CN103114142B - Real-time polymerase chain reaction (PCR) detection primer, kit and detection method for detecting puffer fish ingredients - Google Patents
Real-time polymerase chain reaction (PCR) detection primer, kit and detection method for detecting puffer fish ingredients Download PDFInfo
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Abstract
The invention discloses a real-time polymerase chain reaction (PCR) detection primer, a kit and a detection method for detecting puffer fish ingredients. The puffer fish gene detection primer and probes with the sequences of SEQ ID NO. 1-3 are designed according to the specific gene sequence of puffer fish, and the real-time fluorescent PCR method is adopted to qualitatively detect the puffer fish ingredients of food. The developed real-time fluorescent PCR qualitative detection method for detecting the puffer fish ingredients in food has importance significance in improving the detection efficiency and sensitivity of the puffer fish ingredients in food, lowering the detection cost, detecting related fake products on the market, monitoring food safety and improving a detection technology, and has wide development prospect.
Description
Technical field
The invention belongs to technical field of molecular biology, be specifically related to Puffer fish ingredient in food and detect PCR in real time detection primer, test kit and detection method.
Background technology
Fishery products are important component parts of world economy and trade, are also important food protein sources.Edible fishery products are of a great variety, and the principal item of international trade has 25 large populations, the kind more than 1700 of just having an appointment on the edible fishery products list that only FDA (Food and Drug Adminstration) lists.Along with the development of trade and the raising of level of processing, the variation of particular types product relation between supply and demand, part businessman sells with the high value fishery products of cheap aquatic product personation, obtain economic interests improperly, as by the trout personation Atlantic salmon of cultivation, tilapia, Mahi loach fish are palmed off to red porgy etc.Also once had filefish fillet were sneaked into the case that safe and comfortable fillet cause eater to be poisoned to death.Carried out by American National Instrument Seafood Inspection Laboratory one by a definite date the investigation result of 9 years show: the incorrect mark of species that has other marine food of 37% fish products and 13%.The market survey that recently carry out Europe and North America shows, fishery products raw material error identification rate is at 15-43%, and the error identification rate of red porgy product is unexpectedly up to 75%.Except financial loss, the behavior also frequently faces extinct species, food safety, economic order to protection and brings serious harm.
The species authenticity of aquatic products processing product has become the major issue facing in fish processing industry at present.European Union has set up labeling acts, forces that manufacturer provides that fishery products species prove, the information such as the place of production and production method; The U.S., according to the regulation of the federal bill of food and medicine makeup, forbids the counterfeit behavior of fishery products; Canadian Food Inspection Agency and importor attempt some Imported sea-food to carry out DNA inspection in December, 2011.This detection is mainly for the economic fraud conditions of pretending to be high value fish with Optimization of Low Value Fish.
In order to prevent the generation of economic fraud, guarantee food safety, be necessary to identify for the raw material true and false of aquatic products processing product.In the time that fishery products are complete without processing treatment, outward appearance, conventionally can judge by morphological feature the true and false of kind.As fish gruel, seasoned, sashimi, can, fish meal etc., need to set up the authentication method based on DNA analysis for aquatic products processing product.After globe fish and anglerfish are processed into fillet or fish mud, be difficult to be distinguished form.In order to ensure edible safety, prevent edible globefish poisoning, be necessary to select suitable gene fragment, set up practical, feasible qualification real-time PCR method.
China's Puffer fish ingredient detection method in food is still a blank at present, has a lot of difficulties at aspects such as method for guaranteeing stability, specificitys, and this is also the bottleneck that restriction method is set up.
