The real-time fluorescence of calf-derived Cyclospora in food and feedstuff is detected using single-copy nuclear genePCR detection methodTechnical field
The invention belongs to bioengineering field, specifically, be with regard to it is a kind of using single-copy nuclear gene detection food andThe real-time fluorescence PCR detection method of calf-derived Cyclospora in feedstuff.
Background technology
Since there is bovine spongiform encephalopathy first from Britain in 1987, countries in the world and area have all successively promulgated laws and regulations to controlThe use of animal derived materials processed.2004, China promulgated《Feed products from animal sources safety and sanitation management method》, it is stipulated that prohibitThe only feed products from animal sources used in ruminant feed outside milk and milk productses.At the beginning of 2013, Sweden, Britain etc. are moreIndividual European countries are involved in " Equus caballus (L.) disturbance " scandal, cause concern of the various countries to meat and the adulterated problem of meat productss.
In China, " adulterated " problem is always the focus of consumer's complaint and the focus of social concerns.Some illegal retailersOrder about because of interests with enterprise and the low cost feedstocks such as pig, duck are mixed in the high price meat such as cattle, sheep and meat productss.These behaviors are not only invadedEvil consumer's interests, it is also possible to because causing dispute containing non-Islamic composition in some religion food, if also may be used without quarantineThe propagation of epidemic disease can be caused, be unfavorable for maintaining social stability and people's health, if defective product outlet abroad more canThe overall image of infringement China food enterprise.In order to prevent the transmissions of disease such as bovine spongiform encephalopathy, the infringement of meat adulteration is hit,Consumer rights and life security are safeguarded, the sound development of food industries is ensured, it would be desirable to strict laws and regulations are formulatedConstraint Producer and the behavior of operator, while we need to work out accurate, sensitive, easy detection method.
Common meat and meat productss real and fake discrimination technology mainly include：(1) immunoassay based on protein molecular structureTechnology etc.；Based on the immune analysis method of protein macromolecule structure have high sensitivity, high flux, it is easy to operate the characteristics of, canRealize the quick detection to a large amount of samples.For example, Macedo-Silva etc. is by preparing the albuminous anti-blood of anti-cattle, chicken, pig, horseELISA method is established clearly for the cheap Meat ingredients that differentiate to mix in hamburger, sensitivity is up to 0.6%.But, baseEasily there is cross reaction in the immune analysis method of the Ag-Ab specific binding species nearer for sibship.AlbumenMatter tertiary structure may be destroyed in food preparation process and be affected the important weak point that antibody recognition is also the method.(2) Protocols in Molecular Biology based on nucleic acid, such as PCR, real-time fluorescence PCR and molecular fingerprint technology etc.；It is special based on DNA sequenceDetection target spot that the Protocols in Molecular Biology of property is differentiated using the difference of hereditary information between animal species as meat kind and extensively madeWith.Compared to protein, DNA is high due to heat stability, although there is a certain degree of degraded in process, remains to carryTaking out small pieces segment DNA is used for the analysis such as PCR.Simultaneously DNA has that specificity is high, sensitivity is high, is not limited etc. all by tissue classMany advantages, the species nearer for sibship have higher resolution capability.In recent years, based on DNA detection technology meat andThe research of meat productss verity discrimination method is more and more.All kinds of analysis methods based on PCR are focused primarily upon at present, itsInclude DNA sequencing, multiplex PCR, real-time fluorescence quantitative PCR etc..Particularly Real-Time Fluorescent Quantitative PCR Technique it is animal derived intoThe detection field for dividing, day by day becomes main flow detection technique.
Round pcr is combined with target dna sequence by one section of special oligonucleotide and is synthesized in vitro millions ofDNA copy.DNA fragmentation is expanded by Species-specific primer, separated by sepharose electrophoresis and observe specificityBand is the most basic method that round pcr differentiates Species composition.Mitochondrial DNA number of copies in cell is big, sensitivity is high, enterChange speed fast, with higher inter-species multiformity and relatively low intraspecific variablity, be widely used in design of primers and target sequenceAmplification.Mitochondrial gene as conventional has cytochrome b gene, 12S and 16S ribosomal RNA subunits, D-loop areas.But,As the copy number of mtdna sequence is high and non-constant, the qualitative detection of animal derived materials is can be only applied to, it is difficult to applyIn detection by quantitative.“Species identification and quantification in meat and meatproducts using droplet digital PCR(ddPCR)Analytical Methods”,C.Floren,I.Wiedemann, B.Brenig, E.Sch ü tz, J.Beck, Food Chemistry 173 (2015) 1,054 1058, this articleConclusion specify that primer and probe using mitochondrion source when quantitative can cause result error.
