CN104250667B - Detecting method for horse source elements in meat and meat products and detecting method for donkey source element in meat and meat products - Google Patents
Detecting method for horse source elements in meat and meat products and detecting method for donkey source element in meat and meat products Download PDFInfo
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Abstract
The invention provides a detecting method and kit for detecting horse source elements in meat and meat products. The kit for detecting horse source elements in meat and meat products comprises an upstream primer as shown in SEQ ID No.1 (in the Specification) and a downstream primer as shown in SEQ ID No.2 (in the Specification). The invention further provides a detecting method and kit for detecting donkey source elements in the meat and meat products. The kit for detecting donkey source elements in the meat and meat products comprises an upstream primer as shown in SEQ ID No.3 (in the Specification) and a downstream primer as shown in SEQ ID No.4 (in the Specification). The upstream primer group is selected to perform PCR amplification on DNA templates extracted from the meat and meat products, so that high specificity is achieved, and the high sensitivity and accuracy of the detection methods can be guaranteed.
Description
Technical field
The invention belongs to technical field of food detection, the detection method of more particularly, to a kind of meat and horse derived components in meat productss
And the detection method of donkey derived components.
Background technology
With the development that deepens continuously of market economy, part illegal retailer in order to seek illegitimate benefits, in meat or meat system
Adulterate in product other low-cost meat kinds, occur in that a kind of phenomenon adulterated, mix the spurious with the genuine in meat and meat productss market.
Mixed with other the low-cost animal derived materials beyond nominal in meat or meat productss, have even all nominal beyond
Other low-cost animal derived materials, this greatly compromises the interests of consumer, has upset circulation market order and has been good for
Kang Fazhan, also causes certain potential conflict to ethnic and religious faith simultaneously.
Tradition is typically judged to parameters such as the color and luster of meat or meat productss, abnormal smells from the patient, tissue profile using sense organ method, but
It is that its reliability is poor, especially with the addition of the additives such as pigment and aromatic or the meat through other processed and meat system
For product, sense organ method judgment accuracy is lower.
Content of the invention
In view of this, it is an object of the invention to provide the detection method of horse derived components and donkey source in a kind of meat and meat productss
The detection method of composition, the detection method result that the present invention provides is accurate, reliable.
The invention provides a kind of test kit for detecting meat and horse derived components in meat productss, comprising: such as seq id
Forward primer shown in no.1 and the downstream primer as shown in seq id no.2.
The present invention is based on the cytb gene order of all horses having logged on announcement on genbank, soft with biology
Part dnastar carries out sequence alignment analysis, find out from country variant, area, kind horse, cytb gene kind in conserved sequence
Region, for designing upstream and downstream amplimer, by designed go out each species pcr amplimer compared again, exclusion kind
Between intercrossing it is ensured that amplification specificity.Meanwhile, find out total homologous sequence in the cytb gene order of horse, design suckling
Animal detection primer, in order to be monitored to each detection system.Finally, each species upstream and downstream amplimer sequence obtaining is carried
Hand over gembank, carry out blast confirmation, non-spy is not had to the dna sequence announced at present with the primer sequence designed by guaranteeing
Opposite sex detection amplification, and select forward primer and downstream primer.The present invention is used for from specific forward primer and downstream primer
Horse derived components in detection meat and meat productss, it has good specificity such that it is able to realize the detection to horse derived components.
Pcr expansion is carried out with the forward primer as shown in seq id no.1 and the downstream primer as shown in seq id no.2
Increase, its amplification site and amplified fragments size are as shown in table 1:
The amplification site of table 1 horse derived components and amplified fragments size
The concentration of described forward primer is preferably 0.1 μm～0.5 μm, more preferably 0.5 μm；The concentration of described downstream primer
It is preferably 0.1 μm～0.5 μm, more preferably 0.5 μm.
Dna polymerase, 10 × pcr buffer solution and deionized water, described dna polymerization is also included in described test kit
The concentration of enzyme is preferably 0.5-1u/ μ l.Also include in described test kit, described 10 × pcr buffer solution specifically includes:
100mm tris-hcl (ph8.3), 500mm kcl, 15mm mgcl2.
