CN103114142A - 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 detection PCR in real time detection primer, test kit and detection method in food.
Background technology
Fishery products are important component parts of world economy and trade, are also important food protein sources.The 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 the trout personation Atlantic salmon with cultivation, with tilapia, the red porgy of Mahi loach fish personation etc.Also once had the filefish fillet were sneaked into the case that safe and comfortable fillet cause the eater to be poisoned to death.The investigation result of 9 years by a definite date of being carried out by American National Instrument Seafood Inspection Laboratory shows: the incorrect sign of species that other marine food of 37% fish products and 13% is arranged.The recently market survey carried out of 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 that faces in fish processing industry at present.European Union has set up labeling acts, forces that the manufacturer provides that the fishery products species prove, the information such as the place of production and production method; The U.S. forbids the counterfeit behavior of fishery products according to the regulation of the federal bill of food and medicine makeup; Canadian Food Inspection Agency and importor were attempted the some Imported sea-food is carried out the DNA check 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.Without processing treatment, when outward appearance is complete, usually can judge by morphological feature the true and false of kind when fishery products.For aquatic products processing product such as fish gruel, seasoned, sashimi, can, fish meal etc., need to set up the authentication method based on DNA analysis.After globe fish and anglerfish are processed into fillet or fish mud, be difficult to be distinguished on form.In order to ensure edible safety, prevent edible globefish poisoning, be necessary to select suitable gene fragment, set up practical, feasible evaluation 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 the restriction method is set up.
Safeguarding fair trading, ensuring food safety, protect the importance of the aspects such as endangered species in view of fishery products raw material authenticity, 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 the FINS method of PCR order-checking to identify porgy, tilapia, mackerel, angler, flatfish, salmon.The plan of DNA barcode is the important component part in the FINS technology, and this plan is utilize one section sequence (COI) that is known as the DNA barcode to check order by PCR and analyze a global project with the 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 evaluation of the marine foods such as alec, the filefish flesh of fish, salmon and trout.Rehbein H etc. utilizes the PCR-SSCP method to carry out the evaluation of the multiple fishes such as salmon, sardines, eel, tuna, stripped tuna, sturgeon.Lowenstein J H etc. adopt the RAPD method to be used for genetic analysis and the evaluations such as catfish.Report is arranged recently for the DNA barcode, utilize the 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 TaqMan MGB probe to be used for the evaluation of eel, tuna, salmon and trout, and Bayha K Mort etc. adopt TaqMan LNA probe to be used for the evaluation of 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 to surpass tens of kinds of fresh or finished vertebratess, comprised 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.This does not have advantage aspect two to the PCR sequencing at speed and cost.The analytical procedure of PCR-RFLP method is many, and is quite time-consuming.The highly sensitive of SSCP requires the very repeatability of high level, and experiment condition each time is without difference.The main drawback of PCR-RAPD is the repeatability of the method, and when especially the amount of target dna was degraded less or slightly, this shortcoming was especially outstanding.The AFLP method is relatively time-consuming, there is no large-scale application.The DNA chip is compared with other method, the relatively slow and high cost of sample analysis.The Real-time PCR method has cost characteristics low, easy and simple to handle, is specially adapted to the evaluation to biased sample, is most widely used.But the method can only be analyzed species with a pair of specific primer, is very limited on versatility.
Summary of the invention
In order to solve above-mentioned practical problems, the present invention has set up Puffer fish ingredient detection PCR in real time detection primer, test kit and detection method in a kind of food.Extract sample DNA, take DNA as template, adopt respectively specific detection primer and probe to carry out the 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 the 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, selected 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 sees Table 1.To the consistence of the annealing temperature of primer and probe, the factors such as similarity of GC content have been carried out sufficient consideration in the design of primer, 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 are the 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.
Can also comprise positive control, negative control, blank in this test kit.Wherein, the DNA that positive control can select Puffer fish ingredient to extract, negative control can select not contain the DNA of the sample extraction of 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, sample DNA is carried out real time fluorescent PCR method detect.
For the real-time fluorescence PCR detection method of Puffer fish ingredient mentioned above, the more excellent selection of described real time fluorescent PCR method is as follows:
The reaction system cumulative volume is 25 μ L, wherein: template DNA (10 ~ 100 μ g/mL) 2 μ L, 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 ℃/10s, and 1 circulation; 95 ℃/5s, 52 ℃/10s, 72 ℃/34s, 40 circulations.