The primer and probe of detection animal component is typically all directed to mitochondrial gene design, mitochondrion both at home and abroad at presentCopy number of the gene in cell is copied or more in 5000-6000.But find in practical study work：Using multicopyWhen mitochondrial gene designs a set of primer and probe is detected, its Ct value can be low up to for 13 or so, so low Ct values, whenIn sample, at 0.00000001%, Ct values are still 37 or so for animal derived materials.In the so low detection of target detection thingConcentration, as occurring, Ct values, make testing staff be difficult to judge that actual sample is really to contain target component, or sample are subject toVery slight pollution.So design primer and probe using mitochondrial gene detected, easily occur false positive results orIt is difficult to judge the testing result under low target content.If positive detection report is provided according to so low Ct values, can drawVery big trade dispute.
Therefore, it is necessary to set up that a set of specificity is good, sensitivity is high, can avoid the occurrence of false positive results etc. food andDead fluorescent quantitative PCR detection method when calf-derived Cyclospora in feedstuff.
The content of the invention
The present invention is had found by studying：The animal derived materials in some animal bright flesh sample are detected, using single copyKaryogene design primer and probe detected, its Ct value for 22-24 when, when in sample, animal derived materials existWhen 0.001%, Ct values are 37 or so.Species are carried out using the karyogene of single copy for this present invention and differentiate detection, as a result foundThe situation that false positive results can be avoided the occurrence of using single copy gene detection or judged result is difficult occurs, and using single copyKaryogene detected, moreover it is possible to carry out detection by quantitative, so the karyogene for finding single copy is carried out detecting and set up accordingly and markedQuasi- method seems very necessary.
Primary and foremost purpose of the present invention is to provide a kind of real-time PCR detection side of calf-derived Cyclospora in food and feedstuffMethod, to overcome the shortcomings of being difficult to detection by quantitative, false positive results etc. easily occur existing for prior art.The second of the present inventionIndividual purpose is to provide the application of a kind of detection kit and detection kit.Third object of the present invention is offer onePlant the primer pair and probe of the real-time fluorescence PCR detection method of calf-derived Cyclospora in food and feedstuff.
For achieving the above object, the present invention is employed the following technical solutions：
It is as the first aspect of the invention, a kind of to detect calf-derived Cyclospora in food and feedstuff using single-copy nuclear geneReal-time fluorescence PCR detection method, the method comprises the following steps：
The first step, the DNA with testing sample carry out fluorescent quantitative PCR as template, obtain pcr amplification product；
Second step, detects the fluorescence signal of amplified production；
3rd step, judges whether contain in calf-derived Cyclospora and detection by quantitative sample in sample with the Ct values of testing resultCalf-derived Cyclospora content；
Wherein, for PCR amplification reaction system in containing amplification calf-derived Cyclospora specific primer to and cattle source propertyThe specific probe of composition, the sequence such as SEQ ID NO.1 and SEQ ID of the specific primer pair of the amplification calf-derived CyclosporaShown in NO.2, the sequence of the specific probe of the calf-derived Cyclospora is as shown in SEQ ID NO.3.
According to the present invention, the condition of the PCR amplifications is as follows：
Reaction system is 25 μ l：12.5 μ l of real-time fluorescence PCR reagent (2 ×), primer are each 10 μM, 5 μM of probe, DNA profiling 5μl；
PCR reaction conditions：95℃10min；95℃15s；60℃1min；Totally 45 circulations.
As the second aspect of the invention, a kind of detection kit of calf-derived Cyclospora, the detection kit contain withLower reagent：
A () expands the specific primer pair of calf-derived Cyclospora；
The specific probe of (b) calf-derived Cyclospora；
Wherein, the sequence such as SEQ ID NO.1 and SEQ ID NO.2 of the specific primer pair of the amplification calf-derived CyclosporaShown, the sequence of the specific probe of the calf-derived Cyclospora is as shown in SEQ ID NO.3.
Further, the detection kit also includes：
(c) marker, for the content of the calf-derived Cyclospora in detection by quantitative sample.
Used as the third aspect of the invention, a kind of application of described detection kit, the detection kit are used forCalf-derived Cyclospora in qualitative or quantitative detection food or feedstuff.
As the fourth aspect of the invention, a kind of specific primer of calf-derived Cyclospora to and probe, the specificityThe sequence of primer pair as shown in SEQ ID NO.1 and SEQ ID NO.2, the sequence such as SEQ ID NO.3 of the specific probeIt is shown.