Present invention also offers a kind of meat and the detection method of horse derived components in meat productss, comprise the following steps:
Extract the dna of meat sample product or meat productss sample；
Pcr amplification, the such as seq id of the forward primer in described pcr amplification reaction system are carried out for template with described dna
Shown in no.1, downstream primer is as shown in seq id no.2；
Described pcr amplified production is carried out with electrophoresis, identifies described amplified production.
The present invention extracts the dna in meat sample product or meat productss sample first, and the present invention does not have to the extracting method of described dna
There is particular restriction, can be extracted using commercial reagents box when extracting in a small amount, can carry out according to ctab-nacl method when extracting in a large number
Extract, specifically:
When extracting in a small amount, its extracting method is:
A. by the meat paste weighing, put in mortar, add liquid nitrogen, quickly, be firmly ground to powder after sample freezing completely
Last shape, should be interrupted addition liquid nitrogen to prevent material melts during grinding；
B. mortar is moved into 65 DEG C of water-baths, when sample powder just starts to melt, add 700 μ l's in mortar
The solution a and rnase a of 1.2 μ l, grinds 30 seconds；
C. collect the ground tissue homogenate of 650 μ l to collection tube.As being homogenized volume less than 650 μ l, mend
Fill solution a to 650 μ l.65 DEG C are incubated 10 minutes；
D. add the solution b of 400 μ l, vibration mixes；
E. add the solution c of 4 DEG C of pre-coolings of 1ml, after fully mixing, 12000rpm is centrifuged 2 minutes；
F. discard upper organic phase, add the solution c of 4 DEG C of pre-coolings of 1ml, after fully mixing, 12000rpm from
The heart 2 minutes；
G. discard upper organic phase, then aqueous phase solution (colourless lower floor) is transferred to and is placed on collection tube
Filter cup in, 12000rpm be centrifuged 1 minute；
H. abandon filter cup, filtrate adds the db buffer of 400 μ l, mix homogeneously；
I. the spin column in test kit is placed on collection tube.Mixed solution by aforesaid operations
It is transferred in spin column, 12000rpm is centrifuged 1 minute, abandons filtrate；
J. the rinse a of 500 μ l is added to spin column, 12000rpm is centrifuged 30 seconds, abandons filtrate；
K. the rinse b of 700 μ l is added to spin column, 12000rpm is centrifuged 30 seconds, abandons filtrate；
L. repeat work of drilling；
M. spin column is placed on the centrifuge tube of new 1.5ml, adds in the centre of spin column film
The sterile purified water of 50-200 μ l or elution buffer, room temperature stands 1 minute；
N.12000rpm 1 minute eluting of centrifugation collects dna.
During a large amount of extraction, its extracting method can be:
A. take the sample muscular tissue that 2g is broken, to 50ml centrifuge tube, plus 10ml ctab-nacl lysate, 65 DEG C are shaken
Swing water-bath 1 hour；
B. 10 μ l proteinase k (20mg/ml) are added, 65 DEG C of shaking baths, overnight；
C. take 1ml overnight treatment fluid to eppendorf centrifuge tube, 12000rpm be centrifuged 10min；
D. take 800 μ l centrifugation supernatants to new centrifuge tube, add 600 μ l chloroforms, fully mix, 12000rpm is centrifuged
10min；
E. take 600 μ l upper strata aqueous phases to new centrifuge tube, add the pre- cold isopropanol of 500 μ l, turn upside down centrifuge tube, room
Temperature precipitation dna, 30min；
F.12000rpm be centrifuged 10min, abandon isopropanol, add 1ml 75% ice ethanol to carry out washing dna, 12000rpm from
Heart 2min, abandons most ethanol, air-dries under room temperature, adds appropriate ddw to dissolve dna, and measures od260Value.
