CN102839212B - Mango derived component fast screening reagent kit and detection method in food and beverage - Google Patents

Abstract

The invention belongs to the fast screening reagent kit and detection technique of mango derived component in food and beverage, specifically using the ITS2 gene fragments of real-time fluorescence PCR technology for detection mango.The present invention for mango derived component uses real-time fluorescence PCR technology, rapidly, sensitively, specifically detects and includes gene, so as to screen in food and beverage whether derived component containing mango.The fast screening reagent kit and detection technique are used to detect that anaphylactogen mango composition may to be contained in biased sample, can be used for detecting in sample whether do not contain mango composition.This method is quick, high specificity, high sensitivity, easy to operate, reproducible.

Description

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

Technical field

The invention belongs to plant derived component mango DNA detection techniques in food and beverage, specifically using real-time Fluorescence PCR technology is quickly screened by mango progress in food and beverage and the method for detection, extraction of the method including DNA, Real-time fluorescent PCR amplification and result judgement.Its advantage is quick, high specificity, high sensitivity, easy to operate, reproducible.

Background technology

Mango mango, scientific name Mangifera indica Linn., alias：Mango, vast and boundless fruit, vexed fruit, honey are hoped, hope fruit, face Fruit and Buddhist nunnery POLO fruit, belong to Anacardiaceae (Anacardiaceae) Mangifera.Not only pulp is abundant sweet for mango, and contributes to U.S. Hold beauty treatment and fat-reducing, the reputation for having " tropical fruit king " claims.The whole world there are about individual Different Cultivars of Mangifera indica more than 1000, and mango is often processed Into mango juice, food, ice cream, Yoghourt, sugar etc..Sweet and dilitious mango meat is the tropical fruit (tree) being loved by people.Though mango It is so delicious nutritious, but because its property is damp and hot, more foods are not only unhelpful to be harmful on the contrary, and especially some people have allergy to mango Shape, therefore mango is also one of common fruit anaphylactogen.In recent years because the patient of mango allergy is more and more, there is aphtha, Fruit rash, oral cavity fiber crops itch, and there will be severe the symptoms such as asthma and anaphylactic shock.Mango food processing product are being processed place In after reason, the identifiable morphological feature of mango is often lost, is made troubles for mango food authenticity.With mango product and beverage Constantly expand, illegal manufacturer is cost-effective, is adulterated using food addition raw material, pretends to be pure natural fruit juice manufacture fruit Processed goods, upset processing market.In order to protect public's rights and interests, the detection method for establishing reliable mango is extremely urgent.

Whether mango derived component, still neither one quick decision method are mixed with food.Most of consumer demand Judge the method containing mango derived component, food processing of a few consumers due to being free of mango composition to mango allergy demand Product.The quality of life of consumer needs detection method really up to the mark and according to ensureing.

The content of the invention

The purpose of the present invention is to design a group-specific primers for mango ITS2 genes, establishes one kind in food and beverage In quickly screen and detect mango derived component real-time fluorescence PCR detection method, existed with overcoming based on formalness detection method Limitation in food processing products detection, the method for providing quick detection for mango Product checking in food and beverage.

The present invention cardinal principle be：One group of primer pair and probe that can specifically identify mango sequence is designed, it is perfect Experimental system and reaction condition, target DNA accelerated accumulation is made to 10 by real-time fluorescence PCR reaction⁹~10¹⁰Copy, by fluorescence Amplification curve, Ct values differentiate amplification.

The ripe experimental method established in the present invention can ensure to detect mango source property in the fabricated products such as food, beverage Composition.This method eliminates the processes such as agarose electrophoresis, directly can carry out interpretation with fluorescent PCR observation amplification curve, simply And the unit of a lines is quickly detected especially suitable for some.

The kit of quick detection mango derived component includes two kinds of pcr amplification reaction liquid, positive control dna characteristic portions Point；

Reaction system cumulative volume is 25 μ L, wherein containing：Sample DNA (10 μ g/mL-100 μ g/mL) 2 μ L, mango primer pair (10 μm of ol/L) each 0.5 μ L, 1 μ L, Taq archaeal dna polymerase (5U/ μ L) of mango probe (10 μm of ol/L) 0.5 μ l, dNTP (10 μ Mol/L) the μ L of 2 μ L, 10 × PCR buffer solution 2.5, water complement to the μ L of cumulative volume 25.

