims (2)

1. the test kit of rapid detection mango derived component comprises P C R amplification reaction solution, two kinds of characteristic parts of positive control D N A:

P C R amplification reaction solution TV is 25 μ L; Wherein contain: sample DNA (10 μ g/mL-100 μ g/mL) 2 μ L, mango primer are to (10 μ mol/L) 1 μ L, mango probe (10 μ mol/L) 1 μ L; Taq archaeal dna polymerase (5U/ μ L) 0.5 μ l; DNTP (10 μ mol/L) 2 μ L, 10 * PCR damping fluid, 2.5 μ L, water complement to TV 25 μ L.Also can adopt commercial real-time fluorescence PCR amplification system, reaction premixed liquid Real time Mix 10 μ L, 10 μ mol/L upstream primers, 0.5 μ L, 10 μ mol/L downstream primers, 0.5 μ L, 10 μ mol/L probes, 1 μ L and template composition 2 μ l add ddH ₂O (sterilization distilled water) complements to 25 μ l.

Primer and probe that the real-time fluorescence PCR augmentation detection that the present invention relates to is used, its sequence is following:

(1) upstream primer: 5 '-TCTGAGTTCTCGGTGACGCTTTC-3 ';

(2) downstream primer: 5 '-CCGGTCTCTAGGGTCGAAGAGC-3 ';

(3) probe sequence: 5 '-FAM-ATCCTGTCGTGCGGTTGCGTTCTCC-TAMRA-3 '.

In practical application, the volume ratio of upstream primer, downstream primer and probe is: 1: 1: 2.

Positive control dna is the DNA extraction liquid that contains the mango derived component.

2. mango derived component method for quick comprises that extraction, real-time fluorescence P C R amplification and the result of food D N A judge.

The food DNA extraction:

Adopt the DNA extraction test kit of phenol-chloroform DNA extraction method or adopting by equation same procedure.

Pcr amplification:

In the fluorescent PCR appearance, carry out 40-45 circulation of PCR reaction, program is:

Preparatory 95 ℃ of 3min of sex change, 40-45 circulation: 95 ℃ of 15s, 60 ℃ of 30s.

The result judges:

After the real-time fluorescence PCR reaction finished, the automatic analytical data of instrument drew Ct value and amplification curve.See that totally knee point is clear, it is obvious for index, and the whole collimation of amplification curve is good, and baseline does not have the phenomenon of raising up, and the side is by Ct value judged result.

When the gene test Ct of testing sample value more than or equal to 45, positive control and blank result are normal, then can be judged as and not detect the mango gene in the sample, do not contain the mango derived component in the sample.

Be less than or equal to 36 when the structure specific gene of testing sample detects the Ct value, positive control and blank result are normal, then can be judged as and detect the mango gene in the sample, contain the mango derived component in the sample.

When detected sample structure specific gene detects Ct value greater than 36 less than 45, the real-time fluorescence PCR of should reforming detects, and afterwards the Ct value is still less than 45 if reform, and positive control and blank result are normal, then can be judged as and detect the mango gene in the sample; Back Ct value is greater than 45 if reform, and positive control and blank result are normal, then can be judged as and not detect the mango gene in the sample.

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