CN108531618A - Detect triple fluorescent PCR primer probe groups, kit and the detection method of domestic animals derived component - Google Patents
Detect triple fluorescent PCR primer probe groups, kit and the detection method of domestic animals derived component Download PDFInfo
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Abstract
The invention discloses the triple fluorescent PCR primer probe groups of detection domestic animals derived component, include triple primed probe group A for family ox, buffalo, yak, for triple primed probe group B of sheep, goat, antelope, specifically as shown in Seq.ID No.1 to Seq.ID No.14.The invention belongs to technical field of gene detection, each primer and probe will not cause to interfere with each other, it is only capable of expanding specific objective sequence and excites fluorescence signal, to non-targeted sequence without amplification and fluorescence signal, the accurate detection to above-mentioned 6 breeding class source property may be implemented, have many advantages, such as that specificity is good, standard error is small, detection time is short, save reagent cost, the authenticity for being suitable for domestic animals product detects.
Description
Technical field
The invention belongs to technical field of gene detection, more particularly to detect the triple fluorescent PCR primer of domestic animals derived component
Probe groups, kit and detection method.
Background technology
Currently, the adulteration of animal product frequently occurred, and has such report repeatly both at home and abroad, such as in 2013, European Union
The beef food adulteration incident for pretending to be beef with horseflesh has occurred in many countries.In recent years, China has also persistently exposed many dynamic
The adulteration incident of produce product, including various regions food medicine prison, quality inspection, industrial and commercial administrative bureau discover and seize all kinds of cases announced with news media：With duck
Meat pretends to be mutton, pretends to be beef with pork, chicken, pretends to be the frauds means such as rabbit meat with cat meat.
Family ox, buffalo, yak, between the kinds such as sheep, goat, antelope, product feature is extremely similar and difference in selling prices
It is larger.Many commercially available characteristic meat products, sell using buffalo, yak, sheep, goat derived component as raw material and as product
Point, can consumer during consuming and be edible, its derived component can not be distinguished by sensory evaluation substantially.In addition, yak, antelope
Sheep further relates to the matters such as the supervision protection of rare wild animal, the artificial feeding of specialty economies domestic animals.
In the technique study using the Protocols in Molecular Biology identification animal product true and false and detection of adulterations, pass through PCR skills
Art expands and detects target animal source property gene, is the scientific basis and technological means of current mainstream, obtains in recent years extensive
Using.The technology is broadly divided into two parts：1. extracting sample DNA；2. the PCR of DNA is detected.
The extraction of the DNA of domestic animals product can be divided into automation extraction, such as not yet universal self-test work from mode of operation
It stands；Semi-automation extraction, the instrument for extracting nucleic acid such as gradually popularized；Manual extraction, such as current common commercial kit, certainly
Reagent processed etc..Undressed and roughing food DNA losses are small and more complete, and above-mentioned three kinds of methods are applicable, and usual DNA is pure
Spend OD260/OD280It is 1.6 to 1.8, concentration can slightly dilute or be directly used in PCR detections in L grades of ng/ μ.
Key points and difficulties are deep processing domestic animals product, such as fried food, refining food at present.The DNA of these samples
Extent of the destruction is very big, if extracted according to the sampling amount of instrument for extracting nucleic acid or extracts kit, usually 1g is hereinafter, almost
Can not get can reach the amount of DNA of detection limit；If sampling amount is increased to 10g or more, and can be because nucleic acid can not be applicable in
Extraction apparatus or the operations specification of extracts kit and cause to be difficult to operate.Currently, respectively detection unit generally use increases sampling amount,
Carries out the method for manual extraction with self-control reagent and obtains DNA, these method types extensively, distinguish it is very big, effect also it is irregular not
Together.
First generation round pcr, commonly referred to as regular-PCR technology first design the primer of species specific gene, then by sample
Repeated amplification is carried out to target gene by PCR instrument after DNA and primer and amplifing reagent mixing, then amplification is produced by electrophoresis
Object is identified, to determine whether to contain the species derived component.
Second generation round pcr is real-time quantitative fluorescence PCR, usual abbreviation fluorescent PCR.In addition to design species specific gene
Outside primer, a fluorescence probe is redesigned.Real-time quantitative fluorescence will be passed through after sample DNA, primer, probe and amplifing reagent mixing
PCR instrument (usual abbreviation fluorescent PCR instrument) carries out repeated amplification to target gene, and the fluorescence signal accumulation of probe is same with gene magnification
Step, opposite regular-PCR, had both improved specificity when amplification, and had also obtained testing result after amplification immediately.