In view of fishery products raw material authenticity safeguarding fair trading, ensure food safety, protect the importance of the aspects such as endangered species, a lot of researchists have all launched further investigation to this field, have set up a series of method and have been applied.EspineiraM etc. adopt FINS method qualification porgy, tilapia, mackerel, angler, flatfish, the salmon of PCR order-checking.The plan of DNA barcode is the important component part in FINS technology, and this plan is to utilize one section of sequence (COI) that is known as DNA barcode check order by PCR and analyze a global project of species standard of perfection.Up to the present, existing 93% fresh-water fishes species and 98% marine fish species can accurately be differentiated by the method.Rasmussen R S, Rea S and Hsieh C H utilize respectively PCR-RFLP to carry out the qualification of the marine foods such as alec, the filefish flesh of fish, salmon and trout.Rehbein H etc. utilizes PCR-SSCP method to carry out the qualification of the multiple fishes such as salmon, sardines, eel, tuna, stripped tuna, sturgeon.Lowenstein J H etc. adopt RAPD method for genetic analysis and qualifications such as catfish.There is recently report for DNA barcode, utilize species specificity multiplex PCR successfully to identify shark, flatfish, stripped tuna, mackerel, salmon and trout.Taylor etc. have set up multiple Real-time PCR method according to ATPase6 and ATPase8 gene, are used for identifying three kinds of cods with important commercial value.In addition, Rasmussen, Itoi S and Lopez I adopt respectively the qualification of TaqMan MGB probe for eel, tuna, salmon and trout, and Bayha K Mort etc. adopt the qualification of TaqMan LNA probe for porgy and U.S. snapper.2004, France took the lead in developing the DNA chip FoodExpert-ID system for food and animal-feed detection, can analyze simultaneously and exceed tens of kinds of fresh or finished vertebratess, comprises 15 kinds of fish.Chisholm J etc. compares FoodExpert-ID system and Real-timePCR, shows to have good suitability.Kochzius etc. have set up a kind of DNA chip for 11 kinds of important import fish of European Union.
Every kind of method has advantage and deficiency separately.At speed and cost, this does not have advantage aspect two to PCR sequencing.The analytical procedure of PCR-RFLP method is many, quite time-consuming.The highly sensitive of SSCP requires the very repeatability of high level, and experiment condition is each time without difference.The main drawback of PCR-RAPD is the repeatability of the method, and when especially the amount of target dna is degraded less or slightly, this shortcoming is especially outstanding.AFLP method is relatively time-consuming, there is no large-scale application.DNA chip compared with other method, the relatively slow and high cost of sample analysis.Real-time PCR method has cost feature low, easy and simple to handle, is specially adapted to the qualification to biased sample, is most widely used.But the method can only be analyzed species with a pair of specific primer, in versatility, is very limited.
Summary of the invention
In order to solve above-mentioned practical problems, the present invention has set up Puffer fish ingredient in a kind of food and has detected PCR in real time detection primer, test kit and detection method.Extract sample DNA, taking DNA as template, adopt respectively specific detection primer and probe to carry out real-time fluorescence PCR amplification, directly read the amplification phenomenon of real-time fluorescence PCR, can identify the Puffer fish ingredient in food.
An aspect of of the present present invention is: disclose a kind of Puffer fish ingredient and detect with primer and probe, it comprises base sequence SEQ IDNO.1 ~ 3.
The present invention sets up the distinguishing method between true and false to globe fish species, the present invention has used database (the gene databanks of European Molecular Bioglogy Laboratory, EMBL) and BOL, FishTrace reference sequence database, wherein data between the kind of abundant Puffer fish ingredient have been compared.In some candidate gene fragments, select globe fish cytochrome b gene fragment to design primer and probe, set up the method for real-time PCR method specific detection globe fish DNA, realize the real and fake discrimination to species attribute.Detect with primer and probe for globe fish cytochrome b gene fragment gene sequences Design, this gene has very high species specificity.Adopt DNAMAN8.0 software, designed accordingly the present invention for detection of detection primer and the probe of Puffer fish ingredient, sequence information is in table 1.The consistence of the annealing temperature to primer and probe in the design of primer, the factors such as the similarity of GC content have been carried out sufficient consideration, and primer and probe are synthetic by precious biotechnology (Dalian) company limited.