Also the real-time fluorescent PCR amplification system of commercialization, μ L of reaction premixed liquid Real time Mix 10,10 μ can be used μ L of mol/L sense primers 0.5,10 μm of μ L of ol/L anti-sense primers 0.5,10 μm of μ L of ol/L probes 1 and the μ l of template composition 2, add ddH₂O (sterilizing distilled water) complements to 25 μ l.

The primer and probe of real-time fluorescent PCR amplification detection of the present invention, its sequence are as follows：

(1) sense primer：5’-TCTGAGTTCTCGGTGACGCTTTC-3’；

(2) anti-sense primer：5’-CCGGTCTCTAGGGTCGAAGAGC-3’；

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

In actual applications, the volume ratio of sense primer, anti-sense primer and probe is：1∶1∶2.

Positive control dna is the DNA extract solutions containing mango derived component.

The method of mango composition in real-time PCR detection food processing products, comprise the following steps (1)-(3) successively：

(1) measuring samples DNA extraction

A. 0.1g samples are weighed, are gone in 1.5mL centrifuge tubes.The lysis buffer of 600 μ L preheatings is added, is gently mixed Afterwards, 65 DEG C of water-bath insulation 10min；

B. isometric μ L of phenol/chloroform 600 are added in pipe, abundant mixing of turning upside down, extract 2min；

C.12000g 5min is centrifuged, draws supernatant into a new centrifuge tube, addition and the isometric precipitated liquid of supernatant, is mixed It is even, after room temperature 10min, 12000g centrifugation 5min, supernatant is removed, retains precipitation；

D. 60 μ L RNases are added in precipitation, after 37 DEG C are placed 2min, are fully mixed it with pipette tips, in 37 DEG C of dissolvings Precipitate, 300 μ L buffer solutions are added after 5min, mixing 10 times of turning upside down；

E. centrifugal column is taken out, centrifugal column is placed on 1 2mL sleeve pipe, solution is added in centrifugal column, is placed 2min；

F. centrifugal column and 2mL sleeve pipes one are reinstated into 8000g centrifugation 30sec, discards solution in sleeve pipe, added in centrifugal column 200 μ L washing lotions, 8000g centrifugation 30sec, discard solution；Repeat this step once；

G. 200 μ L70% ethanol are added in centrifugal column, 8000g centrifugation 30sec, discard solution；Repeat this step once；

H.12000g 30sec is centrifuged, removes trace residue solution in centrifugal column；

I. centrifugal column is placed in a new 1.5mL centrifuge tube, it is slow to add 50 μ L elutions in centrifugal column bottom center Fliud flushing, after 37 DEG C are placed 2min, 12000g centrifugations 30sec.Solution in centrifuge tube is the template that can be used as PCR reactions.

(2) PCR is expanded

A. μ L of Real time Mix 10,10 μm of μ L of ol/L sense primers 0.5,10 μm of ol/L are added in amplified reaction pipe μ L of anti-sense primer 0.5,10 μm of μ L of ol/L probes 1, ddH₂O (sterilizing distilled water) complements to 25 μ l, mixes；

B. measuring samples DNA2 μ L (about 200ng) are added in amplified reaction pipe, are mixed；

C. enter performing PCR in fluorescent PCR instrument and react 40-45 circulation, program is：

95 DEG C of 3min of pre-degeneration, 40-45 circulation：95 DEG C of 15s, 60 DEG C of 30s.

(3) result detects

After real-time fluorescence PCR reaction terminates, instrument automatically analyzes data, draws Ct values and amplification curve.Totally see curve Flex point understands that it is obvious for index, and amplification curve entirety collimation is good, and baseline is without the phenomenon that raises up, and side is by Ct value judged results.

Judged according to the Ct values of fluorescence signal, it is positive when the genetic test Ct values of testing sample are more than or equal to 45 Control and blank control result it is normal, then can determine whether not detect mango gene in sample, do not contained in sample mango source property into Point.

When the structural specificity genetic test Ct values of testing sample are less than or equal to 36, positive control and blank control result Normally, then can determine whether, to detect mango gene in sample, to contain mango derived component in sample.

It is less than 45 when detected sample structural specificity genetic test Ct values are more than 36, real-time PCR detection should be reformed, If reforming rear Ct values still less than 45, positive control and blank control result are normal, then can determine whether to detect mango gene in sample； If rear Ct values are reformed more than 45, and positive control and blank control result are normal, then can determine whether not detect mango base in sample Cause.