Multiple real-time quantitative fluorescence PCR (hereinafter referred to as multiple fluorescence PCR) belongs to one kind of fluorescent PCR, for multiple objects
Kind specific gene, design primer and probe, different probe mark the fluorophor of different wave length respectively one by one.By sample DNA,
Repeated amplification is carried out to multiple target gene by multichannel fluorescent PCR instrument after primer, probe and amplifing reagent mixing, passes through prison
Survey the requirement that multiple fluorescence signals reach while detecting multiple target gene.Opposite common fluorescent PCR, often increase a set of primer and
Detection efficiency can be promoted one times and other reagent costs are reduced by one times by probe.
Prior art discloses the multiple fluorescence PCR methods for being related to animal derived materials identification, such as 20151012723.2
《Detect simultaneously in meat and meat products the Taqman-LNA multiple fluorescence quantitative PCRs method of ox pig derived component and primed probe with
And kit》、201610272771.5《Donkey, horse, ox and pig source property nido fluorescent PCR detecting primer in a kind of donkey-hide gelatin, probe,
Kit, detection method and application》、201510527762.1《Identify donkey in cosmetics, the primer of horse and calf-derived Cyclospora, spy
Injection composition, kit and multiple fluorescence PCR detection method》Deng, but how the above method is not directed to by technological means area
This 6 kinds of other ox, buffalo, yak, sheep, goat, antelope.
Invention content
To solve problems of the prior art, the present invention is for family ox (Bos taurus), buffalo (Bubalus
Bubalis), yak (Bos grunniens), sheep (Ovis aries), goat (Capra aegagrus), antelope (Saiga
Tatarica) the primed probe group of the genome setting specificity of this six kinds of Chinese large-sized oxen, sheep, each primer and probe will not be made
It at interfering with each other, is only capable of expanding specific objective sequence and exciting fluorescence signal, to non-targeted sequence without amplification and fluorescence
Signal, may be implemented the accurate detection to family ox, buffalo, yak, sheep, goat, antelope derived component, sheep, goat it is logical
With primer pair without using base is annexed, has many advantages, such as that specificity is good, standard error is small, detection time is short, saves reagent cost,
Be conducive to the abundant detection of different lines domestic animals source property.Meanwhile the present invention is directed in poultry source property fluorescent PCR detection, deep processing
Domestic animals product dna extraction efficiency is low, ropy problem, optimizes the pre-treating method of sample, has that extraction efficiency is higher, carries
The feature that quality is preferable is taken, the requirement of existing DNA extraction method or extracts kit can be met, is detected in follow-up fluorescent PCR
In, Ct values substantially can be up to 25-35, in during threshold value of the most standard without reinspection.
In deep processing domestic animals food, as difficult point in the DNA extraction process of fried food, refining food is more.First, sample
Product DNA extent of the destruction is very big, if extracted according to the sampling amount of instrument for extracting nucleic acid or extracts kit, usually 1g with
Under, it is virtually impossible to the amount of DNA of detection limit can be reached by getting；If sampling amount is increased to 10g or more, and can be because can not fit
Cause to be difficult to operate with instrument for extracting nucleic acid or the operations specification of extracts kit.This method is first with n-hexane by sample surfaces
60 DEG C of drying after cleaning up, cleaned samples amount can according to actual needs or experience anticipation is voluntarily selected within the scope of 20 to 200g
It selects, ensure that enough sample DNA contents.
Secondly, the sample morphology of deep processing domestic animals food is usually in block, item, powdery, and wherein cell each component usually has become
Homogeneous state, and contain a large amount of indigestible or be difficult to the carbonization component degraded or inert fraction.These carbonizations or inert fraction
Exist usually in the form of a large amount of particle, powder, precipitation etc., in DNA extraction process, DNA solution would generally be adsorbed thereon,
Even by centrifugation, also it can be absorbed and can not be efficiently separated by particle/powder/precipitation rapid, high volume in the several seconds after centrifugation.