Table 1 globe fish specific detection primer and probe sequence
5'FAM in table, 3'ECLIPSE is double-tagging fluorescent probe, FAM(Carboxyfluorescein, Fluoresceincarboxylic acid); Another aspect of the present invention is: a kind of Puffer fish ingredient detection kit, it comprises that Puffer fish ingredient mentioned above detects with primer and probe.
In this test kit, can also comprise positive control, negative control, blank.Wherein, the DNA that positive control can select Puffer fish ingredient to extract, negative control can be selected the DNA of the sample extraction that does not contain Puffer fish ingredient, and blank can be selected aqua sterilisa, also can every group two or more parallel reaction systems be set.
Another aspect of the present invention is: a kind of real-time fluorescence PCR detection method of Puffer fish ingredient, it utilizes primer mentioned above and probe, and sample DNA is carried out to real time fluorescent PCR method detection.
For the real-time fluorescence PCR detection method of Puffer fish ingredient mentioned above, described real time fluorescent PCR method is preferably selected as follows:
Reaction system cumulative volume is 25 μ L, wherein: template DNA (10 ~ 100 μ g/mL) 2 μ L, the each 1 μ L of upstream and downstream primer (10 μ mol/L), probe (5 μ mol/L) 1 μ L, Taq archaeal dna polymerase (5U/ μ L) 0.5 μ L, dNTP(10 μ mol/L) 1 μ L, 10 × PCR damping fluid, 2.5 μ L, water 16 μ L.
Reaction conditions is as follows: 95 DEG C/10s, and 1 circulation; 95 DEG C/5s, 52 DEG C/10s, 72 DEG C/34s, 40 circulations.
The quality control standard that judges of its amplification is: when following condition has one not meet, it is invalid that experiment is considered as:
(a) blank: without fluorescence logarithmic growth, corresponding Ct value > 40.0.
(b) negative control: without fluorescence logarithmic growth, corresponding Ct value > 40.0.
(c) positive control: have fluorescence logarithmic growth, and corresponding Ct value < 30.0 appears typical amplification curve, in fluorescence channel.
Result is judged: consistent to the standard of result judgement with the real-time fluorescence PCR GB such as species identification and detection GB, transgenosis detection, this experiment is in the situation that meeting quality control, when test sample detects: 40 circulations are set equally, and Ct value is cycle number.If Ct value≤35.0, show that sample, in the amplification through in 35 circulations, detects amplification curve, be judged to be the test sample positive.As Ct value >=40.0, show that sample, through 40 cyclic amplifications, does not still have amplification curve, be judged to be test sample feminine gender.As 35.0 < Ct value < 40.0, between this, result is suspicious result, repeats once.If Ct value after again increasing is still < 40.0, judge the test sample positive; As Ct value >=40.0 after again increasing, judge test sample feminine gender.
In the real-time fluorescence PCR detection method of Puffer fish ingredient mentioned above, the routine techniques that the preparation method of described sample DNA grasps for those skilled in the art, the difference of state per sample, for example liquid sample, solid sample and take different extracting method, not only can select the obtainable DNA extraction test kit of commercial sources, can also use CTAB method, alkaline lysis etc., concrete operations can also be carried out according to the method described in the embodiment of the present invention 1.
For extract DNA concentration and the mensuration of purity: get the DNA solution thin up that 3. step obtains, to concentration be 10 μ g/mL ~ 100 μ g/mL, A260/A280 ratio is between 1.7 ~ 1.9, for subsequent use.
By above technical scheme, good primer and the probe of specificity that the present invention not only provides a set of Puffer F/Puffer R primer and Puffer P probe to be suitable for the qualification to globe fish species, has also set up the qualitative checking method to Puffer fish ingredient false distinguishing.Must be beneficial to carrying out of qualification Puffer fish ingredient food true and false work, be with a wide range of applications, can also play a positive role to the international trade of China's relevant food.
Beneficial effect:
(1) solved the problem of Puffer fish ingredient context of detection in food.Set up the method that Puffer fish ingredient is discerned the false from the genuine that detects.