The invention has the beneficial effects as follows：
1st, there is provided it is a kind of from karyogene can specificity identification calf-derived Cyclospora primer pair and probe, which is specialProperty it is good, for calf-derived Cyclospora can realize specific amplification, and for other compositions beyond cattle are then unable to specificity expansionIncrease；And, the primer pair and probe have good repeatability, result reliable and stable.
2nd, can be detected quickly, in large quantity using described primer pair and probe and whether there is in food or feedstuff cattle source propertyThe content of composition and detection by quantitative food or the calf-derived Cyclospora in feedstuff, and required sample size is few, it is simple to operate, it is sensitiveDegree is high.
3rd, the real-time fluorescence PCR detection method of calf-derived Cyclospora of the invention, which is applied widely, is particularly well-suited to all kinds ofThe detection of the calf-derived Cyclospora in food or feedstuff, therefore can be to be applicable, the quality to ensureing product, protection consumer are knownFeelings are weighed and right to choose, safeguard that normal economic order etc. provides technical support, are market surveillance department and the inspection inspection of foodEpidemic disease department provides technical support.
Description of the drawings
Amplification curve diagrams of the Fig. 1 for real-time fluorescence PCR primer specific test.Wherein, amplification curve is from left to right distinguishedIt is：Beef DNA and family's cattle DNA standard substance.
Fig. 2 is real-time fluorescence PCR sensitivity test amplification curve diagram.Wherein, amplification curve is from left to right (W/ respectivelyW)：The DNA that 0.1% beef powder, 0.01% beef powder and 0.001% beef powder are extracted.
Quantitation curves of the Fig. 3 for calf-derived Cyclospora.
Specific embodiment
Below in conjunction with specific embodiment, the invention will be further described.It should be understood that following examples are merely to illustrate thisInvention is not for restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally according to normalRule condition, such as《Molecular cloning：Laboratory manual》(New York：Cold Spring Harbor Laboratory Press,1989) condition that the condition or manufacturer described in is provided is carried out.
The experiment material of the present invention is as follows：
(1) family cattle (Bos taurus), donkey (Equus asinus), sheep (Ovis aries), goat (CapraHircus), horse (Equus caballus), chicken (Gallus gallus), duck (Anas platyrhynchos), goose (AnserAnser), turkey (Meleagris gallopavo), pig (Sus scrofa), Carnis Coturnicis japonicae (Coturnix coturnix), camelThe DNA standard substance of (Camelus dromedarius), domestic cat (Felis catus) are purchased from U.S. ZyagenLaboratories companies.
(2) animal component such as beef, bream meat, Stromateoides argenteus meat, Channa argus meat, Oncorhynchi meat is commercially available prod.
(3) big rat meat, little rat meat buy pul-Bi Kai laboratory animals company limited western in Shanghai.
(4) rice, Semen Maydiss, Semen Glyciness, Semen setariae, Semen phaseoli radiati, Radix Ipomoeae, Rhizoma Solani tuber osi, Semen Phaseoli Vulgaris, Fructus Mali pumilae, pears, Sargassum (Thallus Porphyrae), celeryThe plant components such as dish are commercially available prod.
(5) for the chicken and ham roll sausage (import) of actual verification, pork ham sausage (import), beef ham sausage(import), beef pork luncheon meat, pork luncheon meat, beef with brown sauce canned food, dried beef, dried pork slice, beef dumplings, fried curry beef beans, chickenExtractive flavouring, chicken flavor dog food (import), beef flavour dog food (import), tuna fish cat canned food (import) are commercially available prod.
(6) Australian house pet level Carnis Gallus domesticus bone meal is provided by Shanghai customs port.
The design of 1 primer pair of embodiment and probe
(1) sequence (accession number according to cattle (Bos taurus) the β actin delivered in NCBI：EH170825.1), choosing suitable sequence fragment carries out primed probe design.
It is 62bp for the purpose fragment length of calf-derived Cyclospora.Fluorescence PCR primer to and probe sequence it is as follows：
Bovine-62bp-F：5’-GGCCTCGGAGTGTGTATTCAG-3’(SEQ ID NO：1)
Bovine-62bp-R：5’-GCCCCAGAATGAGGTTCACTT-3’(SEQ ID NO：2)
Bovine-62bp-P：FAM-AGGTGCACAGTACGTTC-MGB(SEQ ID NO：3)
Wherein, FAM represents fluorescent reporter group, and MGB represents quenching group.The present invention adopts fluorescence probe method, its detectionPrinciple is come recognition template using fluorescent labeling specific probe.Compared with SYBR dye methods in prior art, fluorescence of the present inventionThe specificity of labelling specific probe is higher, and ambient interferences are lower.