This method is by being added isometric 3d H2O is 37 DEG C on shaking table, 200rpm is extracted overnight, and sample DNA is dissolved to by guarantee as possible
Water phase；Then the squeezer of manually or electrically isolates most liquid phases, and avoiding centrifugation means can not efficiently separate
The shortcomings that.Last 10000rpm, 10min are centrifugally separating to obtain wherein water phase, and 1mL is concentrated into hereinafter, can by being concentrated in vacuo instrument
It is applicable in conventional manual method, commercial kit, DNA extraction apparatus etc. and carries out DNA purifications.
When designer ox, buffalo, yak, sheep, goat specific primer and probe, its taxonomy clear first.
Family ox Bo.taurus domestica, goat C.aegagrus hircus are wild ox Bo.taurus, aegagrus respectively
The domestication kind of C.aegagrus；Buffalo Bu.bubalis, yak Bo.grunniens, the domestication kind of sheep O.aries and original seed
No scientific name difference.According to chemotaxonomy correlative study, for high species between wild subspecies, gene difference is just very small, leads to
Often only Individual genes difference, especially tames subspecies, genome almost with open country non-hibernating eggs indistinction.And in each genomic library
In, the source of species remarks of correlated series are general only to arrive genus and species；It is accurate to subspecies, mutation, serotype, mutant strain, mostly
Certain pathogenic bacteria, virus.Therefore, the present invention is when retrieving target gene, with Bo.taurus, Bu.bubalis,
Bo.grunniens, O.aries, C.aegagrus correlated series are reference frame, do not differentiate between original seed and domestication kind.
When designing the specific primer and probe of antelope, its taxonomy clear first.According to the literature, in taxology
On, there is no specifically refer exclusively to which section or category to antelope；A total of 86 kinds of the animal of ariel belongs to 11 races, 32 categories.
It is mostly rare, wildlife species in these genus and species；Present invention selection has notable medical value, and there is stringent monitoring and protect
The Saiga tatarica S.tatarica of shield is as identification object.
Retrieve NCBI Genbank after obtain Bo.taurus, Bu.bubalis, Bo.grunniens, O.aries,
Tens of of the mitochondria correlated series of C.aegagrus, S.tatarica are used in Lasergene v7.1.0 first
MegAlign softwares compare out conserved sequence, then are sieved after the Primer-Blast checking computations for passing through Oligo v7.0.1 and NCBI
Choosing, inventor have found according to its glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate
Dehydrogenase, GAPDH) gene specific site can design Bo.taurus, Bu.bubalis a pair of universal primer, two
Specific probe, O.aries, C.aegagrus a pair of universal primer, two specific probes, the exclusive primers of Bo.grunniens and spy
Different probe, the exclusive primers of S.tatarica and specific probe.Above-mentioned probe selects NFQ-MGB fluorescent quenching groups respectively, relatively
TAMRA, BHQ etc. can more effectively avoid the non-specific amplification that single base difference is brought.
Primer and probe provided by the invention is compared with current methods, SN/T 4397-2015《Yak source in export food
The detection method real-time fluorescence PCR method of property ingredient》Middle probe is 34bp, and middle probe of the present invention only 20-22bp is reduced in sequence
The non-specific matched probability of Duan Fasheng；With SN/T 2051-2008《Cattle and sheep pig derived component is examined in food, cosmetics and feed
Survey method real-time PCR methodology》It compares, the present invention has refined ox, sheep classification, closer to daily life and actually detected；With SN/
T2980-2011《Ox, goat and the triple real-time fluorescence PCR detection methods of sheep derived component in animal product》It compares, sheep,
The universal primer of goat improves specificity to not using merger base；With SN/T 3730.7-2013《In food and feed often
See the 7th part buffalo composition detection of identification method of domestic animals kind glimmering PCR methods in real time》And SN/T 2051-2008, SN/
T3730.7-2013, SN/T 4397-2015 are compared, and the detection that detection method provided by the invention solves deep processing sample is asked
Topic, the operation is more convenient.
The primer of Bo.taurus, Bu.bubalis, Bo.grunniens, O.aries, C.aegagrus, S.tatarica
Probe groups detection site is exemplified below：
(1) 2437bp in the GAPDH genes of Bo.taurus, detection site such as NCBI in NC007303.6 sequences is extremely
2510bp。
(2) 2416bp in the GAPDH genes of Bu.bubalis, detection site such as NCBI in NW005785176.1 sequences
To 2490bp.