(2) proved by a large amount of result of practical application, the inventive method detects Puffer fish ingredient in food and has fine suitability.
(3) the present invention adopts real-time fluorescence detection system, can Real-Time Monitoring reaction process, and without electrophoresis, stopped pipe operation, prevents from polluting, and can judge fast result.
(4) the present invention not only can be applied to the correct label detection to fishery products, can also protect frequently endanger species and food safety, safeguards normal economic order.Carry out the real-time PCR method of specific detection, this standard succeed in developing and apply detection efficiency and the sensitivity to improving globe fish species, reduction testing cost, carries out food safety monitoring and raising inspection technology is significant, is with a wide range of applications.
Brief description of the drawings
The real-time fluorescence PCR detected result figure that Fig. 1 globe fish species specificity detects; Wherein, ordinate zou fluorescence intensity Delta Rn, X-coordinate is cycle number.1 ~ 9: the DNA sample of corresponding 9 kinds of globe fishes (Lagocephalus inermis, Lagocephalus lagocephalus, Takifugu fasciatus, Takifugu xanthopterus, Takifugu oblongus, Takifugu rubripes, Gastrophysusgloveri, Gastrophysus lunaris, Gastrophysus spadiceus), through PCR in real time amplification, all obtains positive amplification curve.
The sensitivity detected result of Fig. 2 PCR in real time amplification globe fish cytochrome b DNA fragmentation; Wherein: 1:100% filefish fish meal 2:10% filefish fish meal 3:5% filefish fish meal 4:1% filefish fish meal 5:0.5% filefish fish meal 6:0.1% filefish fish meal 7:0.01% filefish fish meal.
Mark for result shown in Fig. 1 and 2: the curve that detects sample in accompanying drawing is colour, presets different colours and represents corresponding testing sample before experiment, when judgment experiment result, can be according to color differentiating, and know whether counter sample obtains curve amplification.
Embodiment
Following non-limiting example can make the present invention of those of ordinary skill in the art's comprehend, but does not limit the present invention in any way.
In checkout procedure, for ensureing the credible of experimental result, establish respectively positive control, negative control, blank group.The positive contrast of DNA of extracting with globe fish, with the negative contrast of DNA of the known sample extraction that does not contain Puffer fish ingredient, taking aqua sterilisa as blank, every group arranges two parallel reaction systems.
1.DNA extracts
Following DNA extraction method, all reagent and solution are conventional route preparation or are obtained by commercial sources.
Sample DNA extracts the high salt method adopting after improvement.Sample adds liquid nitrogen grinding, get sample 150mg after grinding in the centrifuge tube of 1.5mL, the TE(that adds respectively 400 μ L is containing 10mmol/L Tris-HCl and 1mmol/L EDTA), the Proteinase K of 40 μ L10%SDS and 8 μ L20mg/mL, vortex 30s, fully mixes; The constant temperature water bath that centrifuge tube is placed in to 55 ~ 65 DEG C digests 4h, adds the NaCl solution 300 μ L that 6mol/L is saturated, high speed vortex 30s, the centrifugal 30min of 12000r/min; Get supernatant liquor to new 1.5mL centrifuge tube, add isopyknic phenol/chloroform/primary isoamyl alcohol (25:24:1), slowly shake 1min, the centrifugal 10min of 12000r/min; Get supernatant liquor to new centrifuge tube, add chloroform/primary isoamyl alcohol (24:1) and slowly shake to oyster white, the centrifugal 10min of 12000r/min; Get supernatant liquor to new centrifuge tube, add the 3mol/L NaAc of 1/10 volume, isopyknic Virahol, ice bath 30min, the centrifugal 10min of 12000r/min; Then add 800 μ L70% ethanol to wash, discard ethanol, in the baking oven of 56 DEG C, dry, then add 50 μ L TE, dissolving DNA precipitation, preserves at-20 DEG C.