(2) reaction system and response procedures of real-time fluorescence PCR
Real-time fluorescence PCR reaction system is shown in Table 1：
Wherein, real-time fluorescence PCR response procedures are：95℃10min；95℃15s,60℃1min；Totally 45 circulations.
1 calf-derived Cyclospora real-time fluorescence PCR reaction system of table
The preparation of 2 test sample of embodiment
Animal meat sample Jing after tearing up, dry, using refrigeration grinding machine (SPEX 6850) shape of claying into power.Plant sampleProduct and for actual verification sample directly using refrigeration grinding machine (SPEX 6850) shape of claying into power.With Semen Maydis powder as substrate,Add beef powder be configured to content be respectively 0.1% (W/W), 0.01% (W/W), 0.001% (W/W), 0.0001% (W/W) andThe beef powder biased sample of 0.00001% (W/W), for sensitivity technique.
The extracting of embodiment 3DNA
Using Animal genome extracts kit (Tiangeng biochemical technology company limited；Catalog number (Cat.No.)：DP323) extract animal sampleProduct DNA, Plant Genome extracts kit (Tiangeng biochemical technology company limited；Catalog number (Cat.No.)：DP305 plant sample DNA is extracted),Animal derived plant feed genome extracts kit (Tiangeng biochemical technology company limited；Catalog number (Cat.No.)：DP323) extract aggregate sampleProduct and actual verification sample DNA, extracting method refer to test kit operating instruction.DNA solution is placed in -20 DEG C and saves backup.
The specific detection of the primer pair and probe of 4 calf-derived Cyclospora of embodiment
32 kinds using the primer pair and probe of the calf-derived Cyclospora of embodiment 1 to embodiment 3 for examination of the present embodiment moves plantThing material DNA sample detected, detects the fluorescence signal of amplified production.The summary of testing result is shown in Table 2 and Fig. 1.
The specific detection result of 2 calf-derived Cyclospora of table
From Fig. 1 and Biao 2 as can be seen that primer pair and probe are only positive to family cattle DNA and beef sample amplification, there is obvious SType amplification curve, and other multiple species DNA samples are expanded with feminine gender, without obvious S types amplification curve.
Conclusion：The detection method of the present invention has good species specificity.
The sensitivity technique of the primer pair and probe of 5 calf-derived Cyclospora of embodiment
With Niu Hanliang as 0.1% (W/W), 0.01% (W/W), 0.001% (W/W), 0.0001% (W/W) andThe biased sample DNA of 0.00001% (W/W) is template, carries out real-time PCR detection, and test is repeated 6 times.Detect at 6 timesIn, occur amplification curve in 0.1%, 0.01%, 0.001% cattle sample DNA, and 0.0001% and 0.00001% N of sampleThere is not amplification curve, such as Fig. 2 in product DNA.
Conclusion：When template usage amount is 0.001% (W/W), there is obvious S types amplification curve, Sensitivity reaches0.001% (W/W).
The actual verification of the primer pair and probe of 6 calf-derived Cyclospora of embodiment
Using the detection method of embodiment 4, on market, 15 parts of processed meat food stuffs collecting of foreign trade approach and animalThe samples such as feedstuff are detected.The results are shown in Table 3.
The testing result of the calf-derived Cyclospora of 3 15 parts of testing samples of table
| Sequence number | Sample ID | Result of the test |
| 1 | Beef ham sausage | + |
| 2 | Beef pork luncheon meat | + |
| 3 | Beef with brown sauce canned food | + |
| 4 | Dried beef | + |
| 5 | Fried curry beef beans | + |
| 6 | Beef flavour dog food | + |
| 7 | Chicken and ham roll sausage | — |
| 8 | Pork ham sausage | — |
| 9 | Pork luncheon meat | — |
| 10 | Dried pork slice | — |
| 11 | Beef dumplings | — |
| 12 | Chicken essence seasoning | — |
| 13 | Chicken flavor dog food | — |
| 14 | Tuna fish cat canned food | — |
| 15 | Australian house pet level Carnis Gallus domesticus bone meal | — |
From table 3 it can be seen that in 5 parts of processed meat food stuffs (beef ham sausage, beef pork luncheon meat, beef with brown sauce canned food, beefDry, fried curry beef beans) and 1 part of beef flavour dog food in detect calf-derived Cyclospora；In 6 portions of processed meat food stuffs (chicken and ham roll sausage, Carnis Sus domesticasPetaso sausage, pork luncheon meat, dried pork slice, beef dumplings, chicken essence seasoning) and 3 parts of feedstuffs (chicken flavor dog food, tuna fish cat tanksHead, Australian house pet level Carnis Gallus domesticus bone meal) in do not detect calf-derived Cyclospora.