After obtaining the dna in meat sample product or meat productss, pcr amplification, described pcr amplified reaction are carried out as template using it
, as shown in seq id no.1, downstream primer is as shown in seqid no.2 for forward primer in system.
The concentration of described forward primer is preferably 0.1 μm～0.5 μm, more preferably 0.5 μm；The concentration of described downstream primer
It is preferably 0.1 μm～0.5 μm, more preferably 0.5 μm.
Dna polymerase, 10 × pcr buffer solution and deionized water, institute is also included in described pcr amplification reaction system
The concentration stating dna polymerase is preferably: 0.5-1u/ μ l.Described 10 × pcr buffer solution specifically includes: 100mm tris-
Hcl (ph8.3), 500mm kcl, 15mm mgcl2.In described pcr amplification system, the concentration of described template dna is preferably 10～
100ng/μl.
In a specific embodiment, described pcr amplification reaction system may include that
Described pcr Amplification is:
After pcr amplification terminates, the pcr amplified production obtaining is carried out with electrophoresis, identify described amplified production, concrete grammar is such as
Under: preparation 1% concentration agarose gel, take 8 μ l amplified productions, carry out electrophoresis, electrophoresis terminates, eb dyes, observing under ultraviolet has
No non-specific amplification band, if be described in meat or meat productss containing horse derived component, if no illustrate in meat or meat productss not
Containing horse derived component.
In order to further confirm that testing result, the present invention can also carry out enzyme action to the amplified production obtaining further, mirror
Determine digestion products, thus judging whether to contain horse derived components in meat sample product or meat productss.The present invention preferably uses restriction enzyme
Enzyme xhoi carries out enzyme action to described amplified production, and its relevant parameter is as follows:
| Detection system | Expected size (bp) | Checking restricted enzyme | Endonuclease bamhi size (bp) |
| Horse derived component detection system | 395 | xhoi | 142+253 |
After enzyme action finishes, if the endonuclease bamhi obtaining meets target fragment, you can confirm to contain horse in detected sample
Derived component；If not meeting target fragment, endonuclease bamhi can be sequenced further.
Compared with prior art, the present invention is based on polymerase chain reaction (pcr) method in molecular biology, from base
Differentiate because level carries out identification to meat with meat productss dna, extract the dna of meat sample product or meat productss sample first；Then with described
Dna carries out pcr amplification for template, and the forward primer in described pcr amplification reaction system as shown in seq id no.1, draw by downstream
Thing is as shown in seq id no.2；Described pcr amplified production is carried out with enzyme action, identifies described digestion products, thus being produced according to amplification
The length scale of thing and restriction enzyme site information carry out comprehensive analysis, judge the horse derived components in meat and meat productss.The present invention selects
Select above-mentioned primer sets and pcr amplification is carried out to the dna template of meat and extraction in meat productss, there is higher specificity, ensure that
The sensitivity of detection method and accuracy.Test result indicate that, the method that the present invention provides is to meat and horse derived components in meat productss
Monitoring lower-cut be 0.01ng/ μ l, accuracy rate is up to 100%.
The invention provides a kind of test kit for detecting meat and donkey derived components in meat productss, comprising: such as seq id
Forward primer shown in no.3 and the downstream primer as shown in seq id no.4.
The present invention is based on the cytb gene order of all donkeys having logged on announcement on genbank, soft with biology
Part dnastar carries out sequence alignment analysis, finds out and guards sequence in country variant, area, the cytb gene kind of the donkey of kind
Column region, for designing upstream and downstream amplimer, by designed go out each species pcr amplimer compared again, exclusion
Inter-species intercrossing it is ensured that amplification specificity.Meanwhile, find out total homologous sequence in the cytb gene order of donkey, design is fed
Newborn animal detection primer, in order to be monitored to each detection system.Finally, each species upstream and downstream amplimer sequence that will obtain
Submit gembank to, carry out blast confirmation, not non-to the dna sequence announced at present to guarantee designed primer sequence
Specific detection expands, and selectes forward primer and downstream primer.The present invention selects specific forward primer and downstream primer to use
Donkey derived components in detection meat with meat productss, it has good specificity such that it is able to realize the detection to donkey derived components.