The mensuration of DNA concentration and purity:
Get appropriate DNA solution stoste and add after distilled water dilution certain multiple, use nucleic acid-protein analyser or ultraviolet spectrophotometer to survey the absorption value at 260nm and 280nm place.When concentration is 10 μ g/mL～100 μ g/mL, A260/A280 ratio between 1.7 ~ 1.9 time, is suitable for real-time fluorescence PCR amplification.
2. real-time fluorescence PCR detects
Reaction system cumulative volume is 25 μ L, wherein: template DNA (10 ~ 100 μ g/mL) 2 μ L, the each 1 μ L of upstream and downstream primer (10 μ mol/L), probe (5 μ mol/L) 1 μ L, Taq archaeal dna polymerase (5U/ μ L) 0.5 μ L, dNTP(10 μ mol/L) 1 μ L, 10 × PCR damping fluid, 2.5 μ L, water 16 μ L.
Reaction conditions is as follows: 95 DEG C/10s, and 1 circulation; 95 DEG C/5s, 52 DEG C/10s, 72 DEG C/34s, 40 circulations.
According to above-mentioned system, respectively with primer shown in table 1 and probe, the sample composition DNA extracting respectively, negative control and blank carry out real-time fluorescence PCR reaction.
Detecting instrument: ABI7500 real-time fluorescence PCR instrument, American AB I company; Experimental result is with collection of illustrative plates formal output in brief description of the drawings.
Detect according to the method described above sample, real-time fluorescence PCR detected result:
Positive controls, through PCR in real time amplification, all obtains positive amplification curve, and the detected result of negative control and the contrast of blank water is all negative.
1. the PCR in real time specific amplification of globe fish cytochrome b gene fragment:
Get 42 kinds of sample DNAs listed in table 2, use Puffer F/Puffer R primer and Puffer P probe to carry out PCR in real time detection.The PCR in real time amplification collection of illustrative plates of globe fish specific detection as shown in Figure 1.
Sample described in the embodiment of the present invention and source: 3 kinds of anglerfish (Lophius litulon, Lophiomus setigerus, Lophius americanus), 9 kinds of globe fish (Lagocephalus inermis, Lagocephalus lagocephalus, Takifugufasciatus, Takifugu xanthopterus, Takifugu oblongus, Takifugu rubripes, Gastrophysus gloveri, Gastrophysus lunaris, Gastrophysus spadiceus, and other 24 kinds of fish samples, 1 kind of octopus (Octopus), 1 kind of cod crab (Chionoecetes bairdi), 2 kinds of shrimps, 2 kinds of shellfish samples, add up to 42 kinds of samples.Concrete sample title and source are in table 2.
Table 2 sample title and source
| Sequence number | Sample title | Sequence number | Sample |
| 1 | Lophius |
22 | SHISHAMO |
| 2 | Lophiomus setigerus | 23 | Sardines |
| 3 | Lophius americanus | 24 | Oncorhynchi |
| 4 | Lagocephalus inermis | 25 | Large |
| 5 | Lagocephalus lagocephalus | 26 | Small |
| 6 | Takifugu fasciatus | 27 | Salmon |
| 7 | Takifugu xanthopterus | 28 | Flatfish |
| 8 | Takifugu oblongus | 29 | Carp |
| 9 | Takifugu rubripes | 30 | Crucian |
| 10 | Gastroph ysus gloveri | 31 | Mandarin |
| 11 | Gastrophysus lunaris | 32 | |
| 12 | Gastrophysus spadiceus | 33 | Sole |
| 13 | Freeze |
34 | |
| 14 | Freeze southern |
35 | |
| 15 | Freeze |
36 | |
| 16 | Chub mackerel |
37 | Octopus |
| 17 | |
38 | Chionoecetes bairdi |
| 18 | Willow leaf like |
39 | Pandalus borealis |
| 19 | Tuna | 40 | |
| 20 | Salmon | 41 | Variegated |
| 21 | Anchovies fish | 42 | Patinopecten yessoensis |
2. test-results shows: the DNA sample of 9 kinds of globe fishes (Lagocephalus inermis, Lagocephalus lagocephalus, Takifugufasciatus, Takifugu xanthopterus, Takifugu oblongus, Takifugu rubripes, Gastrophysus gloveri, Gastrophysus lunaris, Gastrophysus spadiceus), through PCR in real time amplification, all obtains positive amplification curve.And the DNA sample of 3 kinds of anglerfish (Lophius litulon, Lophiomus setigerus, Lophius americanus), and the DNA sample of other 24 kinds of fish samples, Octopus, Chionoecetes bairdi, Pandalus borealis, Prawn, variegated clam, Patinopecten yessoensis, and the detected result of blank water contrast is all negative.