(3) 102bp in the GAPDH genes of Bo.grunniens, detection site such as NCBI in EU195062.1 sequences is extremely
191bp。
(4) 146bp in the chondriogen of O.aries, detection site such as NCBI in KF469290.1 sequences is extremely
269bp。
(5) 775bp in the chondriogen of C.aegagrus, detection site such as NCBI in GQ240309.1 sequences is extremely
880bp。
(6) 6484bp in the chondriogen of S.tatarica, detection site such as NCBI in KY829450.1 sequences is extremely
6585bp。
The triple fluorescent PCR primer probe groups of six breeding stock derived components of detection provided by the invention include being directed to
Triple primed probe group A of Bo.taurus, Bu.bubalis, Bo.grunniens, for O.aries, C.aegagrus,
Triple primed probe group B of S.tatarica, it is specific as shown in table 1.Preferably, triple primed probe group A, triple both may be selected
Primed probe group B synchronizes detection with raising efficiency, also can individually select to visit using triple primers according to the actually detected needs of sample
Needle group A or triple primed probe groups B is identified to save operation and reagent.
Table 1
The present invention also provides the triple fluorescent PCR kits of six breeding stock derived components of detection, including above-mentioned primed probe
Group, fluorescent PCR reagent, positive control, negative control and blank control.In matched reagent selection, multiple fluorescence PCR is tried
Agent selects the model with temperature-sensitive Taq antibody, inhibits to be caused by the non-specific annealing of primer or primer dimer under cryogenic conditions
Non-specific amplification, extend the kit holding time, allow more number of freezing and thawing；The fluorescent PCR reagent includes ROX
Dyestuff, for needing the multiple fluorescence PCR instrument of ROX dyestuffs that can promote detection accuracy.It is highly preferred that the blank control is 3d
H2O。
Preferably, the general sense primers of the Bo.taurus and Bu.bubalis, downstream primer concentration be respectively 20 μ
The general sense primer of mol/L, O.aries and C.aegagrus, downstream primer concentration be respectively 20 μm of ol/L；It is described
Bo.grunniens sense primers, Bo.grunniens downstream primers, S.tatarica sense primers, the downstreams S.tatarica are drawn
The concentration of object is respectively 10 μm of ol/L, and 6 concentration and probe concentrations are 10 μm of ol/L described in table 1.Preferably, the positive control is to use
The primer sets for including in above-mentioned primed probe group are expanded, and the probe groups for including in above-mentioned primed probe group is used in combination to detect in sun
The hybrid dna segment or genome of property, a concentration of 105L grades of copies/ μ.
Preferably, the negative control is non-poultry source property DNA, and the blank control is ultra-pure water.
In addition, the present invention also provides the triple fluorescent PCR method of six breeding stock derived components of detection, include the following steps：1)
Sample pre-treatments are carried out, sample DNA is extracted；2) foundation includes the multiple fluorescence PCR of primed probe group described in claims 1 or 2
Reaction system, reaction condition are：95 DEG C of 20s-2min or 10-15min；95 DEG C of 5s -1min, 60 DEG C of 20s -2min；40cycle is simultaneously
Fluorescence signal is collected, the judgement of result is detected.When for biological heat sensitive antibody, 95 DEG C of 20s-2min or 10-15min；When
For chemical heat sensitive antibody when, 95 DEG C of 10-15min.
Preferably, the sample is deep processing domestic animals food, and the sample pre-treatments include：With n-hexane by sample surfaces
60 DEG C of drying, are added isometric 3d H after cleaning up2O is 37 DEG C on shaking table, 200rpm is extracted overnight, and is detached by squeezer
After going out liquid phase, 10000rpm, 10min are centrifugally separating to obtain wherein water phase, and 1mL or less is concentrated by being concentrated in vacuo instrument.
Preferably, the judgment method of the testing result includes：
1. quality control standard：In the PCR reaction systems of triple primed probe group A of positive control and triple primed probe group B,
FAM, VIC, NED have the growth of fluorescence logarithm, and value≤30.0 Ct, negative control and the equal unstressed configuration signal of blank control and logarithm
Increase, and value >=40.0 Ct, carries out the judgement of poultry derived component testing result 2.；
2. testing result：In the PCR reaction systems of triple primed probe group A of sample, FAM and/or VIC and/or NED have
Fluorescence logarithm increases, and when and Ct values≤30.0, then contains corresponding family ox and/or buffalo and/or yak derived component；Sample
In the PCR reaction systems of triple primed probe group B, FAM and/or VIC and/or NED have the growth of fluorescence logarithm, and value≤30.0 Ct
When, then contain corresponding sheep and/or goat and/or antelope derived component；If above-mentioned one or more fluorescence no signal and right
Number increases, then is free of corresponding poultry derived component；If 30.0<Ct values<When 40.0, increase template quantity reinspection, if Ct values >=
40.0, then testing result is feminine gender, if Ct values<40.0, then testing result is the positive.