Pcr expansion is carried out with the forward primer as shown in seq id no.3 and the downstream primer as shown in seq id no.4
Increase, its amplification site and amplified fragments size are as shown in table 2:
The amplification site of table 2 donkey derived components and amplified fragments size
The concentration of described forward primer is preferably 0.1 μm～0.5 μm, more preferably 0.5 μm；The concentration of described downstream primer
It is preferably 0.1 μm～0.5 μm, more preferably 0.5 μm.
Dna polymerase, 10 × pcr buffer solution and deionized water, described dna polymerization is also included in described test kit
The concentration of enzyme is preferably 0.5-1u/ μ l.Also include in described test kit, described 10 × pcr buffer solution specifically includes:
100mm tris-hcl (ph8.3), 500mm kcl, 15mm mgcl2.
Present invention also offers a kind of meat and the detection method of donkey derived components in meat productss, comprise the following steps:
Extract the dna of meat sample product or meat productss sample；
Pcr amplification, the such as seq id of the forward primer in described pcr amplification reaction system are carried out for template with described dna
Shown in no.3, shown in downstream primer seq id no.4；
Described pcr amplified production is carried out with electrophoresis, identifies described amplified production.
The present invention extracts the dna in meat sample product or meat productss sample first, and the present invention does not have to the extracting method of described dna
There is particular restriction, can be extracted using commercial reagents box when extracting in a small amount, can carry out according to ctab-nacl method when extracting in a large number
Extract, specifically:
When extracting in a small amount, its extracting method is:
A. by the meat paste weighing, put in mortar, add liquid nitrogen, quickly, be firmly ground to powder after sample freezing completely
Last shape, should be interrupted addition liquid nitrogen to prevent material melts during grinding；
B. mortar is moved into 65 DEG C of water-baths, when sample powder just starts to melt, add 700 μ l's in mortar
The solution a and rnase a of 1.2 μ l, grinds 30 seconds；
C. collect the ground tissue homogenate of 650 μ l to collection tube.As being homogenized volume less than 650 μ l, mend
Fill solution a to 650 μ l.65 DEG C are incubated 10 minutes；
D. add the solution b of 400 μ l, vibration mixes；
E. add the solution c of 4 DEG C of pre-coolings of 1ml, after fully mixing, 12000rpm is centrifuged 2 minutes；
F. discard upper organic phase, add the solution c of 4 DEG C of pre-coolings of 1ml, after fully mixing, 12000rpm from
The heart 2 minutes；
G. discard upper organic phase, then aqueous phase solution (colourless lower floor) is transferred to and is placed on collection tube
Filter cup in, 12000rpm be centrifuged 1 minute；
H. abandon filter cup, filtrate adds the db buffer of 400 μ l, mix homogeneously；
I. the spin column in test kit is placed on collection tube.Mixed solution by aforesaid operations
It is transferred in spin column, 12000rpm is centrifuged 1 minute, abandons filtrate；
J. the rinse a of 500 μ l is added to spin column, 12000rpm is centrifuged 30 seconds, abandons filtrate；
K. the rinse b of 700 μ l is added to spin column, 12000rpm is centrifuged 30 seconds, abandons filtrate；
L. repeat work of drilling；
M. spin column is placed on the centrifuge tube of new 1.5ml, adds in the centre of spin column film
The sterile purified water of 50-200 μ l or elution buffer, room temperature stands 1 minute；
N.12000rpm 1 minute eluting of centrifugation collects dna.