The judgement quality control standard of its amplification is: when following condition had one not satisfy, it is invalid that experiment is considered as:
(a) blank: without the fluorescence logarithmic growth, corresponding Ct value＞40.0.
(b) negative control: without the fluorescence logarithmic growth, corresponding Ct value＞40.0.
(c) positive control: the fluorescence logarithmic growth is arranged, and corresponding Ct value＜30.0 typical amplification curve appears, in fluorescence channel.
Result is judged: consistent to the standard of result judgement with real-time fluorescence PCR GBs such as species identification and detection GB, transgenosis detections, this experiment is in the situation that meet quality control, when test sample detected: 40 circulations are set equally, and the Ct value was cycle number.If Ct value≤35.0, show sample through 35 circulations with interior amplification, amplification curve namely detected, 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 the test sample feminine gender.As 35.0＜Ct value＜40.0, result is suspicious result between this, repeats once.Be still＜40.0 as Ct value after again increasing, judge that test sample is positive; As Ct value 〉=40.0 after again increasing, judge that test sample is negative.
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 described method of the embodiment of the present invention 1.
For the DNA concentration of extracting 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, and is standby.
By above technical scheme, the present invention not only provides a cover Puffer F/Puffer R primer and Puffer P probe to be suitable for good primer and the probe of specificity to the evaluation of globe fish species, has also set up the qualitative checking method to the Puffer fish ingredient false distinguishing.Must be beneficial to and identify carrying out of Puffer fish ingredient food true and false work, be with a wide range of applications, can also the international trade of China's relevant food be played a positive role.
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, in the inventive method detection food, Puffer fish ingredient has fine suitability.
(3) the present invention adopts real-time fluorescence detection system, can the Real-Time Monitoring reaction process, need not electrophoresis, and the 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, the succeeding in developing and apply improving detection efficiency and the sensitivity of globe fish species of this standard, the reduction testing cost is carried out food safety monitoring and the raising inspection technology is significant, is with a wide range of applications.
Description of 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) all obtains positive amplification curve through the PCR in real time amplification.
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.
Sign for result shown in Fig. 1 and 2: in accompanying drawing, the curve of test sample is colour, presets different colours before experiment and represents corresponding testing sample, and judgment experiment can according to color differentiating, know whether counter sample obtains the curve amplification as a result the time.
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 guaranteeing the credible of experimental result, establish respectively positive control, negative control, blank group.With the positive contrast of DNA that globe fish is extracted, with the known negative contrast of DNA that does not contain the sample extraction of Puffer fish ingredient, take aqua sterilisa as blank, every group arranges two parallel reaction systems.
1.DNA extract
Following DNA extraction method, all reagent and solution are the conventional route preparation or are obtained by commercial sources.
Sample DNA extracts the high salt method that adopts after improveing.Sample adds liquid nitrogen and grinds, get sample 150mg after grinding in the centrifuge tube of 1.5mL, add respectively the TE(of 400 μ L to contain 10mmol/L Tris-HCl and 1mmol/L EDTA), the Proteinase K of 40 μ L10%SDS and 8 μ L20mg/mL, vortex 30s, fully mixing; The constant temperature water bath that centrifuge tube is placed in 55 ~ 65 ℃ digests 4h, adds the saturated NaCl solution of 6mol/L 300 μ L, 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, dry in the baking oven of 56 ℃, then add 50 μ L TE, dissolving DNA precipitation is preserved under-20 ℃.
The mensuration of DNA concentration and purity:
Get after appropriate DNA solution stoste adds 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 is suitable for the real-time fluorescence PCR amplification between 1.7 ~ 1.9 the time.
2. real-time fluorescence PCR detects
The reaction system cumulative volume is 25 μ L, wherein: template DNA (10 ~ 100 μ g/mL) 2 μ L, 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 ℃/10s, and 1 circulation; 95 ℃/5s, 52 ℃/10s, 72 ℃/34s, 40 circulations.
According to above-mentioned system, respectively with primer shown in table 1 and probe, the sample composition DNA that extracts respectively, negative control and blank carry out the 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 description of drawings.
