CN102191315B

CN102191315B - Method for detecting provenance of tunny in fish product

Info

Publication number: CN102191315B
Application number: CN 201010150275
Authority: CN
Inventors: 高宏伟; 林超; 孙敏; 刘彩霞; 徐彪
Original assignee: Inspection and Quarantine Technology Center of Shandong Entry Exit Inspection and Quarantine Bureau
Current assignee: Inspection and Quarantine Technology Center of Shandong Entry Exit Inspection and Quarantine Bureau
Priority date: 2010-03-17
Filing date: 2010-03-17
Publication date: 2013-05-08
Anticipated expiration: 2030-03-17
Also published as: CN102191315A

Abstract

The invention belongs to a detection technique for identifying the provenance of tunny in a fish product, in particular to a quick detection method for detecting tunny in food by using a real-time fluorescence polymerase chain reaction (PCR) technique. The method uses PCR amplification reaction solution, an upstream primer Jinqiang230F 5'-CTCCCTCTTTCCTTCTGC-3' and a downstream primer Jinqiang568R 5'-AGGTTGTATTTAGGTTTCG-3'. The tunny quick detection method comprises: extracting DNA from the fish product, performing the real-time fluorescence PCR amplification of a tunny species specific gene and determining the result by using a fusion curve. The method has the advantages of high speed, high specificity, high flexibility and simple and convenient operation.

Description

The detection method of provenance of tunny in fish product

Technical field

The invention belongs to the detection technique that in fish product, source of species is identified, specifically use the method for quick of tuna in real-time fluorescence PCR technology for detection food.

Background technology

The large-scale fish in the ocean of marine fishing are the food that the people of other countries like always, are also the important goods kinds of international trade.The large-scale fish in ocean usually comprise in trade is processed into fish meat sheet or flesh of fish piece, more comprises the internal organ of some fishes or the product of other deep processings.Therefore the raw material fingerling class of these products from not found out to process in appearance need to not be subjected to the restriction of outward appearance and complete processing to confirm the true and false of commodity.

Can carry out species from gene level based on the detection method of DNA identifies.The advantages such as the real-time fluorescence PCR method is easy and simple to handle, sensitive, special, quick with it, good reproducibility, quantitatively accurate, totally-enclosed reaction obtain investigator's generally approval, become the important tool of gene test and evaluation.Also not open to the authentication method of tuna species both at home and abroad at present, the present invention can make up defects, completes the evaluation of provenance of tunny in fish product.

Summary of the invention

The invention belongs to the detection technique that in fish product, source of species is identified, specifically use the method for quick of tuna in real-time fluorescence PCR technology for detection food.Overcome the evaluation difficulty of bringing based on morphologic detection method in the fish products course of processing, for the provenance of determining tuna provides technique means, thereby guarantee the consistence of food labelling.

Cardinal principle of the present invention is: design one group of primer for conserved sequence: upstream primer Jinqiang230F5 '-CTCCCTCTTTCCTTCTGC-3 '; Jinqiang568R:5 '-AGGTTGTATTTAGGTTTCG-3 '.utilize the lysate smudge cells, trichloromethane extracting protein, isopropanol precipitating obtains DNA, carry out pcr amplification take the DNA that extracts as template, when measuring melting curve, add fluorescence dye Eva Green in reaction system, dyestuff and DNA double chain combination are also sent fluorescence, owing to there being a small amount of non-specific polymer (as primer dimer etc.) in reaction, can cause interference, but non-specific polymeric melting temperature (Tm) is low than specific binding, after amplification finishes, slowly heat again, after the DNA sex change, dyestuff is released, fluorescence reduces, and non-specific binding reduces prior to specific binding, fusion processes is carried out continuous dynamic monitoring can get melting curve, by curve, non-specific signal and specificity signal region are branched away, simultaneously to the specificity signal region, the speed that fluorescence reduces is directly proportional to the logarithm of concentration, can determine whether the primer specificity amplified production with this.

The tuna provenance method for quick that the present invention relates to, reagent wherein comprises as follows:

(1) pcr amplification reaction liquid

Comprise 10 * PCR reaction buffer, 0.1-0.4mmol/L dNTP, 2-4mmol/L sal epsom, 1-2U Taq enzyme, 0.2-0.6 μ mol/L upstream primer, 0.2-0.6 μ mol/L downstream primer, 1.25 μ L20 * Eva Green dyestuff (U.S. Biotum company);

Wherein 10 * PCR reaction buffer contains trihydroxy methyl aminomethane-hydrochloric acid, 500mmol/L Repone K and 1% triton x-100 of 100mmol/LpH8.8;

Upstream primer Jinqiang230F 5 '-CTCCCTCTTTCCTTCTGC-3 '; Jinqiang568R:5 '-AGGTTGTATTTAGGTTTCG-3 '.Wherein upstream primer, downstream primer volume ratio are: 1: 1;

Use the mentioned reagent box to detect the method for tuna provenance in food, comprise the following steps successively (1)-(3):

(1) extraction of sample DNA to be checked

DNA extraction can adopt common phenol-chloroform extraction process or use the DNA extraction test kit.The DNA extraction quality is used OD ₂₆₀And OD ₂₈₀Photoabsorption weigh, perhaps use additive method to weigh.