During a large amount of extraction, its extracting method can be:
A. take the sample muscular tissue that 2g is broken, to 50ml centrifuge tube, plus 10ml ctab-nacl lysate, 65 DEG C are shaken
Swing water-bath 1 hour；
B. 10 μ l proteinase k (20mg/ml) are added, 65 DEG C of shaking baths, overnight；
C. take 1ml overnight treatment fluid to eppendorf centrifuge tube, 12000rpm be centrifuged 10min；
D. take 800 μ l centrifugation supernatants to new centrifuge tube, add 600 μ l chloroforms, fully mix, 12000rpm is centrifuged
10min；
E. take 600 μ l upper strata aqueous phases to new centrifuge tube, add the pre- cold isopropanol of 500 μ l, turn upside down centrifuge tube, room
Temperature precipitation dna, 30min；
F.12000rpm be centrifuged 10min, abandon isopropanol, add 1ml 75% ice ethanol to carry out washing dna, 12000rpm from
Heart 2min, abandons most ethanol, air-dries under room temperature, adds appropriate ddw to dissolve dna, and measures od260Value.
After obtaining the dna in meat sample product or meat productss, pcr amplification, described pcr amplified reaction are carried out as template using it
, as shown in seq id no.3, downstream primer is as shown in seq id no.4 for forward primer in system.
The concentration of described forward primer is preferably 0.1 μm～0.5 μm, more preferably 0.5 μm；The concentration of described downstream primer
It is preferably 0.1 μm～0.5 μm, more preferably 0.5 μm.
Dna polymerase, 10 × pcr buffer solution and deionized water, institute is also included in described pcr amplification reaction system
The concentration stating dna polymerase is preferably 0.5-1u/ μ l.Also include in described test kit, described 10 × pcr buffer solution tool
Body includes: 100mm tris-hcl (ph8.3), 500mm kcl, 15mm mgcl2.In described pcr amplification system, described template
The concentration of dna is preferably 10～100ng/ μ l.
In a specific embodiment, described pcr amplification reaction system may include that
Described pcr Amplification is:
After pcr amplification terminates, the pcr amplified production obtaining is carried out with electrophoresis, identify described amplified production, concrete grammar is such as
Under: preparation 1% concentration agarose gel, take 8 μ l amplified productions, carry out electrophoresis, electrophoresis terminates, eb dyes, observing under ultraviolet has
No non-specific amplification band, if be described in meat or meat productss containing donkey derived component, if no illustrate in meat or meat productss not
Containing donkey derived component.
In order to further confirm that testing result, the present invention can also carry out enzyme action to the amplified production obtaining further, mirror
Determine digestion products, thus judging whether to contain donkey derived components in meat sample product or meat productss.The present invention preferably uses restriction enzyme
Enzyme xhoi carries out enzyme action to described amplified production, and its relevant parameter is as follows:
| Detection system | Expected size (bp) | Checking restricted enzyme | Endonuclease bamhi size (bp) |
| Donkey derived component detection system | 229 | xhoi | 103+126 |
After enzyme action finishes, if the endonuclease bamhi obtaining meets target fragment, you can confirm to contain horse in detected sample
Derived component；If not meeting target fragment, endonuclease bamhi can be sequenced further.
Compared with prior art, the present invention is based on polymerase chain reaction (pcr) method in molecular biology, from base
Differentiate because level carries out identification to meat with meat productss dna, extract the dna of meat sample product or meat productss sample first；Then with described
Dna carries out pcr amplification for template, and the forward primer in described pcr amplification reaction system as shown in seq id no.3, draw by downstream
Thing is as shown in seq id no.4；Described pcr amplified production is carried out with enzyme action, identifies described digestion products, thus being produced according to amplification
The length scale of thing and restriction enzyme site information carry out comprehensive analysis, judge the donkey derived components in meat and meat productss.The present invention selects
Select above-mentioned primer sets and pcr amplification is carried out to the dna template of meat and extraction in meat productss, there is higher specificity, ensure that
The sensitivity of detection method and accuracy.Test result indicate that, the method that the present invention provides is to meat and donkey derived components in meat productss
Monitoring lower-cut be 1.0ng/ μ l, accuracy rate is up to 100%.
Brief description
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
Have technology description in required use accompanying drawing be briefly described it should be apparent that, drawings in the following description be only this
Inventive embodiment, for those of ordinary skill in the art, on the premise of not paying creative work, can also basis
The accompanying drawing providing obtains other accompanying drawings.