Conclusion：In addition to processed meat products (beef dumplings), other meat sources for extracting sample are consistent with testing result.SayThere is adulterated possibility in bright processed meat products (beef dumplings).Also explanation can high-volume using the detection method of embodiment 4 simultaneouslyWhether there is calf-derived Cyclospora in ground detection food or feedstuff.
The foundation of 7 quantitation curves of embodiment
(1) detection by quantitative of calf-derived Cyclospora primer pair and probe
The family cattle DNA that embodiment 3 extracts acquisition is diluted to into 100ng/well, 10ng/well, 1ng/well, 0.1ng/Well, 0.01ng/well, totally five Concentraton gradient, are detected using the primer pair and probe of embodiment 1, each concentration 6Repeat, obtain Ct values.Negative control is water.As shown in table 3, wherein Ct values are the meansigma methodss of 6 repetition tests.
The Ct values of 3 real-time fluorescence PCR detection by quantitative of table test
| Template consumption (ng/well) | Ct values |
| 100 | 26.39 |
| 10 | 30.00 |
| 1 | 33.64 |
| 0.1 | 37.27 |
| 0.01 | 40.90 |
| Negative control | - |
Conclusion：It can be seen that the concentration with calf-derived Cyclospora is reduced, amplification Ct values increase in gradient, and in certain lineSexual intercourse (R2=0.997).
(2) process and calculating of data
As a result as shown in figure 3, with the logarithm value of DNA concentration as abscissa, with putting down for the Ct values of 6 repetition tests of sampleAverage is vertical coordinate, takes and a little carries out curve fitting, and obtains quantitative criterion straight line, and linear equation is y=3.6273x+22.76, and R2=0.997.For quantitative sample is needed, the respective Ct values of testing sample are measured every time, phase is tried to achieve using calibration curve formulaThe x values answered.The content of the cattle source constituent of testing sample can be drawn.
(3) checking of quantitation curves
DNA is extracted as described in Example 3, quantitative 6 independent samples are diluted respectively, and concentration is respectively 50ng/wellWith 0.5ng/well, respective Ct values are detected, and the content of cattle source constituent therein, use are calculated by quantitation curvesTo verify the repeatability of the method.
As a result show, the meansigma methodss of 50ng sample measured values are 53.7ng, the meansigma methodss of 0.5ng sample measured values are0.4ng。
Conclusion：Illustrate the ability that there is accurate quantification using the method for the present embodiment in certain dynamic range.So as to demonstrate,proveThe actually measured value of the bright method of the present invention is consistent with theoretical value, reproducible.
The present invention establishes the real-time fluorescence PCR side that calf-derived Cyclospora in food and feedstuff is detected using single-copy nuclear geneMethod, for the karyogene design primer pair and probe of cattle species, it is only necessary to reference to real-time fluorescence amplification curve diagram, little less than twoWhen just accurately can detect with the presence or absence of cattle source constituent in sample, its sensitivity is 0.001% (W/W).
In sum, the present invention design cattle source constituent real-time fluorescent PCR amplification specific primer to and probe notOnly specificity is good, and sensitivity is high, whether provides a kind of good inspection containing calf-derived Cyclospora rapidly and accurately to distinguishSurvey method, in the detection of field of food safety shutting, has a good application prospect.And, the specific primer toProbe can adopt method as known in the art, further make detection kit, and the detection kit is except described specialProperty primer pair and probe outside, can also include marker, this will be apparent to the person skilled in the art.
Ultimate principle, principal character and the advantages of the present invention of the present invention has been shown and described above.The technology of the industryPersonnel it should be appreciated that the present invention is not restricted to the described embodiments, the simply explanation described in above-described embodiment and description thisThe principle of invention, of the invention without departing from the spirit and scope of the present invention also to have various changes and modifications, these changesChange and improvement is both fallen within scope of the claimed invention.The claimed scope of the invention by appending claims and itsEquivalent is defined.
SEQUENCE LISTING
<110>Technical Center for Animal, Plant and Food Inspection and Quarantine, Shanghai Entry-Exit Inspection and Quarantine Bureau
<120>The real time fluorescent PCR method of calf-derived Cyclospora in food and feedstuff is detected using single-copy nuclear gene
<130> 161053
<160> 3
<170> PatentIn version 3.3
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