Brief description of the drawings

The ITS2 genetic fragment real-time fluorescence PCR collection of illustrative plates of the multiple kind mango of Fig. 1.

The real-time fluorescent PCR amplification figure and 2 kinds of unknown concentration mango juice beverages of Fig. 2 difference dilution factor platform awns DNA solutions Amplification figure.

Embodiment

With reference to embodiment, the present invention will be further described.

Embodiment 1

Detected according to following procedure：

(1) extraction to be measured that mango components Sample DNA may be contained

A. 0.1g samples are weighed, are gone in 1.5mL centrifuge tubes.The lysis buffer of 600 μ L preheatings is added, is gently mixed Afterwards, 65 DEG C of water-bath insulation 10min；

B. isometric μ L of phenol/chloroform 600 are added in pipe, abundant mixing of turning upside down, extract 2min；

C.12000g 5min is centrifuged, draws supernatant into a new centrifuge tube, addition and the isometric precipitated liquid of supernatant, is mixed It is even, after room temperature 10min, 12000g centrifugation 5min, supernatant is removed, retains precipitation；

D. 60 μ L RNases are added in precipitation, after 37 DEG C are placed 2min, are fully mixed it with pipette tips, in 37 DEG C of dissolvings Precipitate, 300 μ L buffer solutions are added after 5min, mixing 10 times of turning upside down；

E. centrifugal column is taken out, centrifugal column is placed on 1 2mL sleeve pipe, solution is added in centrifugal column, is placed 2min；

F. centrifugal column and 2mL sleeve pipes one are reinstated into 8000g centrifugation 30sec, discards solution in sleeve pipe, added in centrifugal column 200 μ L washing lotions, 8000g centrifugation 30sec, discard solution；Repeat this step once；

G. 200 μ L70% ethanol are added in centrifugal column, 8000g centrifugation 30sec, discard solution；Repeat this step once；

H.12000g 30sec is centrifuged, removes trace residue solution in centrifugal column；

I. centrifugal column is placed in a new 1.5mL centrifuge tube, it is slow to add 50 μ L elutions in centrifugal column bottom center Fliud flushing, after 37 DEG C are placed 2min, 12000g centrifugations 30sec.Solution in centrifuge tube is the template that can be used as PCR reactions.

(2) PCR of component beverage containing mango amplifications to be measured

A. μ L of Fluorescence PCR premixed liquid Real time Mix 10,10 μm of ol/L upstreams are added in amplified reaction pipe μ L of primer 0.5,10 μm of μ L of ol/L anti-sense primers 0.5,10 μm of μ L of ol/L probes 1, ddH₂O (sterilizing distilled water) complements to 25 μ l, Mix；

B. the μ L (about 200ng) of measuring samples DNA 2 are added in amplified reaction pipe, are mixed；

C. performing PCR reaction is entered in the fluorescent PCR instrument of ABI 7900, program is：

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

(3) result detects

After real-time fluorescence PCR reaction terminates, instrument automatically analyzes data, draws Ct values and amplification curve.Totally see curve Flex point understands that it is obvious for index, and amplification curve entirety collimation is good, and baseline is without the phenomenon that raises up, by Ct value judged results.

Judged according to the Ct values of fluorescence signal, the genetic test Ct values of testing sample are positive right between 20-36 According to normal with blank control result, it is judged as detecting mango gene in sample.

Claims (1)

1. the kit of quick detection mango derived component, including P C R amplification reaction solutions, positive control D N two kinds of features of A Property part：

P C R amplification reaction solutions cumulative volume is 25 μ L, wherein containing：10 μ g/mL-100 μ g/mL of sample DNA 2 μ L, 10 μm of ol/L Mango sense primer has primer each 1 μ L, 10 μm of 1 μ L, 5U/ μ L Taq archaeal dna polymerases of ol/L mango probe 0.5 μ l, 10 μ with The μ L of 2 μ L, 10 × PCR buffer solutions of mol/L dNTP 2.5, water complement to the μ L of cumulative volume 25；

The primer and probe for the real-time fluorescent PCR amplification detection being related to, its sequence are as follows：

(1) sense primer：5’-TCTGAGTTCTCGGTGACGCTTTC-3’；

(2) anti-sense primer：5’-CCGGTCTCTAGGGTCGAAGAGC-3’；

(3) probe sequence：5’-FAM-ATCCTGTCGTGCGGTTGCGTTCTCC-TAMRA-3’；

Positive control dna is the DNA extract solutions containing mango derived component.