The electrophoresis result of the amplified production that Fig. 1 obtains for the embodiment of the present invention 1；
Fig. 2 is the electrophoresis result of the amplified production that the embodiment of the present invention 1 and comparative example 1～8 obtain；
Fig. 3 is horse provided in an embodiment of the present invention and the electrophoresis result of donkey standard endonuclease bamhi；
The electrophoresis result of the donkey amplified production that Fig. 4 obtains for the embodiment of the present invention 4；
Fig. 5 is the electrophoresis result of the amplified production that the embodiment of the present invention 4 and comparative example 9～17 obtain；
The electrophoresis result of the amplified production that Fig. 6 obtains for the embodiment of the present invention 7～16；
The electrophoresis result of the amplified production that Fig. 7 obtains for the embodiment of the present invention 17～26.
Specific embodiment
Below in conjunction with the embodiment in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear,
It is fully described by it is clear that described embodiment is only a part of embodiment of the present invention, rather than whole embodiments.Base
Embodiment in the present invention, those of ordinary skill in the art obtained under the premise of not making creative work all its
His embodiment, broadly falls into the scope of protection of the invention.
Embodiment 1
The method extracted in a small amount as described above extracts the dna of Mongolian horse；
To extract the dna obtaining as template, expanded according to following Amplification in following pcr amplification reaction system
Increase, obtain amplified production；
Pcr amplification reaction system may include that
Described pcr Amplification is:
Wherein, forward primer is as shown in seq id no.1, shown in downstream primer seq id no.2；
After pcr amplification terminates, the pcr amplified production obtaining is carried out with electrophoresis, identify described amplified production, concrete grammar is such as
Under: preparation 1% concentration agarose gel, take 8 μ l amplified productions, carry out electrophoresis, electrophoresis terminates, eb dyes, observe under ultraviolet, knot
The electrophoresis result of the amplified production that fruit obtains for the embodiment of the present invention 1 referring to Fig. 1, Fig. 1, wherein, 1 amplification obtaining for embodiment
The electrophoresis result of product, m is dl2000.As shown in Figure 1, the method that the present invention provides can expand and obtain genes of interest, therefore
Can be used in the detection of horse derived components.
Embodiment 2
According to method same as Example 1, Equus caballus (L.) is detected, with difference is, extract the dna of Yili horse, result is joined
See Fig. 1, the electrophoresis result of the amplified production that Fig. 1 obtains for the embodiment of the present invention 1, wherein, 2 amplifications obtaining for embodiment 2 are produced
The electrophoresis result of thing, m is dl2000.As shown in Figure 1, the method that the present invention provides is capable of the horse derived components to different cultivars
Detection.
Comparative example 1～8
According to method same as Example 1 respectively to Carnis Leporis, Carnis Anseris domestica, duck meat, Carnis Gallus domesticus, Carnis caprae seu ovis, Carnis Equi Asini, beef and Carnis Sus domestica
Detected, result referring to Fig. 2, the electrophoresis result of the amplified production that Fig. 2 is the embodiment of the present invention 1 and comparative example 1～8 obtains,
Wherein m is dl2000.As shown in Figure 2, the method that the present invention provides has preferable specificity.
Embodiment 3
Obtain after amplified production according to method same as Example 1, enzyme is carried out to it using restricted enzyme xhoi
Cut, result is dl2000 referring to Fig. 3, wherein m, and Fig. 3 is the electrophoresis result of endonuclease bamhi provided in an embodiment of the present invention.