Compared with prior art, beneficial effects of the present invention include：
(1) primed probe group is arranged for family ox, buffalo, yak, sheep, goat, antelope specific gene in the present invention, respectively
Primed probe will not cause to interfere with each other, and be only capable of expanding specific objective sequence and exciting fluorescence signal, to non-targeted sequence
For row without amplification and fluorescence signal, specificity is good, and detection sensitivity is high.
(2) detection method provided by the invention is conducive to the abundant detection of different lines domestic animals source property, opposite multiplex PCR side
Method has standard error smaller, and (standard 3kbp plasmid initial concentrations are 105When grade, quantitative criterion error is 104Grade), detection time
Shorter (upper machine 1h or so provides testing result) generates the excellent of poisonous and harmful substance less (fluorescent dye dosage is L grades of ng/ μ)
Point；Relative fluorescence PCR method has the advantages that while detecting multiple target genes, saves reagent cost；Opposite regular-PCR side
Method has both above two advantage.
(3) primer provided by the invention, probe, kit and method have been applied in the routine testing of our unit (detection
Limit 0.01% mass fraction), it also has verified that and passes through in another detection unit of the same trade, batch samples up to a hundred are without missing inspection, flase drop
Situation is identical as standard method testing result；Practice have shown that relative standard's method 70% or more operating time of saving of the present invention,
70% or more reagent cost is saved, there is preferable practical value.
Description of the drawings
The testing result figure of Fig. 1 embodiment of the present invention three；Wherein, 1-6 is the positive control of initial survey, and 15-26 is initial survey
Negative control and blank control；7-12 is the positive control of reinspection, and 27-38 is negative control and the blank control of reinspection, and 13 are
The family ox source property fluorescence signal of initial survey, 14 be the family ox source property fluorescence signal of reinspection.
Specific implementation mode
The present invention is described in further details with reference to the accompanying drawings and examples.
In the present invention, involved reagent and material are conventional commercial product, or can pass through the ordinary skill in the art
Means obtain.
Embodiment one detects structure and the verification of the triple fluorescent PCR kit of six breeding stock derived components
1. primed probe group：As shown in table 1, it can be synthesized by the company with primer and probe synthesis capability, this implementation
The synthesis of example selection Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, is diluted to 100 μm of ol/L by primer and probe dry powder and makees
For storing solution, it is configured to 10 μm of ol/L according to table 2 and is used as using liquid, black 1.5mL centrifuge tubes is used in combination to be stored at -20 DEG C.
2 100 μm of ol/L storing solutions of table are formulated as 10 μm of ol/L and use liquid
2. fluorescent PCR reagent：Common commercial samples fluorescent PCR reagent, the present embodiment select ABIPath-IDTM
qPCR Master Mix。
3. positive control：Whole primer storing solutions are taken, are diluted to 10 μm of ol/L respectively.6 breeding class DNA standard items are extracted,
It is expanded on regular-PCR instrument with corresponding primer and regular-PCR reagent, TaKaRa will be transferred to after purpose band gel extraction
T-Vector pMDTM20 carriers are replicated with competence E.coli cells JM109, are extracted pMT-20 plasmids, are diluted to respectively
106L grades of concentration of copies/ μ use 3d H after respectively taking 10 μ L mixings2O is settled to 100 μ L, and final concentration is 105L grades of copies/ μ.
4. negative control：Non- poultry source property DNA.
5. blank control：Ultra-pure water.
6. multichannel fluorescent PCR instrument selects ABI Quantstudio 5, verification sample feature such as table 3 is carried out instead by table 4
Liquid is answered to prepare, reacted by table 5.
3 sample characteristic of table
4 30 μ L reaction system (units of table：μL)
5 reaction condition of table
Note*：Detect fluorescence signal.