Conclusion: the Puffer F/Puffer R primer that the present invention is designed and Puffer P probe have the specificity of good globe fish species qualification, can go out cytochrome b DNA fragmentation by specific amplification to 9 kinds of globe fish DNA of 3 genus of globe fish [comprising that Lagocephalus belongs to (Lagocephalus), Fugu belongs to (Takifugu), ventral spine Puffer genus (Gastrophysus)].
The sensitivity of embodiment 3 detection methods
Fish products, through processing treatment process, can cause fracture and the loss of DNA, also can affect the sensitivity of detection.In order to study the impact of the course of processing on detection sensitivity, this experiment, taking anglerfish as matrix, is added globe fish, does the interpolation test of simulation converted products.
Get respectively globe fish and anglerfish sample after mincer rubs, 80 DEG C of bakings are spent the night, and are ground into powder.Filefish fish meal and safe and comfortable fish meal sample are 100% by the mass ratio of globe fish, 10%, 5%, 1%, 0.5%, 0.1%, 0.01%, 0.001% mixes, obtain the fish meal biased sample of serial per-cent, extract the biased sample DNA of different content, adopt Puffer F/Puffer R primer and Puffer P probe according to method described in embodiment 1, carry out the analysis of PCR in real time detection sensitivity.Result as shown in Figure 2.The process of spending the night through 80 DEG C of bakings from sample, to DNA extraction and PCR in real time amplification, all sensitivity experiments have all carried out 6 times to be repeated.
The sensitivity of Fig. 2 PCR in real time amplification globe fish cytochrome b DNA fragmentation: 1:100% filefish fish meal 2:10% filefish fish meal 3:5% filefish fish meal 4:1% filefish fish meal 5:0.5% filefish fish meal 6:0.1% filefish fish meal 7:0.01% filefish fish meal, Fig. 2 result shows that the detectability of real-time PCR method detection globe fish cytochrome b DNA can reach 0.01%.
Claims (1)
1. a real-time fluorescence PCR detection kit for Puffer fish ingredient in food, is characterized in that: the detection primer and the probe that comprise Puffer fish ingredient in described food:
Upstream primer: SEQ ID NO.1
Downstream primer: SEQ ID NO.2
Probe: SEQ ID NO.3.
2. a real-time fluorescence PCR detection method for Puffer fish ingredient in food, is characterized in that: utilize the test kit described in claim 1, sample DNA is carried out to the detection of real-time fluorescence PCR method.
3. the real-time fluorescence PCR detection method of Puffer fish ingredient in food according to claim 2, is characterized in that: described to sample DNA carry out real-time fluorescence PCR method detect be:
1. real-time fluorescence PCR reaction system:
Reaction system cumulative volume is 25 μ L, wherein:
Concentration is the sample DNA 2 μ L of 10 ~ 100 μ g/mL
The each 1 μ L of 10 μ mol/L upstream and downstream primer
5 μ mol/L probe 1 μ L
5 U/ μ L Taq archaeal dna polymerase 0.5 μ L
10 μmol/L dNTP 1 μL
10 × PCR damping fluid, 2.5 μ L
Water 16 μ L
2. real-time fluorescence PCR reaction parameter: 95 DEG C, 10 s, 1 circulation; 95 DEG C, 5 s; 52 DEG C, 10s; 72 DEG C, 34s; 40 circulations.
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