Embodiment 4
The method that a small amount of as described above is extracted extracts the dna of Region in Guanzhong Donkey；
To extract the dna obtaining as template, expanded according to following Amplification in following pcr amplification reaction system
Increase, obtain amplified production；
Pcr amplification reaction system may include that
Described pcr Amplification is:
Wherein, forward primer is as shown in seq id no.3, shown in downstream primer seq id no.4；
After pcr amplification terminates, the pcr amplified production obtaining is carried out with electrophoresis, identify described amplified production, concrete grammar is such as
Under: preparation 1% concentration agarose gel, take 8 μ l amplified productions, carry out electrophoresis, electrophoresis terminates, eb dyes, observe under ultraviolet, knot
The electrophoresis result of the amplified production that fruit obtains for the embodiment of the present invention 4 referring to Fig. 4, Fig. 4, wherein, 1 expansion obtaining for embodiment 4
The electrophoresis result of volume increase thing, m is dl2000.As shown in Figure 4, the method that the present invention provides can expand and obtain genes of interest, because
This can be used in the detection of donkey derived components.
Embodiment 5
According to method same as Example 4, Carnis Equi Asini is detected, with difference is, extract the dna of Dezhou donkey, result is joined
See Fig. 4, the electrophoresis result of the amplified production that Fig. 4 obtains for the embodiment of the present invention 4, wherein, 2 amplifications obtaining for embodiment 5 are produced
The electrophoresis result of thing, m is dl2000.As shown in Figure 4, the method that the present invention provides is capable of the donkey derived components to different cultivars
Detection.
Comparative example 9～17
According to method same as Example 4 respectively to Carnis Leporis, Carnis Anseris domestica, duck meat, Carnis Gallus domesticus, Carnis caprae seu ovis, Equus caballus (L.), beef and Carnis Sus domestica
Detected, result referring to Fig. 5, the electrophoresis result of the amplified production that Fig. 5 is the embodiment of the present invention 4 and comparative example 9～17 obtains,
Wherein m is dl2000.As shown in Figure 5, the method that the present invention provides has preferable specificity.
Embodiment 6
Obtain after amplified production according to method same as Example 1, enzyme is carried out to it using restricted enzyme xhoi
Cut, result is dl2000 referring to Fig. 3, wherein m, and Fig. 3 is the electrophoresis result of endonuclease bamhi provided in an embodiment of the present invention
Embodiment 7～16
According to method same as Example 1, Equus caballus (L.) is detected, with difference is, the concentration of dna template is respectively
0.01st, 0.1,1.0,10,50,100,200,300,400 and 500ng/ μ l, result referring to Fig. 6, Fig. 6 be the embodiment of the present invention 7～
The electrophoresis result of 16 amplified productions obtaining, wherein m is for dl2000. it will be appreciated from fig. 6 that the method for present invention offer is to horse derived components
Monitoring lower-cut be 0.01ng/ μ l.
Embodiment 17～26
According to method same as Example 4, Carnis Equi Asini is detected, with difference is, the concentration of dna template is respectively
0.01st, 0.1,1.0,10,50,100,200,300,400 and 500ng/ μ l, referring to Fig. 7, Fig. 7 is the embodiment of the present invention 17 to result
The electrophoresis result of～26 amplified productions obtaining, for dl2000. as shown in Figure 7, the method that the present invention provides is to Ma Yuancheng for wherein m
The Monitoring lower-cut dividing is 1.0ng/ μ l.
Embodiment 27
Detected, difference is according to method same as Example 1, detection object is the mixing of Equus caballus (L.) and Carnis Equi Asini
Thing, detection is all capable of detecting when for 20 times the accuracy rate of the method that it contains Equus caballus (L.) composition, and the present invention provides up to 100%.
Embodiment 28
Detected, difference is according to method same as Example 1, detection object is Carnis Equi Asini, detected 20 times all no
Method detects the accuracy rate of the method that it contains Equus caballus (L.) composition, and the present invention provides up to 100%.
Embodiment 29
Detected, difference is according to method same as Example 4, detection object is the mixing of Equus caballus (L.) and Carnis Equi Asini
Thing, detection is all capable of detecting when for 20 times the accuracy rate of the method that it contains Carnis Equi Asini composition, and the present invention provides up to 100%.
Embodiment 30
Detected, difference is according to method same as Example 4, detection object is Equus caballus (L.), detected 20 times all no
Method detects the accuracy rate of the method that it contains Carnis Equi Asini composition, and the present invention provides up to 100%.