7. reaction result is as shown in table 6：
6 sample detection result of table：It surveys Ct values and whether there is or not S type curves
8. verification result：Send microorganism (containing molecular biology) detection to detection unit of the same trade inside the province real kit
Room is tested, as a result unanimously.By positive control, mark-on control, negative control, blank control setting, show that Kit components have
Effect, and specificity is good, standard error is small, detection time is short, saves reagent cost.
Two people of embodiment entrust detection sample --- fermented soya beans, salted or other wise juice chicken claw, numbed and hot duck neck, roasted goose, Spiced beef, buffalo milk, yak
The detection of jerky, fertile sheep volume, sliced mutton, Cornu Saigae Tataricae powder
Transfer personal commission detection sample fermented soya beans, salted or other wise juice chicken claw, numbed and hot duck neck, roasted goose, Spiced beef, buffalo milk, dried yak beef, fertilizer
Sheep volume, sliced mutton, Cornu Saigae Tataricae powder, number is #1, #2, #3, #4, #5, #6, #7, #8, #9 sample respectively.By solid sample ball milling
It crushes, liquid sample direct sample, is existed using outsourcing DNA extraction kit (Qiagen MagAttract Hmw DNA kit)
Sample DNA is extracted on small magnetic frame (Qiagen MagAttract Magnetic Rack), uses 3d H2O dilutions (or it is true
Sky concentration) to 50ng/ μ L concentration, the examination in embodiment one is pressed in primer needle group, positive control, negative control, blank control etc.
The fluorescent PCR reagent of agent box ingredient, outsourcing isMultiplex PCR Kit, upper machine testing (ABI fluorescent PCRs
Instrument, model 7500Fast).
Testing result is shown in Table 7：1. quality control standard：FAM, VIC, NED of the system A and system B of positive control have fluorescence
Logarithm increases, and value≤30.0 Ct；Negative control and the equal unstressed configuration signal of blank control and logarithm increase, and value >=40.0 Ct.
2. in system A in the NED of VIC, #6 of FAM, #5 of #4 and system B the NED of VIC, #9 of FAM, #8 of #7 have fluorescence signal and
Logarithm increases and value≤30.0 Ct, other unstressed configuration signals and logarithm increase and value >=40.0 Ct, show Spiced beef, buffalo milk,
Dried yak beef, fertile sheep volume, sliced mutton, Cornu Saigae Tataricae powder contain family ox, buffalo, yak, sheep, goat, antelope derived component respectively,
Other samples are free of above-mentioned derived component.3. comparison result：With according to or with reference to SN/T 2980-2011, SN/T 3730.7-
2013, the standards such as SN/T 4397-2015 are consistent to the result of sample progress correlated source composition detection.
7 sample detection result of table：It surveys Ct values and whether there is or not S type curves
Commission detection sample --- the detection of translucent beef slices silk of three enterprise of embodiment
Enterprise's commission detection sample, 1 part of translucent beef slices silk are transferred, sample size 200g is set as 10# samples, will with n-hexane
60 DEG C of drying after sample surfaces clean up, are added isometric 3d H2O is 37 DEG C on shaking table, 200rpm is extracted overnight, and passes through electricity
After dynamic pressure crusher isolates liquid phase, 10000rpm, 10min are centrifugally separating to obtain wherein water phase, are concentrated by being concentrated in vacuo instrument
1mL, by Nucleic acid purification kits (Beckman Agencourt AMPure XP) in small magnetic frame (Agencourt
Spristand 6position tube magnet) on purifying be concentrated into 50 μ L.The fluorescent PCR reagent of outsourcing selects environment sample
Product optimize reagent A BI TaqManTMEnvironmental Master Mix 2.0, DNA extracting solution applied sample amount select 5 μ L.Primer
The Kit components in embodiment one, upper machine testing (ABI fluorescence are pressed in needle group, positive control, negative control, blank control etc.
PCR instrument, model Quantstudio 5).
Testing result is shown in Fig. 1 and table 8：1. quality control standard：FAM, VIC, NED of the system A and system B of positive control have
Fluorescence logarithm increases, and value≤30.0 Ct；Negative control and the equal unstressed configuration signal of blank control and logarithm increase, and Ct values >=
40.0.2. the 30.0 of the FAM of #10 in system A<Ct values<40.0, other unstressed configuration signals and logarithm increase and value >=40.0 Ct；
Increase Ct values after DNA extracting solutions applied sample amount is rechecked to 10 μ L<40.0, judge that testing result for the positive, shows that it contains an ox source
Property ingredient, other samples be free of above-mentioned derived component.3. comparison result：With according to or with reference to SN/T 2980-2011, SN/
The result that the standards such as T3730.7-2013, SN/T 4397-2015 carry out sample correlated source composition detection is consistent.