Test sample according to the method described above, the real-time fluorescence PCR detected result:
Positive controls all obtains positive amplification curve through the PCR in real time amplification, 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 and detect.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 see Table 2.
Table 2 sample title and source
| Sequence number | The sample title | Sequence number | The |
| 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 | |
| 9 | Takifugu? |
30 | Crucian |
| 10 | Gastroph?ysus? |
31 | The Mandarin |
| 11 | Gastrophysus?lunaris | 32 | |
| 12 | Gastrophysus?spadiceus | 33 | Sole |
| 13 | Freeze the |
34 | |
| 14 | Freeze southern |
35 | |
| 15 | Freeze |
36 | |
| 16 | The chub mackerel |
37 | |
| 17 | |
38 | Chionoecetes? |
| 18 | Willow leaf like |
39 | Pandalus?borealis |
| 19 | Tuna | 40 | |
| 20 | |
41 | |
| 21 | The Anchovies |
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) all obtains positive amplification curve through the PCR in real time amplification.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 that good globe fish species are identified, can go out the 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 can cause fracture and the loss of DNA through the processing treatment process, also can affect the sensitivity of detection.In order to study the course of processing to the impact of detection sensitivity, this experiment is added globe fish take anglerfish as matrix, does the interpolation test of simulation converted products.
Get respectively globe fish and anglerfish sample after mincer rubs, 80 ℃ 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 the described method of embodiment 1, carry out the analysis of PCR in real time detection sensitivity.Result as shown in Figure 2.From the process that sample spends the night through 80 ℃ of bakings, 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 show that the detectability of real-time PCR method detection globe fish cytochrome b DNA can reach 0.01%.
Claims (4)
1. in a food, the detection of Puffer fish ingredient with primer and probe, is characterized in that, comprises following base sequence:
Upstream primer: SEQ ID NO.1
Downstream primer: SEQ ID NO.2
Probe: SEQ ID NO.3.
2. the real-time fluorescence PCR assay kit of Puffer fish ingredient in a food is characterized in that: the detection that comprises Puffer fish ingredient in the described food of claim 1 is with primer and probe.
In a food Puffer fish ingredient real-time fluorescence PCR detection method, it is characterized in that: utilize primer claimed in claim 1 and probe, sample DNA is carried out real time fluorescent PCR method detect.
In food according to claim 3 Puffer fish ingredient real-time fluorescence PCR detection method, it is characterized in that: describedly sample DNA is carried out real time fluorescent PCR method detect and to be:
1. real-time fluorescence PCR reaction system:
The reaction system cumulative volume is 25 μ L, wherein:
2. real-time fluorescence PCR reaction parameter: 95 ℃, 10s, 1 circulation; 95 ℃, 5s; 52 ℃, 10s; 72 ℃, 34s; 40 circulations.
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| CN104894254A (en) * | 2015-05-30 | 2015-09-09 | 福建出入境检验检疫局检验检疫技术中心 | Species identification method of globefishes |
| CN107894410A (en) * | 2017-10-26 | 2018-04-10 | 南昌大学 | A kind of fluorescence probe and its synthetic method for cell surface molecule imaging |
| CN109439761A (en) * | 2018-06-26 | 2019-03-08 | 中国计量大学 | Application of the COI sequence in Rapid identification river Puffer and its fish product |
| CN109777865A (en) * | 2019-03-07 | 2019-05-21 | 青岛市疾病预防控制中心（青岛市预防医学研究院） | Food poisoning source DNA bar code data library and its quick Testing and appraisal source tracing method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104894254A (en) * | 2015-05-30 | 2015-09-09 | 福建出入境检验检疫局检验检疫技术中心 | Species identification method of globefishes |
| CN107894410A (en) * | 2017-10-26 | 2018-04-10 | 南昌大学 | A kind of fluorescence probe and its synthetic method for cell surface molecule imaging |
| CN109439761A (en) * | 2018-06-26 | 2019-03-08 | 中国计量大学 | Application of the COI sequence in Rapid identification river Puffer and its fish product |
| CN109777865A (en) * | 2019-03-07 | 2019-05-21 | 青岛市疾病预防控制中心（青岛市预防医学研究院） | Food poisoning source DNA bar code data library and its quick Testing and appraisal source tracing method |
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