The above is only the preferred embodiment of the present invention it is noted that ordinary skill people for the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (6)
1. a kind of test kit for detecting meat and horse derived components in meat productss is it is characterised in that include: such as seq id no.1
Shown forward primer and the downstream primer as shown in seq id no.2.
2. test kit according to claim 1 it is characterised in that described forward primer concentration be 0.1 μm～0.5 μm,
The concentration of described downstream primer is 0.1 μm～0.5 μm.
3. a kind of meat and the detection method of horse derived components in meat productss, comprises the following steps:
Extract the dna of meat sample product or meat productss sample；
Pcr amplification is carried out for template with described dna, the such as seq id no.1 institute of the forward primer in described pcr amplification reaction system
Show, downstream primer is as shown in seq id no.2；
Described pcr amplified production is carried out with electrophoresis, identifies described amplified production.
4. detection method according to claim 3 is it is characterised in that also include:
Enzyme action is carried out to described amplified production.
5. the detection method according to claim 3 or 4 is it is characterised in that in described pcr amplification reaction system, described on
The concentration of trip primer is 0.1 μm～0.5 μm, and the concentration of described downstream primer is 0.1 μm～0.5 μm.
6. detection method according to claim 5 is it is characterised in that in described pcr amplification reaction system, described upstream is drawn
The concentration of thing is 0.5 μm, and the concentration of described downstream primer is 0.5 μm.
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| CN201410528241.3A CN104250667B (en) | 2014-10-09 | 2014-10-09 | Detecting method for horse source elements in meat and meat products and detecting method for donkey source element in meat and meat products |
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| CN201410528241.3A CN104250667B (en) | 2014-10-09 | 2014-10-09 | Detecting method for horse source elements in meat and meat products and detecting method for donkey source element in meat and meat products |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105671034A (en) * | 2015-04-29 | 2016-06-15 | 中华人民共和国汕头出入境检验检疫局 | Locked nucleic acid probe fluorescent quantitative PCR method for quick identification on adulteration of horse-sourced component in meat products, and primer and locked nucleic acid probe sequence thereof |
| CN105586420B (en) * | 2016-01-29 | 2020-08-28 | 湖南省药品检验研究院 | Specific primer pair and method for identifying donkey-derived component in donkey-hide gelatin raw material |
| CN107245520A (en) * | 2017-06-09 | 2017-10-13 | 北京农学院 | A kind of detection method and kit for being used to detect meat and donkey derived components in meat products |
| CN109280708A (en) * | 2017-07-19 | 2019-01-29 | 成都市食品药品检验研究院 | It is a kind of for detecting the kit and method of donkey derived component |
| CN109825610B (en) * | 2019-04-11 | 2021-03-19 | 中国农业大学 | Kit for rapidly detecting horse-derived components in food and application thereof |
| CN109852705B (en) * | 2019-04-11 | 2021-01-01 | 中国农业大学 | Method and kit for rapidly detecting horse-derived components in food |
| CN111763714B (en) * | 2020-07-21 | 2023-06-13 | 山东省农业科学院畜牧兽医研究所 | Kit for rapidly identifying donkey and horse-derived components and donkey and horse-derived components in product and application of kit |
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| CN102876805A (en) * | 2012-10-25 | 2013-01-16 | 苏州市红冠庄国药饮片有限公司 | Primer system for PCR (polymerase chain reaction) identification for deer, pig, cow, sheep, horse, donkey, rabbit and chicken |
| CN103154268A (en) * | 2010-07-23 | 2013-06-12 | 耶迪特普大学 | A kit for detection of donkey meat in meat products |
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| CN103154268A (en) * | 2010-07-23 | 2013-06-12 | 耶迪特普大学 | A kit for detection of donkey meat in meat products |
| CN102876805A (en) * | 2012-10-25 | 2013-01-16 | 苏州市红冠庄国药饮片有限公司 | Primer system for PCR (polymerase chain reaction) identification for deer, pig, cow, sheep, horse, donkey, rabbit and chicken |
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