8 sample initial survey of table and reinspection result：Survey Ct values
The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it cannot be said that
The specific implementation of the present invention is confined to these explanations.For those of ordinary skill in the art to which the present invention belongs, exist
Under the premise of not departing from present inventive concept, a number of simple deductions or replacements can also be made, all shall be regarded as belonging to the present invention's
Protection domain.
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<120>Detect triple fluorescent PCR primer probe groups, kit and the detection method of domestic animals derived component
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Claims (9)
1. detecting the triple fluorescent PCR primer probe groups of domestic animals derived component, it is characterised in that：Including being directed to an ox, buffalo, yak
Triple primed probe group A of ox and triple primed probe group B for sheep, goat；Triple primed probe group A include：
Family ox and general sense primer Seq.ID No.1 of buffalo, family ox and buffalo general reverse primer Seq.ID No.2, yak upstream
Primer Seq.ID No.3, yak downstream primer Seq.ID No.4, family ox fluorescence probe Seq.ID No.9, buffalo fluorescence probe
Seq.ID No.10, yak fluorescence probe Seq.ID No.11；Triple primed probe group B include：Sheep and goat is general
Sense primer Seq.ID No.5, sheep and goat general reverse primer Seq.ID No.6, antelope sense primer Seq.ID
No.7, antelope downstream primer Seq.ID No.8, sheep fluorescence probe Seq.ID No.12, goat fluorescence probe Seq.ID
No.13, antelope fluorescence probe Seq.ID No.14.
2. the triple fluorescent PCR primer probe groups of detection domestic animals derived component according to claim 1, it is characterised in that：
5 ' ends of described ox fluorescence probe are modified with FAM, and 5 ' ends of the buffalo fluorescence probe are modified with VIC, the yak fluorescence
5 ' ends of probe are modified with NED, and 5 ' ends of the sheep fluorescence probe are modified with FAM, and 5 ' ends of the goat fluorescence probe are used
VIC is modified, and 5 ' ends of the antelope fluorescence probe are modified with NED, and 3 ' ends of the probe are modified with NFQ-MGB respectively.
3. detecting the triple fluorescent PCR kit of domestic animals derived component, it is characterised in that：Draw including as claimed in claim 1 or 2
Object probe groups, fluorescent PCR reagent, positive control, negative control, blank control.
4. the triple fluorescent PCR kit of detection domestic animals derived component according to claim 3, it is characterised in that：It is described
The concentration of family ox and the general sense primer of buffalo, downstream primer is respectively 20 μm of ol/L, the yak sense primer, downstream primer
Concentration be respectively 10 μm of ol/L, the general sense primer of the sheep and goat, downstream primer concentration be respectively 20 μm of ol/L,
The antelope sense primer, downstream primer concentration be respectively 10 μm of ol/L, the concentration of the probe is respectively 10 μm of ol/L.
5. the triple fluorescent PCR kit of detection domestic animals derived component according to claim 3, it is characterised in that：It is described
Positive control is the mixing for triple fluorescent PCR primer probe groups as claimed in claim 1 or 2 expand simultaneously tests positive
DNA fragmentation or genome, a concentration of 105L grades of copies/ μ.
6. the triple fluorescent PCR kit of detection domestic animals derived component according to claim 3, it is characterised in that：It is described
Negative control is non-poultry source property DNA；The blank control is ultra-pure water.
7. detecting the triple fluorescent PCR method of domestic animals derived component, it is characterised in that：Include the following steps：1) before carrying out sample
Sample DNA is extracted in processing；2) foundation includes the multiple fluorescence PCR reaction system of primed probe group described in claims 1 or 2, instead
The condition is answered to be：95 DEG C of 20s-2min or 10-15min；95 DEG C of 5s -1min, 60 DEG C of 20s -2min；40cycle simultaneously collects fluorescence letter
Number, it is detected the judgement of result.
8. the triple fluorescent PCR method of the detection domestic animals derived component described according to claim 6 or 7, it is characterised in that：It is described
Sample is deep processing domestic animals food, and the sample pre-treatments include：60 DEG C of drying after being cleaned up sample surfaces with n-hexane,
Isometric 3d H are added2O is 37 DEG C on shaking table, 200rpm is extracted overnight, after expression separation goes out liquid phase, 10000rpm, 10min from
The isolated wherein water phase of the heart is concentrated into 1mL or less by being concentrated in vacuo instrument.