(2) real-time fluorescence PCR of tuna provenance amplification

A. add 1 μ L sample DNA to be checked, mixing in the reaction tubes that 24 μ L pcr amplification reaction liquid A are housed.

B. establish respectively positive control, blank in testing process.Use has the positive criteria material of traceability as positive control, makes negative control with the known sample that does not contain the fish composition, replaces template DNA to make blank with isopyknic water.

C. the PCR reaction tubes is put into quantitative real time PCR Instrument, completes pcr amplification by following reaction conditions:

94-95 ℃ of 2-4min, 1 circulation;

94-95 ℃ of 10-60s, 61 ℃ of 10-60s, totally 40 circulations;

95 ℃ of 15s, 60 ℃ of 15s are warming up to 95 ℃ in 20min, 95 ℃ of 15s.

(3) use melting curve analysis pcr amplification result.

The peak value of tuna amplified production solubility curve 83 ± 0.5 ℃ and 89 ± 0.5 ℃ of appearance bimodal.

The present invention has set up real-time fluorescence PCR detection method and the detection method of tuna provenance.The present invention adopts the real-time fluorescence PCR technology, and this technology high specificity, highly sensitive, easy and simple to handle is specially adapted to the detection of the provenance of tunny in fish product of some complicated components.

Description of drawings

Fig. 1 is the melting curve figure of tuna sample P CR product in embodiment 1.

Fig. 2 is the melting curve figure of tuna sample P CR product in embodiment 2.

Embodiment

The present invention will be further described below in conjunction with embodiment.

Embodiment 1

Detect as follows commercially available tuna:

Comprise 10 * PCR reaction buffer, 0.2mmol/L dNTP, 2mmol/L sal epsom 1.5U Taq enzyme, 0.4 μ mol/L upstream primer Jinqiang230F:5 '-CTCCCTCTTTCCTTCTGC-3 '; Jinqiang568R:5 '-AGGTTGTATTTAGGTTTCG-3 '.Wherein 10 * PCR reaction buffer contains trihydroxy methyl aminomethane-hydrochloric acid, 500mmol/L Repone K and 1% triton x-100 of 100mmol/LpH8.8;

Detect according to following program:

(1) extraction of flesh of fish sample DNA to be measured

A. take the 0.5g flesh of fish, shred, go in the 1.5mL centrifuge tube.Add 500 μ L extracting lysis buffers (0.05M Tris-HCL (trihydroxy methyl aminomethane-hydrochloric acid), 0.1M EDTA (ethylenediamine tetraacetic acid (EDTA)), 0.2M sodium-chlor, pH value 7.6 sterilization distilled waters), fully vibration;

B. add Proteinase K 60 μ L, 10%SDS 2 μ L put upside down mixing.57 ℃ of water-bath 10h are clear liquid to raw material digestion, and the centrifugal 2min of 4000rpm gets supernatant;

C. add the 500 isopyknic Tris-phenol of μ L, put upside down mixing 10min, the centrifugal 10min of 12000rpm gets supernatant;

D. repeat the step;

E. add Tris-phenol, chloroform and primary isoamyl alcohol (volume ratio is 25: 24: 1) in supernatant liquor, put upside down mixing 10min, the centrifugal 10min of 12000rpm gets supernatant;

F. add chloroform and primary isoamyl alcohol (volume ratio is 24: 1) in supernatant liquor, put upside down mixing 10min, the centrifugal 10min of 12000rpm gets supernatant;

G. add the two volumes dehydrated alcohol, the sodium-acetate of 1/5 volume (4M) is put upside down mixing 10min, precipitation DNA; The centrifugal 10min of 12000rpm abandons supernatant;

H. add 600 μ L ethanol (70%) washing DNA, the centrifugal supernatant of abandoning is uncapped, and dries;

I. add 100 μ L sterilization distilled water dissolving DNAs.

J.DNA extracts quality: purity OD ₂₆₀/ OD ₂₈₀=1.83, concentration is 101ng/ μ L.Satisfy the condition of PCR reaction.