9. the triple fluorescent PCR method of detection domestic animals derived component according to claim 7, it is characterised in that：The inspection
Survey result judgement include：
1. quality control standard：In the PCR reaction systems of triple primed probe group A of positive control and triple primed probe group B, FAM,
VIC, NED have the growth of fluorescence logarithm, and value≤30.0 Ct, negative control and the equal unstressed configuration signal of blank control and logarithm increase
It is long, and value >=40.0 Ct, carry out the judgement of poultry derived component testing result 2.；
2. testing result：In the PCR reaction systems of triple primed probe group A of sample, FAM and/or VIC and/or NED have fluorescence
Logarithm increases, and when and Ct values≤30.0, then contains corresponding family ox and/or buffalo and/or yak derived component；Sample it is triple
In the PCR reaction systems of primed probe group B, FAM and/or VIC and/or NED have a growth of fluorescence logarithm, when and Ct values≤30.0,
Then contain corresponding sheep and/or goat and/or antelope derived component；If above-mentioned one or more fluorescence no signal and logarithm increase
It is long, then be free of corresponding poultry derived component；If 30.0<Ct values<When 40.0, increase template quantity reinspection, if value >=40.0 Ct,
Testing result is feminine gender, if Ct values<40.0, then testing result is the positive.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1490609A (en) * | 2002-10-15 | 2004-04-21 | 曹际娟 | Probe sequence and reagent box for real time fluorescent PCR oringinal determination of ox and sheep |
| CN104946790A (en) * | 2015-07-28 | 2015-09-30 | 中国计量学院 | PCR method for tracking source identification of origin components of eight kinds of animals |
| CN104946788A (en) * | 2015-07-25 | 2015-09-30 | 舟山市食品药品检验检测研究院 | PCR primer and kit for identifying eight kinds of animal-derived components |
| CN105274099A (en) * | 2015-10-23 | 2016-01-27 | 山东省农业科学院生物技术研究中心 | Primers, probe composition and kit for rapid identification of nine animal origin ingredients in food or feed, detection method for identification of nine animal origin ingredients in food or feed and application of primers, probe composition, kit and detection method |
| CN107058498A (en) * | 2017-01-24 | 2017-08-18 | 贵州省产品质量监督检验院 | A kind of detection ox, sheep, triple real time fluorescent PCR methods of pig derived component |
| CN107663543A (en) * | 2017-11-09 | 2018-02-06 | 贵州省产品质量监督检验院 | Detect triple fluorescent PCR primer probe groups, kit and the method for six kinds of avian compositions |
-
2018
- 2018-05-30 CN CN201810540219.9A patent/CN108531618A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1490609A (en) * | 2002-10-15 | 2004-04-21 | 曹际娟 | Probe sequence and reagent box for real time fluorescent PCR oringinal determination of ox and sheep |
| CN104946788A (en) * | 2015-07-25 | 2015-09-30 | 舟山市食品药品检验检测研究院 | PCR primer and kit for identifying eight kinds of animal-derived components |
| CN104946790A (en) * | 2015-07-28 | 2015-09-30 | 中国计量学院 | PCR method for tracking source identification of origin components of eight kinds of animals |
| CN105274099A (en) * | 2015-10-23 | 2016-01-27 | 山东省农业科学院生物技术研究中心 | Primers, probe composition and kit for rapid identification of nine animal origin ingredients in food or feed, detection method for identification of nine animal origin ingredients in food or feed and application of primers, probe composition, kit and detection method |
| CN107058498A (en) * | 2017-01-24 | 2017-08-18 | 贵州省产品质量监督检验院 | A kind of detection ox, sheep, triple real time fluorescent PCR methods of pig derived component |
| CN107663543A (en) * | 2017-11-09 | 2018-02-06 | 贵州省产品质量监督检验院 | Detect triple fluorescent PCR primer probe groups, kit and the method for six kinds of avian compositions |
Non-Patent Citations (1)
| Title |
|---|
| RENE: "Multiplex real-time PCR for the detection and quantification of DNA from duck, goose, chicken, turkey and pork", 《EUR FOOD RES TECHNOL》 * |
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