(2) real-time fluorescence PCR of fillet DNA to be measured amplification

A. add 24 μ L PCR reaction solution A and 1 μ L sample DNA in the PCR reaction tubes, mixing.

B. the PCR reaction tubes is put into quantitative real time PCR Instrument, completes pcr amplification by following reaction conditions:

94-95 ℃ of 2-4min, 1 circulation;

94-95 ℃ of 10-60s, 61 ℃ of 10-60s, totally 40 circulations;

95 ℃ of 15s, 60 ℃ of 15s are warming up to 95 ℃ in 20min, 95 ℃ of 15s.

(3) use the quantitative real time PCR Instrument accompanying software, analyze the melting curve result.

Non-tuna sample: melting curve at 81 ± 1 ℃ without simple spike; The tuna sample: the peak value of product solubility curve 83 ± 0.5 ℃ and 89 ± 0.5 ℃ of appearance bimodal.Bimodal in figure is the melting curve of tuna positive criteria product, sees Fig. 1.

Embodiment 2

Whether detect as follows commercially available fish viscera is the tuna source:

Comprise 10 * PCR reaction buffer, 0.2mmol/L dNTP, 2mmol/L sal epsom 1.5U Taq enzyme, 0.4 μ mol/L upstream primer Jinqiang230F:5 '-CTCCCTCTTTCCTTCTGC-3 '; Jinqiang568R:5 '-AGGTTGTATTTAGGTTTCG-3 '.Wherein 10 * PCR reaction buffer contains trihydroxy methyl aminomethane-hydrochloric acid, 500mmol/L Repone K and 1% triton x-100 of 100mmol/L pH8.8;

Detect according to following program:

(1) extraction of fish viscera sample DNA to be measured

A. take the 0.5g fish viscera, shred, go in the 1.5mL centrifuge tube.Add 500 μ L extracting lysis buffers (0.05M Tris-HCL (trihydroxy methyl aminomethane-hydrochloric acid), 0.1M EDTA (ethylenediamine tetraacetic acid (EDTA)), 0.2M sodium-chlor, pH value 7.6 sterilization distilled waters), fully vibration;

D. repeat the step;

I. add 100 μ L sterilization distilled water dissolving DNAs.

J.DNA extracts quality: purity OD ₂₆₀/ OD ₂₈₀=1.87, concentration is 127ng/ μ L.Satisfy the condition of PCR reaction.

(2) real-time fluorescence PCR of fillet DNA to be measured amplification

94-95 ℃ of 2-4min, 1 circulation;

94-95 ℃ of 10-60s, 61 ℃ of 10-60s, totally 40 circulations;

95 ℃ of 15s, 60 ℃ of 15s are warming up to 95 ℃ in 20min, 95 ℃ of 15s.

The melting curve of sample not 83 ± 0.5 ℃ and 89 ± 0.5 ℃ of appearance bimodal.Can judge that accordingly this sample is not the tuna source.Simple spike is respectively the melting curve of tuna positive criteria product.The line that does not go out the peak is sample to be checked and blank, sees Fig. 2.

The nucleotides sequence list

＜110〉the super Liu's Sun Min rosy clouds of high grand woods Xu Biao

＜120〉detection method of provenance of tunny in fish product

<160>2

<210>1

<211>18

<212>DNA

＜213〉artificial sequence

<221>prim_bind

<222>(1)…(18)

<400>1

ctccctctttccttctgc 18

<210>2

<211>19

<212>DNA

＜213〉artificial sequence

<221>prim_bind

<222>(1)…(19)

<400>2

aggttgtatttaggtttcg 19

Claims

1. the method for quick identified of tuna species is characterized in that primer wherein:

(1) upstream primer Jinqiang230F 5 '-CTCCCTCTTTCCTTCTGC-3 ';

(2) downstream primer Jinqiang568R:5 '-AGGTTGTATTTAGGTTTCG-3 ';

The method comprises the following steps:

(1) DNA extraction of sample to be checked;

(2) real-time fluorescence PCR of tuna CO I gene amplification:

Add the sample DNA to be checked of 1 μ L, mixing in the reaction tubes of the pcr amplification reaction liquid that 24 μ L are housed;

Complete pcr amplification according to following step:

94-95 ℃ of 2-4min, 1 circulation;

94-95 ℃ of 10-60s, 61 ℃ of 10-60s, totally 40 circulations;

95 ℃ of 15s, 60 ℃ of 15s are warming up to 95 ℃ in 20min, 95 ℃ of 15s;

(3) use melting curve analysis PCR product;

The peak value of tuna amplified production melting curve 83 ± 0.5 ℃ and 89 ± 0.5 ℃ of appearance bimodal.

2. a right to use requires 1 described detection method whether to contain the application of tuna composition in detecting food.